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Sample records for allele specific primers

  1. Microarrays for high-throughput genotyping of MICA alleles using allele-specific primer extension.

    PubMed

    Baek, I C; Jang, J-P; Choi, H-B; Choi, E-J; Ko, W-Y; Kim, T-G

    2013-10-01

    The role of major histocompatibility complex (MHC) class I chain-related gene A (MICA), a ligand of NKG2D, has been defined in human diseases by its allele associations with various autoimmune diseases, hematopoietic stem cell transplantation (HSCT) and cancer. This study describes a practical system to develop MICA genotyping by allele-specific primer extension (ASPE) on microarrays. From the results of 20 control primers, strict and reliable cut-off values of more than 30,000 mean fluorescence intensity (MFI) as positive and less than 3000 MFI as negative, were applied to select high-quality specific extension primers. Among 55 allele-specific primers, 44 primers could be initially selected as optimal primer. Through adjusting the length, six primers were improved. The other failed five primers were corrected by refractory modification. MICA genotypes by ASPE on microarrays showed the same results as those by nucleotide sequencing. On the basis of these results, ASPE on microarrays may provide high-throughput genotyping for MICA alleles for population studies, disease-gene associations and HSCT.

  2. Determination of DQB1 alleles using PCR amplification and allele-specific primers.

    PubMed

    Lepage, V; Ivanova, R; Loste, M N; Mallet, C; Douay, C; Naoumova, E; Charron, D

    1995-10-01

    Molecular genotyping of HLA class II genes is commonly carried out using polymerase chain reaction (PCR) in combination with sequence-specific oligotyping (PCR-SSO) or a combination of the PCR and restriction fragment length polymorphism methods (PCR-RFLP). However, the identification of the DQB1 type by PCR-SSO and PCR-RFLP is very time-consuming which is disadvantageous for the typing of cadaveric organ donors. We have developed a DQB1 typing method using PCR in combination with allele-specific amplification (PCR-ASA), which allows the identification of the 17 most frequent alleles in one step using seven amplification mixtures. PCR allele-specific amplification HLA-DQB1 typing is easy to perform, and the results are easy to interpret in routine clinical practice. The PCR-ASA method is therefore better suited to DQB1 typing for organ transplantation than other methods.

  3. Identification of new primer binding site mutations at TH01 and D13S317 loci and determination of their corresponding STR alleles by allele-specific PCR.

    PubMed

    Li, Fengrui; Xuan, Jinfeng; Xing, Jiaxin; Ding, Mei; Wang, Baojie; Pang, Hao

    2014-01-01

    Several commercial multiplex PCR kits for the amplification of short tandem repeat (STR) loci have been extensively applied in forensic genetics. Consequently, large numbers of samples have been genotyped, and the number of discordant genotypes observed has also increased. We observed allele dropout with two novel alleles at the STR loci TH01 and D13S317 during paternity testing using the AmpFℓSTR Identifiler PCR Amplification Kit. The lost alleles reappeared when alternative PCR primer pairs were used. A sequence analysis revealed a G-to-A substitution 82 bases downstream of the last TCAT motif of the repeat region at the TH01 locus (GenBank accession: D00269) and a G-to-T substitution 90 bases upstream of the first TATC motif of the repeat region at the D13S317 locus (GenBank accession: G09017). The frequencies of these two point mutations were subsequently investigated in the Chinese population using sequence-specific primer PCR (SSP-PCR), but neither of these mutations was detected in any of the samples tested. In addition, the DNA samples in which the mutations were identified were amplified to type the point mutations by SSP-PCR to determine the corresponding STR alleles at the two loci. Subsequently, the amplified PCR products with different point mutations and STR repeat numbers were directly sequenced because this strategy overcomes the appearance overlapping peaks generated by different STR alleles and accurately characterizes genotypes. Thus, our findings not only provide useful information for DNA databases and forensic identification but also establish an effective strategy for typing STR alleles with primer binding site mutations.

  4. New primer for specific amplification of the CAG repeat in Huntington disease alleles

    SciTech Connect

    Bond, C.E.; Hodes, M.E.

    1994-09-01

    Huntington disease is an autosomal dominant neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat near the 5{prime} end of the gene for Huntington disease (IT15). The CAG repeat is flanked by a variable-length CCG repeat that is included in the amplification product obtained with most currently used primer sets and PCR protocols. Inclusion of this adjacent CCG repeat complicates the accurate assessment of CAG repeat length and interferes with the genotype determination of those individuals carrying alleles in the intermediate range between normal and expanded sized. Due to the GC-rich nature of this region, attempts at designing a protocol for amplification of only the CAG repeat have proved unreliable and difficult to execute. We report here the development of a compatible primer set and PCR protocol that yields consistent amplification of the CAG-repeat region. PCR products can be visualized in ethidium bromide-stained agarose gels for rapid screening or in 6% polyacrylamide gels for determination of exact repeat length. This assay produces bands that can be sized accurately, while eliminating most nonspecific products. Fifty-five specimens examined showed consistency with another well-known method, but one that amplifies the CCG repeats as well. The results we obtained also matched the known carrier status of the donors.

  5. A novel one cycle allele specific primer extension--molecular beacon displacement method for DNA point mutation detection with improved specificity.

    PubMed

    Li, Xiaomin; Huang, Yong; Guan, Yuan; Zhao, Meiping; Li, Yuanzong

    2007-02-12

    We report here a new method for the real-time detection of DNA point mutations with molecular beacon as the fluorescence tracer and 3' (exo-) Bst DNA polymerase large fragment as the polymerase. The method is based on the mechanism of allele specific primer extension-strand displacement (ASPE-SD). To improve the specificity of the method only one cycle of the allele specific polymerase chain reaction (PCR) was used that could largely eliminate the non-specific reactions between the primers and template of the "wrong" genotype. At first, the primer and molecular beacon both hybridize to the DNA template, and the molecular beacon emits intensive fluorescence. The role of 3' exonuclease excision of Bst DNA polymerase large fragment is utilized for primer extension. When 3'-termini matches its corresponding template, the primer would efficiently extend and replace the molecular beacon that would simultaneously return to its closed form leading to the quenching of the fluorescence. However, when 3'-termini of the primer mismatches its corresponding template primer extension and molecular beacon displacement would not happen and fluorescence of the hybridized molecular beacon holds the line without fluorescence quenching. This approach was fully demonstrated in synthetic template systems and applied to detect point mutation at codon 259, a possible point mutation site in exon 7 of p53 gene, obtained from human genomic DNA samples with unambiguous differentiation power.

  6. Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment.

    PubMed

    Waterfall, Christy M; Eisenthal, Robert; Cobb, Benjamin D

    2002-12-20

    A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR. PMID:12470637

  7. Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment.

    PubMed

    Waterfall, Christy M; Eisenthal, Robert; Cobb, Benjamin D

    2002-12-20

    A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR.

  8. Human Platelet Antigen Alleles in 998 Taiwanese Blood Donors Determined by Sequence-Specific Primer Polymerase Chain Reaction

    PubMed Central

    Burnouf, Thierry; Chen, Jen-Wei; Lin, Liang-In

    2013-01-01

    Polymorphism of human platelet antigens (HPAs) leads to alloimmunizations and immune-mediated platelet disorders including fetal-neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura (PTP), and platelet transfusion refractoriness (PTR). HPA typing and knowledge of antigen frequency in a population are important in particular for the provision of HPA-matched blood components for patients with PTR. We have performed allele genotyping for HPA-1 through -6 and -15 among 998 platelet donors from 6 blood centers in Taiwan using sequence-specific primer polymerase chain reaction. The HPA allele frequency was 99.55, and 0.45% for HPA-1a and -1b; 96.49, and 3.51% for HPA-2a and -2b; 55.81, and 44.19% for HPA-3a and -3b; 99.75, and 0.25% for HPA-4a and -4b; 98.50, and 1.50% for HPA-5a and -5b; 97.75 and 2.25% for HPA-6a and -6b; 53.71 and 46.29% for HPA-15a and -15b. HPA-15b and HPA-3a, may be considered the most important, followed by HPA-2, -6, -1, -5, and -4 systems, as a cause of FNAIT, PTP, and PTR based on allele frequency. HPA-4b and HPA-5b role cannot be excluded based on their immunogenicity. A larger-scale study will now be conducted to confirm these hypotheses and to establish an apheresis donor database for the procurement of HPA-matched apheresis platelets for patients with PTR. PMID:23865077

  9. Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species

    PubMed Central

    2010-01-01

    Background Genotype analysis using multiple single nucleotide polymorphisms (SNPs) is a useful but labor-intensive or high-cost procedure in plant research. Here we describe an alternative genotyping method that is suited to multi-sample or multi-locus SNP genotyping and does not require electrophoresis or specialized equipment. Results We have developed a simple method for multi-sample or multi-locus SNP genotyping using allele-specific primers (ASP). More specifically, we (1) improved the design of allele-specific primers, (2) established a method to detect PCR products optically without electrophoresis, and (3) standardized PCR conditions for parallel genomic assay using various allele-specific primers. As an illustration of multi-sample SNP genotyping using ASP, we mapped the locus for lodging resistance in a typhoon (lrt5). Additionally, we successfully tested multi-locus ASP-PCR analysis using 96 SNPs located throughout the genomes of rice (Oryza sativa) cultivars 'Koshihikari' and 'Kasalath', and demonstrated its applicability to other diverse cultivars/subspecies, including wild rice (O. rufipogon). Conclusion Our ASP methodology allows characterization of SNPs genotypes without electrophoresis, expensive probes or specialized equipment, and is highly versatile due to the flexibility in the design of primers. The method could be established easily in any molecular biology laboratory, and is applicable to diverse organisms. PMID:20409329

  10. The allele-specific probe and primer amplification assay, a new real-time PCR method for fine quantification of single-nucleotide polymorphisms in pooled DNA.

    PubMed

    Billard, A; Laval, V; Fillinger, S; Leroux, P; Lachaise, H; Beffa, R; Debieu, D

    2012-02-01

    The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.

  11. Absolute quantification of the alleles in somatic point mutations by bioluminometric methods based on competitive polymerase chain reaction in the presence of a locked nucleic acid blocker or an allele-specific primer.

    PubMed

    Iliadi, Alexandra; Petropoulou, Margarita; Ioannou, Penelope C; Christopoulos, Theodore K; Anagnostopoulos, Nikolaos I; Kanavakis, Emmanuel; Traeger-Synodinos, Jan

    2011-09-01

    In somatic (acquired) point mutations, the challenge is to quantify minute amounts of the mutant allele in the presence of a large excess of the normal allele that differs only in a single base pair. We report two bioluminometric methods that enable absolute quantification of the alleles. The first method exploits the ability of a locked nucleic acid (LNA) oligonucleotide to bind to and inhibit effectively the polymerase chain reaction (PCR) amplification of the normal allele while the amplification of the mutant allele remains unaffected. The second method employs allele-specific PCR primers, thereby allowing the amplification of the corresponding allele only. DNA internal standards (competitors) are added to the PCR mixture to compensate for any sample-to-sample variation in the amplification efficiency. The amplification products from the two alleles and the internal standards are quantified by a microtiter well-based bioluminometric hybridization assay using the photoprotein aequorin as a reporter. The methods allow absolute quantification of less than 300 copies of the mutant allele even in samples containing less than 1% of the mutant allele.

  12. Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube.

    PubMed

    Chen, Dong; Yang, Zhao; Xia, Han; Huang, Jun-Fu; Zhang, Yang; Jiang, Tian-Nun; Wang, Gui-Yu; Chuai, Zheng-Ran; Fu, Wei-Ling; Huang, Qing

    2014-01-01

    Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques. PMID:24705376

  13. Enhanced Specificity of TPMT*2 Genotyping Using Unidirectional Wild-Type and Mutant Allele-Specific Scorpion Primers in a Single Tube

    PubMed Central

    Chen, Dong; Yang, Zhao; Xia, Han; Huang, Jun-Fu; Zhang, Yang; Jiang, Tian-Nun; Wang, Gui-Yu; Chuai, Zheng-Ran; Fu, Wei-Ling; Huang, Qing

    2014-01-01

    Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques. PMID:24705376

  14. Swine Leukocyte Antigen (SLA) class I allele typing of Danish swine herds and identification of commonly occurring haplotypes using sequence specific low and high resolution primers.

    PubMed

    Pedersen, Lasse Eggers; Jungersen, Gregers; Sorensen, Maria Rathmann; Ho, Chak-Sum; Vadekær, Dorte Fink

    2014-12-15

    The swine major histocompatibility complex (MHC) genomic region (SLA) is extremely polymorphic comprising high numbers of different alleles, many encoding a distinct MHC class I molecule, which binds and presents endogenous peptides to circulating T cells of the immune system. Upon recognition of such peptide-MHC complexes (pMHC) naïve T cells can become activated and respond to a given pathogen leading to its elimination and the generation of memory cells. Hence SLA plays a crucial role in maintaining overall adaptive immunologic resistance to pathogens. Knowing which SLA alleles that are commonly occurring can be of great importance in regard to future vaccine development and the establishment of immune protection in swine through broad coverage, highly specific, subunit based vaccination against viruses such as swine influenza, porcine reproductive and respiratory syndrome virus, vesicular stomatitis virus, foot-and-mouth-disease virus and others. Here we present the use of low- and high-resolution PCR-based typing methods to identify individual and commonly occurring SLA class I alleles in Danish swine. A total of 101 animals from seven different herds were tested, and by low resolution typing the top four most frequent SLA class I alleles were those of the allele groups SLA-3*04XX, SLA-1*08XX, SLA-2*02XX, and SLA-1*07XX, respectively. Customised high resolution primers were used to identify specific alleles within the above mentioned allele groups as well as within the SLA-2*05XX allele group. Our studies also suggest the most common haplotype in Danish pigs to be Lr-4.0 expressing the SLA-1*04XX, SLA-2*04XX, and SLA-3*04XX allele combination.

  15. [Detection of JAK2V617F mutation rate by real-time fluorescent quantitative PCR using allele specific primer and TaqMan-MGB probe for dual inhibiting amplification of wild type alleles].

    PubMed

    Liang, Guo-Wei; Shao, Dong-Hua; He, Mei-Ling; Cao, Qing-Yun

    2012-12-01

    This study was purposed to develop a real-time PCR assay for sensitive quantification of JAK2V617F allele burden in peripheral blood and to evaluate the clinical value of this method. Both allele-specific mutant reverse primer and wild-type TaqMan-MGB probe were used for dual-inhibiting amplification of wild-type alleles in a real-time PCR, and then the JAK2V617F mutant alleles were amplified specially. The standard curve for quantification of JAK2V617F was established by percentages of JAK2V617F alleles with threshold cycle (Ct) values in a real-time PCR. Furthermore, 89 apparent healthy donors were tested by this method. The results showed that the quantitative lower limit of this method for JAK2V617F was 0.1%, and the intra- and inter-assay average variability for quantifying percentage of JAK2V617F in total DNA was 4.1% and 6.1%, respectively. Two JAK2V617F-positive individuals were identified (the percentage of JAK2V617F alleles were 0.64% and 0.98%, respectively) using this method in blood from 89 apparently healthy donors. It is concluded that the developed method with highly sensitive and reproducible quantification of JAK2V617F mutant burden can be used clinically for diagnosis and evaluation of disease prognosis and efficacy of therapy in patients with myeloproliferative neoplasms. Moreover, this technique can be also used for quantitative detection of variety of single nucleotide mutation.

  16. Quantitative polymerase chain reaction analysis with allele-specific oligonucleotide primers for individual IgH VDJ regions to evaluate tumor burden in myeloma patients.

    PubMed

    Sata, Hiroshi; Shibayama, Hirohiko; Maeda, Ikuhiro; Habuchi, Yoko; Nakatani, Eiji; Fukushima, Kentaro; Fujita, Jiro; Ezoe, Sachiko; Tadokoro, Seiji; Maeda, Tetsuo; Mizuki, Masao; Kosugi, Satoru; Nakagawa, Masashi; Ueda, Shuji; Iida, Masato; Tokumine, Yukihiro; Azenishi, Yasuhiko; Mitsui, Hideki; Oritani, Kenji; Kanakura, Yuzuru

    2015-05-01

    Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+ CD38- B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+ CD38- B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+ CD38- populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis. PMID:25591497

  17. Quantitative polymerase chain reaction analysis with allele-specific oligonucleotide primers for individual IgH VDJ regions to evaluate tumor burden in myeloma patients.

    PubMed

    Sata, Hiroshi; Shibayama, Hirohiko; Maeda, Ikuhiro; Habuchi, Yoko; Nakatani, Eiji; Fukushima, Kentaro; Fujita, Jiro; Ezoe, Sachiko; Tadokoro, Seiji; Maeda, Tetsuo; Mizuki, Masao; Kosugi, Satoru; Nakagawa, Masashi; Ueda, Shuji; Iida, Masato; Tokumine, Yukihiro; Azenishi, Yasuhiko; Mitsui, Hideki; Oritani, Kenji; Kanakura, Yuzuru

    2015-05-01

    Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+ CD38- B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+ CD38- B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+ CD38- populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis.

  18. Rapid DNA typing for HLA-C using sequence-specific primers (PCR-SSP): identification of serological and non-serologically defined HLA-C alleles including several new alleles.

    PubMed

    Bunce, M; Welsh, K I

    1994-01-01

    Detection of HLA-C antigens by complement mediated cytotoxicity using human alloantisera is often difficult. Between 20 to 40% of individuals in every race have undetectable HLA-C locus antigens and 9 out of the 29 sequenced HLA-C alleles so far published encode serologically undetected antigens. In addition, HLA-C molecules are expressed at the cell surface at about 10% of the levels of HLA-A and HLA-B. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable and rapid method for typing HLA-DR, HLA-DQA and HLA-DQB genes. PCR-SSP takes two hours to perform and is therefore suitable for the genotyping of cadaveric donors. We have designed a set of primers which will positively identify the HLA-C alleles corresponding to the serologically defined series HLA-Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7 and Cw8. The serologically undetectable alleles have also been detected in groups according to sequence homology. In addition, three new unsequenced variants have been identified. DNA samples from 56 International Histocompatibility Workshop reference cell lines and 103 control individuals have been typed by the HLA-C PCR-SSP technique. 4/56 cell line types and 11/103 normal control individuals types were discrepant with the reported serological types. All combinations of serologically detectable and most of the serologically blank HLA-C antigens can be readily identified. DNA typing for HLA-Cw by PCR-SSP can take as little as 130 minutes from start to finish, including DNA preparation.

  19. URPD: a specific product primer design tool

    PubMed Central

    2012-01-01

    Background Polymerase chain reaction (PCR) plays an important role in molecular biology. Primer design fundamentally determines its results. Here, we present a currently available software that is not located in analyzing large sequence but used for a rather straight-forward way of visualizing the primer design process for infrequent users. Findings URPD (yoUR Primer Design), a web-based specific product primer design tool, combines the NCBI Reference Sequences (RefSeq), UCSC In-Silico PCR, memetic algorithm (MA) and genetic algorithm (GA) primer design methods to obtain specific primer sets. A friendly user interface is accomplished by built-in parameter settings. The incorporated smooth pipeline operations effectively guide both occasional and advanced users. URPD contains an automated process, which produces feasible primer pairs that satisfy the specific needs of the experimental design with practical PCR amplifications. Visual virtual gel electrophoresis and in silico PCR provide a simulated PCR environment. The comparison of Practical gel electrophoresis comparison to virtual gel electrophoresis facilitates and verifies the PCR experiment. Wet-laboratory validation proved that the system provides feasible primers. Conclusions URPD is a user-friendly tool that provides specific primer design results. The pipeline design path makes it easy to operate for beginners. URPD also provides a high throughput primer design function. Moreover, the advanced parameter settings assist sophisticated researchers in performing experiential PCR. Several novel functions, such as a nucleotide accession number template sequence input, local and global specificity estimation, primer pair redesign, user-interactive sequence scale selection, and virtual and practical PCR gel electrophoresis discrepancies have been developed and integrated into URPD. The URPD program is implemented in JAVA and freely available at http://bio.kuas.edu.tw/urpd/. PMID:22713312

  20. Delimiting Allelic Imbalance of TYMS by Allele-Specific Analysis

    PubMed Central

    Balboa-Beltrán, Emilia; Cruz, Raquel; Carracedo, Angel; Barros, Francisco

    2015-01-01

    Abstract Allelic imbalance of thymidylate synthase (TYMS) is attributed to polymorphisms in the 5′- and 3′-untranslated region (UTR). These polymorphisms have been related to the risk of suffering different cancers, for example leukemia, breast or gastric cancer, and response to different drugs, among which are methotrexate glutamates, stavudine, and specifically 5-fluorouracil (5-FU), as TYMS is its direct target. A vast literature has been published in relation to 5-FU, even suggesting the sole use of these polymorphisms to effectively manage 5-FU dosage. Estimates of the extent to which these polymorphisms influence in TYMS expression have in the past been based on functional analysis by luciferase assays and quantification of TYMS mRNA, but both these studies, as the association studies with cancer risk or with toxicity or response to 5-FU, are very contradictory. Regarding functional assays, the artificial genetic environment created in luciferase assay and the problems derived from quantitative polymerase chain reactions (qPCRs), for example the use of a reference gene, may have distorted the results. To avoid these sources of interference, we have analyzed the allelic imbalance of TYMS by allelic-specific analysis in peripheral blood mononuclear cells (PBMCs) from patients. Allelic imbalance in PBMCs, taken from 40 patients with suspected myeloproliferative haematological diseases, was determined by fluorescent fragment analysis (for the 3′-UTR polymorphism), Sanger sequencing and allelic-specific qPCR in multiplex (for the 5′-UTR polymorphisms). For neither the 3′- nor the 5′-UTR polymorphisms did the observed allelic imbalance exceed 1.5 fold. None of the TYMS polymorphisms is statistically associated with allelic imbalance. The results acquired allow us to deny the previously established assertion of an influence of 2 to 4 fold of the rs45445694 and rs2853542 polymorphisms in the expression of TYMS and narrow its allelic imbalance to 1.5 fold

  1. Allele-specific enzymatic amplification of. beta. -globin genomic DNA for diagnosis of sickle cell anemia

    SciTech Connect

    Wu, D.Y.; Ugozzoli, L.; Pal, B.K.; Wallace, B. )

    1989-04-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell {beta}-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3{prime} nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.

  2. Enhancing allele-specific PCR for specifically detecting short deletion and insertion DNA mutations.

    PubMed

    Wang, Yiran; Rollin, Joseph A; Zhang, Y-H Percival

    2010-02-01

    Allele-specific PCR (AS-PCR) has been widely used for the detection of single nucleotide polymorphism. But there are some challenges in using AS-PCR for specifically detecting DNA variations with short deletions or insertions. The challenges are associated with designing selective allele-specific primers as well as the specificity of AS-PCR in distinguishing some types of single base-pair mismatches. In order to address such problems and enhance the applicability of AS-PCR, a general primer design method was developed to create a multiple base-pair mismatch between the primer 3'-terminus and the template DNA. This approach can destabilize the primer-template complex more efficiently than does a single base-pair mismatch, and can dramatically increase the specificity of AS-PCR. As a proof-of-principle demonstration, the method of primer design was applied in colony PCR for identifying plasmid DNA deletion or insertion mutants after site-directed mutagenesis. As anticipated, multiple base-pair mismatches achieved much more specific PCR amplification than single base-pair mismatches. Therefore, with the proposed primer design method, the detection of short nucleotide deletion and insertion mutations becomes simple, accurate and more reliable.

  3. Allelic divergence and cultivar-specific SSR alleles revealed by capillary electrophoresis using fluorescence-labeled SSR markers in sugarcane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Though sugarcane cultivars (Saccharum spp. hybrids) are complex aneu-polyploid hybrids, genetic evaluation and tracking of clone- or cultivar-specific alleles become possible due to capillary electrophoregrams (CE) using fluorescence-labeled SSR primer pairs. Twenty-four sugarcane cultivars, 12 each...

  4. Allele-specific H3K79 Di- versus trimethylation distinguishes opposite parental alleles at imprinted regions.

    PubMed

    Singh, Purnima; Han, Li; Rivas, Guillermo E; Lee, Dong-Hoon; Nicholson, Thomas B; Larson, Garrett P; Chen, Taiping; Szabó, Piroska E

    2010-06-01

    Imprinted gene expression corresponds to parental allele-specific DNA CpG methylation and chromatin composition. Histone tail covalent modifications have been extensively studied, but it is not known whether modifications in the histone globular domains can also discriminate between the parental alleles. Using multiplex chromatin immunoprecipitation-single nucleotide primer extension (ChIP-SNuPE) assays, we measured the allele-specific enrichment of H3K79 methylation and H4K91 acetylation along the H19/Igf2 imprinted domain. Whereas H3K79me1, H3K79me2, and H4K91ac displayed a paternal-specific enrichment at the paternally expressed Igf2 locus, H3K79me3 was paternally biased at the maternally expressed H19 locus, including the paternally methylated imprinting control region (ICR). We found that these allele-specific differences depended on CTCF binding in the maternal ICR allele. We analyzed an additional 11 differentially methylated regions (DMRs) and found that, in general, H3K79me3 was associated with the CpG-methylated alleles, whereas H3K79me1, H3K79me2, and H4K91ac enrichment was specific to the unmethylated alleles. Our data suggest that allele-specific differences in the globular histone domains may constitute a layer of the "histone code" at imprinted genes.

  5. S-genotype identification based on allele-specific PCR in Japanese pear

    PubMed Central

    Nashima, Kenji; Terakami, Shingo; Nishio, Sogo; Kunihisa, Miyuki; Nishitani, Chikako; Saito, Toshihiro; Yamamoto, Toshiya

    2015-01-01

    Gametophytic self-incompatibility in Japanese pear (Pyrus pyrifolia Nakai) is controlled by the single, multi-allelic S-locus. Information about the S-genotypes is important for breeding and the selection of pollen donors for fruit production. Rapid and reliable S-genotype identification system is necessary for efficient breeding of new cultivars in Japanese pear. We designed S allele-specific PCR primer pairs for ten previously reported S-RNase alleles (S1–S9 and Sk) as simple and reliable method. Specific nucleotide sequences were chosen to design the primers to amplify fragments of only the corresponding S alleles. The developed primer pairs were evaluated by using homozygous S-genotypes (S1/S1–S9/S9 and S4sm/S4sm) and 14 major Japanese pear cultivars, and found that S allele-specific primer pairs can identify S-genotypes effectively. The S allele-specific primer pairs developed in this study will be useful for efficient S-genotyping and for marker-assisted selection in Japanese pear breeding programs. PMID:26175617

  6. Allelic variation contributes to bacterial host specificity.

    PubMed

    Yue, Min; Han, Xiangan; De Masi, Leon; Zhu, Chunhong; Ma, Xun; Zhang, Junjie; Wu, Renwei; Schmieder, Robert; Kaushik, Radhey S; Fraser, George P; Zhao, Shaohua; McDermott, Patrick F; Weill, François-Xavier; Mainil, Jacques G; Arze, Cesar; Fricke, W Florian; Edwards, Robert A; Brisson, Dustin; Zhang, Nancy R; Rankin, Shelley C; Schifferli, Dieter M

    2015-01-01

    Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype diversity is conventionally associated with gene gain or loss, the role of pathoadaptive nonsynonymous single-nucleotide polymorphisms (nsSNPs) has not been systematically evaluated. Here, our genome-wide analysis of core genes within Salmonella enterica serovar Typhimurium genomes reveals a high degree of allelic variation in surface-exposed molecules, including adhesins that promote host colonization. Subsequent multinomial logistic regression, MultiPhen and Random Forest analyses of known/suspected adhesins from 580 independent Typhimurium isolates identifies distinct host-specific nsSNP signatures. Moreover, population and functional analyses of host-associated nsSNPs for FimH, the type 1 fimbrial adhesin, highlights the role of key allelic residues in host-specific adherence in vitro. Together, our data provide the first concrete evidence that functional differences between allelic variants of bacterial proteins likely contribute to pathoadaption to diverse hosts. PMID:26515720

  7. Allelic variation contributes to bacterial host specificity

    SciTech Connect

    Yue, Min; Han, Xiangan; Masi, Leon De; Zhu, Chunhong; Ma, Xun; Zhang, Junjie; Wu, Renwei; Schmieder, Robert; Kaushik, Radhey S.; Fraser, George P.; Zhao, Shaohua; McDermott, Patrick F.; Weill, François-Xavier; Mainil, Jacques G.; Arze, Cesar; Fricke, W. Florian; Edwards, Robert A.; Brisson, Dustin; Zhang, Nancy R.; Rankin, Shelley C.; Schifferli, Dieter M.

    2015-10-30

    Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype diversity is conventionally associated with gene gain or loss, the role of pathoadaptive nonsynonymous single-nucleotide polymorphisms (nsSNPs) has not been systematically evaluated. Here, our genome-wide analysis of core genes within Salmonella enterica serovar Typhimurium genomes reveals a high degree of allelic variation in surface-exposed molecules, including adhesins that promote host colonization. Subsequent multinomial logistic regression, MultiPhen and Random Forest analyses of known/suspected adhesins from 580 independent Typhimurium isolates identifies distinct host-specific nsSNP signatures. Moreover, population and functional analyses of host-associated nsSNPs for FimH, the type 1 fimbrial adhesin, highlights the role of key allelic residues in host-specific adherence in vitro. In conclusion, together, our data provide the first concrete evidence that functional differences between allelic variants of bacterial proteins likely contribute to pathoadaption to diverse hosts.

  8. Allelic variation contributes to bacterial host specificity

    DOE PAGES

    Yue, Min; Han, Xiangan; Masi, Leon De; Zhu, Chunhong; Ma, Xun; Zhang, Junjie; Wu, Renwei; Schmieder, Robert; Kaushik, Radhey S.; Fraser, George P.; et al

    2015-10-30

    Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype diversity is conventionally associated with gene gain or loss, the role of pathoadaptive nonsynonymous single-nucleotide polymorphisms (nsSNPs) has not been systematically evaluated. Here, our genome-wide analysis of core genes within Salmonella enterica serovar Typhimurium genomes reveals a high degree of allelic variation in surface-exposed molecules, including adhesins that promote host colonization. Subsequent multinomial logistic regression, MultiPhen and Random Forest analyses of known/suspected adhesins from 580 independent Typhimurium isolates identifies distinct host-specific nsSNP signatures. Moreover, population andmore » functional analyses of host-associated nsSNPs for FimH, the type 1 fimbrial adhesin, highlights the role of key allelic residues in host-specific adherence in vitro. In conclusion, together, our data provide the first concrete evidence that functional differences between allelic variants of bacterial proteins likely contribute to pathoadaption to diverse hosts.« less

  9. Allelic variation contributes to bacterial host specificity

    PubMed Central

    Yue, Min; Han, Xiangan; Masi, Leon De; Zhu, Chunhong; Ma, Xun; Zhang, Junjie; Wu, Renwei; Schmieder, Robert; Kaushik, Radhey S.; Fraser, George P.; Zhao, Shaohua; McDermott, Patrick F.; Weill, François-Xavier; Mainil, Jacques G.; Arze, Cesar; Fricke, W. Florian; Edwards, Robert A.; Brisson, Dustin; Zhang, Nancy R.; Rankin, Shelley C.; Schifferli, Dieter M.

    2015-01-01

    Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype diversity is conventionally associated with gene gain or loss, the role of pathoadaptive nonsynonymous single-nucleotide polymorphisms (nsSNPs) has not been systematically evaluated. Here, our genome-wide analysis of core genes within Salmonella enterica serovar Typhimurium genomes reveals a high degree of allelic variation in surface-exposed molecules, including adhesins that promote host colonization. Subsequent multinomial logistic regression, MultiPhen and Random Forest analyses of known/suspected adhesins from 580 independent Typhimurium isolates identifies distinct host-specific nsSNP signatures. Moreover, population and functional analyses of host-associated nsSNPs for FimH, the type 1 fimbrial adhesin, highlights the role of key allelic residues in host-specific adherence in vitro. Together, our data provide the first concrete evidence that functional differences between allelic variants of bacterial proteins likely contribute to pathoadaption to diverse hosts. PMID:26515720

  10. A novel measurement of allele discrimination for assessment of allele-specific silencing by RNA interference.

    PubMed

    Takahashi, Masaki; Hohjoh, Hirohiko

    2014-11-01

    Allele-specific silencing by RNA interference (ASP-RNAi) is an atypical RNAi that is capable of discriminating target alleles from non-target alleles, and may be therapeutically useful for specific inhibition of disease-causing alleles without affecting their corresponding normal alleles. However, it is difficult to design and select small interfering RNA (siRNAs) that confer ASP-RNAi. A major problem is that there are few appropriate measures in determining optimal allele-specific siRNAs. Here we show two novel formulas for calculating a new measure of allele-discrimination, named "ASP-score". The formulas and ASP-score allow for an unbiased determination of optimal siRNAs, and may contribute to characterizing such allele-specific siRNAs.

  11. SNP-Based Quantification of Allele-Specific DNA Methylation Patterns by Pyrosequencing®.

    PubMed

    Busato, Florence; Tost, Jörg

    2015-01-01

    The analysis of allele-specific DNA methylation patterns has recently attracted much interest as loci of allele-specific DNA methylation overlap with known risk loci for complex diseases and the analysis might contribute to the fine-mapping and interpretation of non-coding genetic variants associated with complex diseases and improve the understanding between genotype and phenotype. In the presented protocol, we present a method for the analysis of DNA methylation patterns on both alleles separately using heterozygous Single Nucleotide Polymorphisms (SNPs) as anchor for allele-specific PCR amplification followed by analysis of the allele-specific DNA methylation patterns by Pyrosequencing(®). Pyrosequencing is an easy-to-handle, quantitative real-time sequencing method that is frequently used for genotyping as well as for the analysis of DNA methylation patterns. The protocol consists of three major steps: (1) identification of individuals heterozygous for a SNP in a region of interest using Pyrosequencing; (2) analysis of the DNA methylation patterns surrounding the SNP on bisulfite-treated DNA to identify regions of potential allele-specific DNA methylation; and (3) the analysis of the DNA methylation patterns associated with each of the two alleles, which are individually amplified using allele-specific PCR. The enrichment of the targeted allele is re-enforced by modification of the allele-specific primers at the allele-discriminating base with Locked Nucleic Acids (LNA). For the proof-of-principle of the developed approach, we provide assay details for three imprinted genes (IGF2, IGF2R, and PEG3) within this chapter. The mean of the DNA methylation patterns derived from the individual alleles corresponds well to the overall DNA methylation patterns and the developed approach proved more reliable compared to other protocols for allele-specific DNA methylation analysis.

  12. Homolog-specific PCR primer design for profiling splice variants

    PubMed Central

    Srivastava, Gyan Prakash; Hanumappa, Mamatha; Kushwaha, Garima; Nguyen, Henry T.; Xu, Dong

    2011-01-01

    To study functional diversity of proteins encoded from a single gene, it is important to distinguish the expression levels among the alternatively spliced variants. A variant-specific primer pair is required to amplify each alternatively spliced variant individually. For this purpose, we developed a new feature, homolog-specific primer design (HSPD), in our high-throughput primer and probe design software tool, PRIMEGENS-v2. The algorithm uses a de novo approach to design primers without any prior information of splice variants or close homologs for an input query sequence. It not only designs primer pairs but also finds potential isoforms and homologs of the input sequence. Efficiency of this algorithm was tested for several gene families in soybean. A total of 187 primer pairs were tested under five different abiotic stress conditions with three replications at three time points. Results indicate a high success rate of primer design. Some primer pairs designed were able to amplify all splice variants of a gene. Furthermore, by utilizing combinations within the same multiplex pool, we were able to uniquely amplify a specific variant or duplicate gene. Our method can also be used to design PCR primers to specifically amplify homologs in the same gene family. PRIMEGENS-v2 is available at: http://primegens.org. PMID:21415011

  13. Allele Workbench: transcriptome pipeline and interactive graphics for allele-specific expression.

    PubMed

    Soderlund, Carol A; Nelson, William M; Goff, Stephen A

    2014-01-01

    Sequencing the transcriptome can answer various questions such as determining the transcripts expressed in a given species for a specific tissue or condition, evaluating differential expression, discovering variants, and evaluating allele-specific expression. Differential expression evaluates the expression differences between different strains, tissues, and conditions. Allele-specific expression evaluates expression differences between parental alleles. Both differential expression and allele-specific expression have been studied for heterosis (hybrid vigor), where the hybrid has improved performance over the parents for one or more traits. The Allele Workbench software was developed for a heterosis study that evaluated allele-specific expression for a mouse F1 hybrid using libraries from multiple tissues with biological replicates. This software has been made into a distributable package, which includes a pipeline, a Java interface to build the database, and a Java interface for query and display of the results. The required input is a reference genome, annotation file, and one or more RNA-Seq libraries with optional replicates. It evaluates allelic imbalance at the SNP and transcript level and flags transcripts with significant opposite directional allele-specific expression. The Java interface allows the user to view data from libraries, replicates, genes, transcripts, exons, and variants, including queries on allele imbalance for selected libraries. To determine the impact of allele-specific SNPs on protein folding, variants are annotated with their effect (e.g., missense), and the parental protein sequences may be exported for protein folding analysis. The Allele Workbench processing results in transcript files and read counts that can be used as input to the previously published Transcriptome Computational Workbench, which has a new algorithm for determining a trimmed set of gene ontology terms. The software with demo files is available from https://code.google.com/p/allele

  14. Allele Workbench: Transcriptome Pipeline and Interactive Graphics for Allele-Specific Expression

    PubMed Central

    Soderlund, Carol A.; Nelson, William M.; Goff, Stephen A.

    2014-01-01

    Sequencing the transcriptome can answer various questions such as determining the transcripts expressed in a given species for a specific tissue or condition, evaluating differential expression, discovering variants, and evaluating allele-specific expression. Differential expression evaluates the expression differences between different strains, tissues, and conditions. Allele-specific expression evaluates expression differences between parental alleles. Both differential expression and allele-specific expression have been studied for heterosis (hybrid vigor), where the hybrid has improved performance over the parents for one or more traits. The Allele Workbench software was developed for a heterosis study that evaluated allele-specific expression for a mouse F1 hybrid using libraries from multiple tissues with biological replicates. This software has been made into a distributable package, which includes a pipeline, a Java interface to build the database, and a Java interface for query and display of the results. The required input is a reference genome, annotation file, and one or more RNA-Seq libraries with optional replicates. It evaluates allelic imbalance at the SNP and transcript level and flags transcripts with significant opposite directional allele-specific expression. The Java interface allows the user to view data from libraries, replicates, genes, transcripts, exons, and variants, including queries on allele imbalance for selected libraries. To determine the impact of allele-specific SNPs on protein folding, variants are annotated with their effect (e.g., missense), and the parental protein sequences may be exported for protein folding analysis. The Allele Workbench processing results in transcript files and read counts that can be used as input to the previously published Transcriptome Computational Workbench, which has a new algorithm for determining a trimmed set of gene ontology terms. The software with demo files is available from https://code.google.com/p/allele

  15. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

    PubMed

    Lee, Han B; Schwab, Tanya L; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L; Cervera, Roberto Lopez; McNulty, Melissa S; Bostwick, Hannah S; Clark, Karl J

    2016-06-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  16. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

    PubMed

    Lee, Han B; Schwab, Tanya L; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L; Cervera, Roberto Lopez; McNulty, Melissa S; Bostwick, Hannah S; Clark, Karl J

    2016-06-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  17. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping

    PubMed Central

    Lee, Han B.; Schwab, Tanya L.; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L.; Cervera, Roberto Lopez; McNulty, Melissa S.; Bostwick, Hannah S.; Clark, Karl J.

    2016-01-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98–100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  18. Intragenic allele pyramiding combines different specificities of wheat Pm3 resistance alleles.

    PubMed

    Brunner, Susanne; Hurni, Severine; Streckeisen, Philipp; Mayr, Gabriele; Albrecht, Mario; Yahiaoui, Nabila; Keller, Beat

    2010-11-01

    Some plant resistance genes occur as allelic series, with each member conferring specific resistance against a subset of pathogen races. In wheat, there are 17 alleles of the Pm3 gene. They encode nucleotide-binding (NB-ARC) and leucine-rich-repeat (LRR) domain proteins, which mediate resistance to distinct race spectra of powdery mildew. It is not known if specificities from different alleles can be combined to create resistance genes with broader specificity. Here, we used an approach based on avirulence analysis of pathogen populations to characterize the molecular basis of Pm3 recognition spectra. A large survey of mildew races for avirulence on the Pm3 alleles revealed that Pm3a has a resistance spectrum that completely contains that of Pm3f, but also extends towards additional races. The same is true for the Pm3b and Pm3c gene pair. The molecular analysis of these allelic pairs revealed a role of the NB-ARC protein domain in the efficiency of effector-dependent resistance. Analysis of the wild-type and chimeric Pm3 alleles identified single residues in the C-terminal LRR motifs as the main determinant of allele specificity. Variable residues of the N-terminal LRRs are necessary, but not sufficient, to confer resistance specificity. Based on these data, we constructed a chimeric Pm3 gene by intragenic allele pyramiding of Pm3d and Pm3e that showed the combined resistance specificity and, thus, a broader recognition spectrum compared with the parental alleles. Our findings support a model of stepwise evolution of Pm3 recognition specificities.

  19. [Evaluation of minimal residual disease using allele (mutation) -specific PCR].

    PubMed

    Matsuda, Kazuyuki

    2014-06-01

    For patients with hematological malignancies, monitoring minimal residual disease (MRD) provides useful information to evaluate the therapeutic response and risk of relapse. The currently available quantitative MRD assays are fluorescence in situ hybridization of chromosomal aberrations, multiparameter flow cytometry of leukemia-associated immunophenotypes, and quantitative polymerase chain reaction (qPCR) analysis of fusion genes, immunoglobulin/T-cell receptor gene rearrangements, genetic alterations, or over-expressed genes. Single nucleotide mutations associated with leukemogenesis can be considered as applicable MRD markers. Allele-specific qPCR (AS-qPCR) using primers including mismatched bases and locked nucleic acids (LNA) can quantify not only the insertion and duplication of several nucleotides, but also single nucleotide mutation in the presence of an excess amount of wild-type nucleotides. The AS-qPCR for analyzing single nucleotide mutations contributes to the monitoring of MRD in patients without recurrent fusion genes throughout the clinical course and, thus, broadens the spectrum of patients in whom MRD can be monitored. In addition to the evaluation of MRD, AS-qPCR can provide insight into the development of leukemia and the sequential acquisition of gene mutations.

  20. Detection of Fusarium oxysporum f. sp. vasinfectum race 3 by single-base extension method and allele-specific polymerase chain reaction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed allele specific (AS) SNP primers for rapid detection of Fusarium oxysporum f.sp vasinfectum (FOV) race 3. FOV_BT_SNP_R3 and FOV_BT_AS_R3 primers were designed based on single nucleotide polymorphisms of partial sequence alignment of the ß-tubulin (BT) gene from several FOV races. These ...

  1. Allele-specific MMP-3 transcription under in vivo conditions

    SciTech Connect

    Zhu Chaoyong; Odeberg, Jacob; Hamsten, Anders; Eriksson, Per . E-mail: Per.Eriksson@ki.se

    2006-09-29

    A common matrix metalloproteinases-3 (MMP-3) -1612 5A/6A promoter polymorphism is associated with risk for cardiovascular disease, rheumatoid arthritis, and other diseases. Here we used the haplotype chromatin immunoprecipitation method to study allele-specific MMP-3 expression under in vivo conditions in heterozygous THP-1 cells. Pyrosequencing was used to analyse the ratio of 5A-allele to 6A-allele after chromatin immunoprecipitation using an antibody against phosphorylated active RNA polymerase II. There was no allele-specific difference in transcriptional activity during basal conditions, i.e., in unstimulated monocytic THP-1 cells. However, after stimulation of MMP-3 expression by monocyte differentiation or incubation with IL-1{beta}, the haplotype containing the 5A-allele was associated with higher transcriptional activity compared with the 6A-containing haplotype. Electromobility shift assay demonstrated increased binding of nuclear proteins to the 5A-allele after monocyte differentiation. In conclusion, the common MMP-3 5A/6A promoter polymorphism appears to be functional only during specific environmental conditions involving inflammation.

  2. Sequence-specific DNA primer effects on telomerase polymerization activity.

    PubMed Central

    Lee, M S; Blackburn, E H

    1993-01-01

    The ribonucleoprotein enzyme telomerase synthesizes one strand of telomeric DNA by copying a template sequence within the RNA moiety of the enzyme. Kinetic studies of this polymerization reaction were used to analyze the mechanism and properties of the telomerase from Tetrahymena thermophila. This enzyme synthesizes TTGGGG repeats, the telomeric DNA sequence of this species, by elongating a DNA primer whose 3' end base pairs with the template-forming domain of the RNA. The enzyme was found to act nonprocessively with short (10- to 12-nucleotide) primers but to become processive as TTGGGG repeats were added. Variation of the 5' sequences of short primers with a common 3' end identified sequence-specific effects which are distinct from those involving base pairing of the 3' end of the primer with the RNA template and which can markedly induce enzyme activity by increasing the catalytic rate of the telomerase polymerization reaction. These results identify an additional mechanistic basis for telomere and DNA end recognition by telomerase in vivo. Images PMID:8413255

  3. Disagreement in genotyping results of drug resistance alleles of the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene by allele-specific PCR (ASPCR) assays and Sanger sequencing.

    PubMed

    Sharma, Divya; Lather, Manila; Dykes, Cherry L; Dang, Amita S; Adak, Tridibes; Singh, Om P

    2016-01-01

    The rapid spread of antimalarial drug resistance in Plasmodium falciparum over the past few decades has necessitated intensive monitoring of such resistance for an effective malaria control strategy. P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr) genes act as molecular markers for resistance against the antimalarial drugs sulphadoxine and pyrimethamine, respectively. Resistance to pyrimethamine which is used as a partner drug in artemisinin combination therapy (ACT) is associated with several mutations in the Pfdhfr gene, namely A16V, N51I, C59R, S108N/T and I164L. Therefore, routine monitoring of Pfdhfr-drug-resistant alleles in a population may help in effective drug resistance management. Allele-specific PCR (ASPCR) is one of the commonly used methods for molecular genotyping of these alleles. In this study, we genotyped 55 samples of P. falciparum for allele discrimination at four codons of Pfdhfr (N51, C59, S108 and I164) by ASPCR using published methods and by Sanger's DNA sequencing method. We found that the ASPCR identified a significantly higher number of mutant alleles as compared to the DNA sequencing method. Such discrepancies arise due to the non-specificity of some of the allele-specific primer sets and due to the lack of sensitivity of Sanger's DNA sequencing method to detect minor alleles present in multiple clone infections. This study reveals the need of a highly specific and sensitive method for genotyping and detecting minor drug-resistant alleles present in multiple clonal infections.

  4. Disagreement in genotyping results of drug resistance alleles of the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene by allele-specific PCR (ASPCR) assays and Sanger sequencing.

    PubMed

    Sharma, Divya; Lather, Manila; Dykes, Cherry L; Dang, Amita S; Adak, Tridibes; Singh, Om P

    2016-01-01

    The rapid spread of antimalarial drug resistance in Plasmodium falciparum over the past few decades has necessitated intensive monitoring of such resistance for an effective malaria control strategy. P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr) genes act as molecular markers for resistance against the antimalarial drugs sulphadoxine and pyrimethamine, respectively. Resistance to pyrimethamine which is used as a partner drug in artemisinin combination therapy (ACT) is associated with several mutations in the Pfdhfr gene, namely A16V, N51I, C59R, S108N/T and I164L. Therefore, routine monitoring of Pfdhfr-drug-resistant alleles in a population may help in effective drug resistance management. Allele-specific PCR (ASPCR) is one of the commonly used methods for molecular genotyping of these alleles. In this study, we genotyped 55 samples of P. falciparum for allele discrimination at four codons of Pfdhfr (N51, C59, S108 and I164) by ASPCR using published methods and by Sanger's DNA sequencing method. We found that the ASPCR identified a significantly higher number of mutant alleles as compared to the DNA sequencing method. Such discrepancies arise due to the non-specificity of some of the allele-specific primer sets and due to the lack of sensitivity of Sanger's DNA sequencing method to detect minor alleles present in multiple clone infections. This study reveals the need of a highly specific and sensitive method for genotyping and detecting minor drug-resistant alleles present in multiple clonal infections. PMID:26407876

  5. Allele-Specific DNA Methylation Detection by Pyrosequencing®.

    PubMed

    Kristensen, Lasse Sommer; Johansen, Jens Vilstrup; Grønbæk, Kirsten

    2015-01-01

    DNA methylation is an epigenetic modification that plays important roles in healthy as well as diseased cells, by influencing the transcription of genes. In spite the fact that human somatic cells are diploid, most of the currently available methods for the study of DNA methylation do not provide information on the methylation status of individual alleles of genes. This information may be of importance in many situations. In particular, in cancer both alleles of tumour suppressor genes generally need to be inactivated for a phenotypic effect to be observed. Here, we present a simple and cost-effective protocol for allele-specific DNA methylation detection based on Pyrosequencing(®) of methylation-specific PCR (MSP) products including a single nucleotide polymorphism (SNP) within the amplicon. PMID:26103906

  6. Allele-specific DNA methylation reinforces PEAR1 enhancer activity.

    PubMed

    Izzi, Benedetta; Pistoni, Mariaelena; Cludts, Katrien; Akkor, Pinar; Lambrechts, Diether; Verfaillie, Catherine; Verhamme, Peter; Freson, Kathleen; Hoylaerts, Marc F

    2016-08-18

    Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 5'-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation. PMID:27313330

  7. Assessment of allele-specific gene silencing by RNA interference with mutant and wild-type reporter alleles.

    PubMed

    Ohnishi, Yusuke; Tokunaga, Katsushi; Kaneko, Kiyotoshi; Hohjoh, Hirohiko

    2006-02-28

    Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically suppressing the expression of alleles associated with disease. To realize such allele-specific RNAi (ASPRNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital, but is also difficult. Here, we show ASP-RNAi against the Swedish- and London-type amyloid precursor protein (APP) variants related to familial Alzheimer's disease using two reporter alleles encoding the Photinus and Renilla luciferase genes and carrying mutant and wild-type allelic sequences in their 3'-untranslated regions. We examined the effects of siRNA duplexes against the mutant alleles in allele-specific gene silencing and off-target silencing against the wild-type allele under heterozygous conditions, which were generated by cotransfecting the reporter alleles and siRNA duplexes into cultured human cells. Consistently, the siRNA duplexes determined to confer ASP-RNAi also inhibited the expression of the bona fide mutant APP and the production of either amyloid beta 40- or 42-peptide in Cos-7 cells expressing both the full-length Swedish- and wild-type APP alleles. The present data suggest that the system with reporter alleles may permit the preclinical assessment of siRNA duplexes conferring ASP-RNAi, and thus contribute to the design and selection of the most suitable of such siRNA duplexes.

  8. [Study on identification of cistanche hebra and its adulterants by PCR amplification of specific alleles based on ITS sequences].

    PubMed

    Li, Zhen-Hua; Long, Ping; Zou, De-Zhi; Li, Yue; Cui, Zhan-Hu; Li, Min-Hui

    2014-10-01

    To explore the new method of discriminating Cistanche deserticola, Cynomorium songaricum and Orobanche pycnostachya by using PCR amplification of specific alleles. 30 samples of the different C. deserticola, 21 samples of C. songaricum and O. pycnostachya were collected. The total DNA of the samples were extracted, the ITS sequences from C. deserticola, C. songaricum and O. pycnostachya were amplified by PCR and sequenced unidirectionally. These sequences were aligned by using ClustulW. Specific primer was designed according to the ITS sequences of specific alleles, and PCR reaction system was optimized. Additionally, compare with the identification of specific PCR method and DNA sequence analysis method. The result showed that the 331 bp identification band for C. deserticola and the adulterants not amplified bands by a single PCR reaction, which showed good identification ability to the three species. PCR amplification of specific alleles can be used to identify C. deserticola, C. songaricum and O. pycnostachya successfully.

  9. Extensive allele-specific translational regulation in hybrid mice.

    PubMed

    Hou, Jingyi; Wang, Xi; McShane, Erik; Zauber, Henrik; Sun, Wei; Selbach, Matthias; Chen, Wei

    2015-08-07

    Translational regulation is mediated through the interaction between diffusible trans-factors and cis-elements residing within mRNA transcripts. In contrast to extensively studied transcriptional regulation, cis-regulation on translation remains underexplored. Using deep sequencing-based transcriptome and polysome profiling, we globally profiled allele-specific translational efficiency for the first time in an F1 hybrid mouse. Out of 7,156 genes with reliable quantification of both alleles, we found 1,008 (14.1%) exhibiting significant allelic divergence in translational efficiency. Systematic analysis of sequence features of the genes with biased allelic translation revealed that local RNA secondary structure surrounding the start codon and proximal out-of-frame upstream AUGs could affect translational efficiency. Finally, we observed that the cis-effect was quantitatively comparable between transcriptional and translational regulation. Such effects in the two regulatory processes were more frequently compensatory, suggesting that the regulation at the two levels could be coordinated in maintaining robustness of protein expression.

  10. Description of a novel HLA-B allele, B*5613, identified during HLA-typing using sequence-specific oligonucleotide hybridization and sequence-specific amplification.

    PubMed

    Hoppe, B; Heymann, G A; Schoenemann, C; Nagy, M; Kiesewetter, H; Salama, A

    2004-11-01

    Here, we report on the characterization of a novel human leukocyte antigen (HLA)-B allele, B*5613. The allele was identified in an adult male from North Africa who was suffering from sickle cell anemia. HLA-B*5613 most closely matches to B*5601 differing only by a substitution of three nucleotides of codon 180. Due to this substitution, low-resolution HLA-typing using sequence-specific oligonucleotide hybridization or amplification using sequence-specific primers gave inconclusive results. DNA sequencing confirmed a variation of codon 180 (CTG-->GAC) resulting in an amino acid substitution Leu156Asp. PMID:15496207

  11. A modified approach to identification of the sickle cell anemia mutation by means of allele-specific polymerase chain reaction.

    PubMed

    Birikh, K R; Plutalov, O V; Schwartz, E I; Devi, P S; Berlin, Y A

    1992-01-01

    The allele-specific PCR approach has been modified by introducing a second mismatch at the 3'-penultimate link of the primer and used to identify the sickle cell anemia mutation (A-->T transversion in the sixth codon of the human beta-globin gene causing Glu-->Val substitution in the protein), thus obviating the problem of an interpretationally ambiguous 3'-terminal mismatch including T residue. PMID:1301951

  12. A modified approach to identification of the sickle cell anemia mutation by means of allele-specific polymerase chain reaction.

    PubMed

    Birikh, K R; Plutalov, O V; Schwartz, E I; Devi, P S; Berlin, Y A

    1992-01-01

    The allele-specific PCR approach has been modified by introducing a second mismatch at the 3'-penultimate link of the primer and used to identify the sickle cell anemia mutation (A-->T transversion in the sixth codon of the human beta-globin gene causing Glu-->Val substitution in the protein), thus obviating the problem of an interpretationally ambiguous 3'-terminal mismatch including T residue.

  13. Genomic landscape of human allele-specific DNA methylation.

    PubMed

    Fang, Fang; Hodges, Emily; Molaro, Antoine; Dean, Matthew; Hannon, Gregory J; Smith, Andrew D

    2012-05-01

    DNA methylation mediates imprinted gene expression by passing an epigenomic state across generations and differentially marking specific regulatory regions on maternal and paternal alleles. Imprinting has been tied to the evolution of the placenta in mammals and defects of imprinting have been associated with human diseases. Although recent advances in genome sequencing have revolutionized the study of DNA methylation, existing methylome data remain largely untapped in the study of imprinting. We present a statistical model to describe allele-specific methylation (ASM) in data from high-throughput short-read bisulfite sequencing. Simulation results indicate technical specifications of existing methylome data, such as read length and coverage, are sufficient for full-genome ASM profiling based on our model. We used our model to analyze methylomes for a diverse set of human cell types, including cultured and uncultured differentiated cells, embryonic stem cells and induced pluripotent stem cells. Regions of ASM identified most consistently across methylomes are tightly connected with known imprinted genes and precisely delineate the boundaries of several known imprinting control regions. Predicted regions of ASM common to multiple cell types frequently mark noncoding RNA promoters and represent promising starting points for targeted validation. More generally, our model provides the analytical complement to cutting-edge experimental technologies for surveying ASM in specific cell types and across species. PMID:22523239

  14. Genomic landscape of human allele-specific DNA methylation

    PubMed Central

    Fang, Fang; Hodges, Emily; Molaro, Antoine; Dean, Matthew; Hannon, Gregory J.; Smith, Andrew D.

    2012-01-01

    DNA methylation mediates imprinted gene expression by passing an epigenomic state across generations and differentially marking specific regulatory regions on maternal and paternal alleles. Imprinting has been tied to the evolution of the placenta in mammals and defects of imprinting have been associated with human diseases. Although recent advances in genome sequencing have revolutionized the study of DNA methylation, existing methylome data remain largely untapped in the study of imprinting. We present a statistical model to describe allele-specific methylation (ASM) in data from high-throughput short-read bisulfite sequencing. Simulation results indicate technical specifications of existing methylome data, such as read length and coverage, are sufficient for full-genome ASM profiling based on our model. We used our model to analyze methylomes for a diverse set of human cell types, including cultured and uncultured differentiated cells, embryonic stem cells and induced pluripotent stem cells. Regions of ASM identified most consistently across methylomes are tightly connected with known imprinted genes and precisely delineate the boundaries of several known imprinting control regions. Predicted regions of ASM common to multiple cell types frequently mark noncoding RNA promoters and represent promising starting points for targeted validation. More generally, our model provides the analytical complement to cutting-edge experimental technologies for surveying ASM in specific cell types and across species. PMID:22523239

  15. D9S1120, a simple STR with a common Native American-specific allele: forensic optimization, locus characterization and allele frequency studies.

    PubMed

    Phillips, C; Rodriguez, A; Mosquera-Miguel, A; Fondevila, M; Porras-Hurtado, L; Rondon, F; Salas, A; Carracedo, A; Lareu, M V

    2008-12-01

    The simple tetrameric STR D9S1120 exhibits a common population-specific allele of 9 repeats (9RA) reported to have an average frequency of 0.36 in Native Americans from both North and South of the continent. Apart from the presence of 9RA in two northeast Siberian populations, D9S1120 shows variability exclusive to, and universal in all American populations studied to date. This STR therefore provides an informative forensic marker applicable in countries with significant proportions of Native American populations or ancestry. We have re-designed PCR primers that reduce the amplified product sizes reported in NCBI UniSTS by more than a third and have characterized the repeat structure of D9S1120. The 9RA allele shares the same repeat structure as the majority of other D9S1120 alleles and so originates from a slippage-diminution mutation rather than an independent deletion. We confirm the previously reported allele frequencies from a range of populations indicating a global heterozygosity range for D9S1120 of 66-75% and estimate the proportion of Native American-diagnostic genotypes to average 53%, underlining the potential usefulness of this STR in both forensic identification and in population genetics studies of the Americas.

  16. Allele-Specific Reduction of the Mutant Huntingtin Allele Using Transcription Activator-Like Effectors in Human Huntington's Disease Fibroblasts.

    PubMed

    Fink, Kyle D; Deng, Peter; Gutierrez, Josh; Anderson, Joseph S; Torrest, Audrey; Komarla, Anvita; Kalomoiris, Stefanos; Cary, Whitney; Anderson, Johnathon D; Gruenloh, William; Duffy, Alexandra; Tempkin, Teresa; Annett, Geralyn; Wheelock, Vicki; Segal, David J; Nolta, Jan A

    2016-01-01

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of CAG repeats. Although pathogenesis has been attributed to this polyglutamine expansion, the underlying mechanisms through which the huntingtin protein functions have yet to be elucidated. It has been suggested that postnatal reduction of mutant huntingtin through protein interference or conditional gene knockout could prove to be an effective therapy for patients suffering from HD. For allele-specific targeting, transcription activator-like effectors (TALE) were designed to target single-nucleotide polymorphisms (SNP) in the mutant allele and packaged into a vector backbone containing KRAB to promote transcriptional repression of the disease-associated allele. Additional TALEs were packaged into a vector backbone containing heterodimeric FokI and were designed to be used as nucleases (TALEN) to cause a CAG-collapse in the mutant allele. Human HD fibroblasts were treated with each TALE-SNP or TALEN. Allele-expression was measured using a SNP-genotyping assay and mutant protein aggregation was quantified with Western blots for anti-ubiquitin. The TALE-SNP and TALEN significantly reduced mutant allele expression (p < 0.05) when compared to control transfections while not affecting expression of the nondisease allele. This study demonstrates the potential of allele-specific gene modification using TALE proteins, and provides a foundation for targeted treatment for individuals suffering from Huntington's or other genetically linked diseases. PMID:26850319

  17. Self-(in)compatibility inheritance and allele-specific marker development in yellow mustard (Sinapis alba).

    PubMed

    Zeng, Fangqin; Cheng, Bifang

    2014-01-01

    Yellow mustard (Sinapis alba) has a sporophytic self-incompatibility reproduction system. Genetically stable self-incompatible (SI) and self-compatible (SC) inbred lines have recently been developed in this crop. Understanding the S haplotype of different inbred lines and the inheritance of the self-(in)compatibility (SI/SC) trait is very important for breeding purposes. In this study, we used the S-locus gene-specific primers in Brassica rapa and Brassica oleracea to clone yellow mustard S-locus genes of SI lines Y514 and Y1130 and SC lines Y1499 and Y1501. The PCR amplification results and DNA sequences of the S-locus genes revealed that Y514 carried the class I S haplotype, while Y1130, Y1499, and Y1501 had the class II S haplotype. The results of our genetic studies indicated that self-incompatibility was dominant over self-compatibility and controlled by a one-gene locus in the two crosses of Y514 × Y1499 and Y1130 × Y1501. Of the five S-locus gene polymorphic primer pairs, Sal-SLGI and Sal-SRKI each generated one dominant marker for the SI phenotype of Y514; Sal-SLGII and Sal-SRKII produced dominant marker(s) for the SC phenotype of Y1501 and Y1499; Sal-SP11II generated one dominant marker for Y1130. These markers co-segregated with the SI/SC phenotype in the F2 populations of the two crosses. In addition, co-dominant markers were developed by mixing the two polymorphic primer pairs specific for each parent in the multiplex PCR, which allowed zygosity to be determined in the F2 populations. The SI/SC allele-specific markers have proven to be very useful for the selection of the desirable SC genotypes in our yellow mustard breeding program.

  18. Assignment of SNP allelic configuration in polyploids using competitive allele-specific PCR: application to citrus triploid progeny

    PubMed Central

    Cuenca, José; Aleza, Pablo; Navarro, Luis; Ollitrault, Patrick

    2013-01-01

    Background Polyploidy is a major component of eukaryote evolution. Estimation of allele copy numbers for molecular markers has long been considered a challenge for polyploid species, while this process is essential for most genetic research. With the increasing availability and whole-genome coverage of single nucleotide polymorphism (SNP) markers, it is essential to implement a versatile SNP genotyping method to assign allelic configuration efficiently in polyploids. Scope This work evaluates the usefulness of the KASPar method, based on competitive allele-specific PCR, for the assignment of SNP allelic configuration. Citrus was chosen as a model because of its economic importance, the ongoing worldwide polyploidy manipulation projects for cultivar and rootstock breeding, and the increasing availability of SNP markers. Conclusions Fifteen SNP markers were successfully designed that produced clear allele signals that were in agreement with previous genotyping results at the diploid level. The analysis of DNA mixes between two haploid lines (Clementine and pummelo) at 13 different ratios revealed a very high correlation (average = 0·9796; s.d. = 0·0094) between the allele ratio and two parameters [θ angle = tan−1 (y/x) and y′ = y/(x + y)] derived from the two normalized allele signals (x and y) provided by KASPar. Separated cluster analysis and analysis of variance (ANOVA) from mixed DNA simulating triploid and tetraploid hybrids provided 99·71 % correct allelic configuration. Moreover, triploid populations arising from 2n gametes and interploid crosses were easily genotyped and provided useful genetic information. This work demonstrates that the KASPar SNP genotyping technique is an efficient way to assign heterozygous allelic configurations within polyploid populations. This method is accurate, simple and cost-effective. Moreover, it may be useful for quantitative studies, such as relative allele-specific expression analysis and bulk segregant analysis

  19. Substrate specificity of allelic variants of the TAP peptide transporter.

    PubMed

    Heemels, M T; Ploegh, H L

    1994-12-01

    The transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum (ER). An important determinant for the specificity of translocation is the identity of the C-terminal residue of the peptide substrate. In the rat, a suitable C terminus is necessary but not always sufficient for a peptide to be selected for translocation. Here we show that sequence constraints within a peptide of optimal length (9 residues) may interfere with transport; that the transporter selectively translocates shorter derivatives of a 16-mer peptide rather than the 16-mer itself; and that the transporter cimb allele, which is most selective in the C termini it will tolerate, is more relaxed in peptide length preference than is the clma variant. PMID:7895166

  20. Substrate specificity of allelic variants of the TAP peptide transporter.

    PubMed

    Heemels, M T; Ploegh, H L

    1994-12-01

    The transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum (ER). An important determinant for the specificity of translocation is the identity of the C-terminal residue of the peptide substrate. In the rat, a suitable C terminus is necessary but not always sufficient for a peptide to be selected for translocation. Here we show that sequence constraints within a peptide of optimal length (9 residues) may interfere with transport; that the transporter selectively translocates shorter derivatives of a 16-mer peptide rather than the 16-mer itself; and that the transporter cimb allele, which is most selective in the C termini it will tolerate, is more relaxed in peptide length preference than is the clma variant.

  1. Polymorphism analysis of Chinese Theileria sergenti using allele-specific polymerase chain reaction of the major piroplasm surface protein gene.

    PubMed

    Liu, Ai Hong; Guan, Gui Quan; Liu, Jun Long; Liu, Zhi Jie; Leblanc, Neil; Li, You Quan; Gao, Jin Liang; Ma, Mi Ling; Niu, Qing Li; Ren, Qiao Yun; Bai, Qi; Yin, Hong; Luo, Jian Xun

    2011-02-01

    Theileria sergenti is a tick-borne parasite found in many parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a marker for epidemiological and phylogenetic studies of benign Theileria species. In this study, Chinese species of T. sergenti were characterized by allele-specific polymerase chain reaction (PCR) and DNA sequence analysis of the MPSP gene. Using universal or allele-specific primer sets for PCR amplification of the MPSP gene, 98 of 288 cattle blood samples, collected from 6 provinces in China, were found to be positive. Among the positive samples, only 3 allelic MPSP gene types (Chitose [C]-, Ikeda [I]-, and buffeli [B]-type) were successfully amplified. Moreover, the results revealed that the majority of the parasites sampled in this study were C- and I-type (prevalence of 84 and 69%, respectively), whereas the B-type was less common (prevalence of 36%). Co-infections with C-, I-, and B-type T. sergenti also were found. An additional known allele, Thai-type, was not detected. Phylogenetic analysis based on the MPSP gene sequences, including 3 standard stocks generated in the laboratory ( T. sergenti Wenchuan, T. sergenti Ningxian, and T. sergenti Liaoyang), revealed that the isolates of Chinese sergenti were comprised of at least 4 allelic MPSP gene types, i.e., C-, I-, B1-, and B2-type, and these parasites with 6 MPSP types 1-5 and 7 were present in China.

  2. Allele-specific chemical genetics: concept, strategies, and applications.

    PubMed

    Islam, Kabirul

    2015-02-20

    The relationship between DNA and protein sequences is well understood, yet because the members of a protein family/subfamily often carry out the same biochemical reaction, elucidating their individual role in cellular processes presents a challenge. Forward and reverse genetics have traditionally been employed to understand protein functions with considerable success. A fundamentally different approach that has gained widespread application is the use of small organic molecules, known as chemical genetics. However, the slow time-scale of genetics and inherent lack of specificity of small molecules used in chemical genetics have limited the applicability of these methods in deconvoluting the role of individual proteins involved in fast, dynamic biological events. Combining the advantages of both the techniques, the specificity achieved with genetics along with the reversibility and tunability of chemical genetics, has led to the development of a powerful approach to uncover protein functions in complex biological processes. This technique is known as allele-specific chemical genetics and is rapidly becoming an essential toolkit to shed light on proteins and their mechanism of action. The current review attempts to provide a comprehensive description of this approach by discussing the underlying principles, strategies, and successful case studies. Potential future implications of this technology in expanding the frontiers of modern biology are discussed.

  3. GSP: A web-based platform for designing genome-specific primers in polyploids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The sequences among subgenomes in a polyploid species have high similarity. This makes difficult to design genome-specific primers for sequence analysis. We present a web-based platform named GSP for designing genome-specific primers to distinguish subgenome sequences in the polyploid genome backgr...

  4. [Microchip electrophoresis coupled with multiplex allele-specific am-plification for typing multiple single nucleotide polymorphisms (SNPs) simultaneously].

    PubMed

    Wang, Wei-Peng; Zhou, Guo-Hua

    2009-02-01

    A new method of DNA adapter ligation-mediated allele-specific amplification (ALM-ASA) was developed for typing multiple single nucleotide polymorphisms (SNPs) on the platform of microchip electrophoresis. Using seven SNPs of 794C>T, 1274C>T, 2143T>C, 2766T>del, 3298G>A, 5200G>A, and 5277C>T in the interleukin 1B (IL1B) gene as a target object, a long DNA fragment containing the seven SNPs of interest was pre-amplified to enhance the specificity. The pre-amplified DNA fragment was digested by a restriction endonuclease to form sticky ends; and then the adapter was ligated to either end of the digested fragment. Using the adapter-ligated fragments as templates, a 7-plex allele-specific amplification was performed by 7 allele-specific primers and a universal primer in one tube. The allele-specific products amplified were separated by chip electrophoresis and the types of SNPs were easily discriminated by the product sizes. The seven SNPs in IL1B gene in 48 healthy Chinese were successfully typed by microchip electrophoresis and the results coincided with those by PCR-restriction fragment length polymorphism and sequencing method. The method established was accurate and can be used to type multiple SNPs simultaneously. In combination with microchip electrophoresis for readout, ALM-ASA assay can be used for fast SNP detection with a small amount of sample. Using self-prepared gel matrix and reused chips for analysis, the SNP can be typed at an ultra low cost.

  5. BatchPrimer3: A high throughput web application for PCR and sequencing primer design

    PubMed Central

    You, Frank M; Huo, Naxin; Gu, Yong Qiang; Luo, Ming-cheng; Ma, Yaqin; Hane, Dave; Lazo, Gerard R; Dvorak, Jan; Anderson, Olin D

    2008-01-01

    Background Microsatellite (simple sequence repeat – SSR) and single nucleotide polymorphism (SNP) markers are two types of important genetic markers useful in genetic mapping and genotyping. Often, large-scale genomic research projects require high-throughput computer-assisted primer design. Numerous such web-based or standard-alone programs for PCR primer design are available but vary in quality and functionality. In particular, most programs lack batch primer design capability. Such a high-throughput software tool for designing SSR flanking primers and SNP genotyping primers is increasingly demanded. Results A new web primer design program, BatchPrimer3, is developed based on Primer3. BatchPrimer3 adopted the Primer3 core program as a major primer design engine to choose the best primer pairs. A new score-based primer picking module is incorporated into BatchPrimer3 and used to pick position-restricted primers. BatchPrimer3 v1.0 implements several types of primer designs including generic primers, SSR primers together with SSR detection, and SNP genotyping primers (including single-base extension primers, allele-specific primers, and tetra-primers for tetra-primer ARMS PCR), as well as DNA sequencing primers. DNA sequences in FASTA format can be batch read into the program. The basic information of input sequences, as a reference of parameter setting of primer design, can be obtained by pre-analysis of sequences. The input sequences can be pre-processed and masked to exclude and/or include specific regions, or set targets for different primer design purposes as in Primer3Web and primer3Plus. A tab-delimited or Excel-formatted primer output also greatly facilitates the subsequent primer-ordering process. Thousands of primers, including wheat conserved intron-flanking primers, wheat genome-specific SNP genotyping primers, and Brachypodium SSR flanking primers in several genome projects have been designed using the program and validated in several laboratories

  6. Development of allele-specific PCR and RT-PCR assays for clustered resistance genes using a potato late blight resistance transgene as a model.

    PubMed

    Millett, B P; Bradeen, J M

    2007-02-01

    Members of the NBS-LRR gene family impart resistance to a wide variety of pathogens and are often found clustered within a plant genome. This clustering of homologous sequences can complicate PCR-based characterizations, especially the study of transgenes. We have developed allele-specific PCR and RT-PCR assays for the potato late blight resistance gene RB. Our assay utilizes two approaches toward primer design, allowing discrimination between the RB transgene and both the endogenous RB gene and numerous RB homeologs. First, a reverse primer was designed to take advantage of an indel present in the RB transgene but absent in rb susceptibility alleles, enhancing specificity for the transgene, though not fully discriminating against RB homeologs. Second, a forward primer was designed according to the principles of mismatch amplification mutation assay (MAMA) PCR, targeting SNPs introduced during the cloning of RB. Together, the indel reverse primer and the MAMA forward primer provide an assay that is highly specific for the RB transgene, being capable of distinguishing the transgene from all RB endogenous gene copies and from all RB paralogs in a diverse collection of wild and cultivated potato genotypes. These primers have been successfully multiplexed with primers of an internal control. The multiplexed assay is useful for both PCR and RT-PCR applications. Double MAMA-PCR, in which both PCR primers target separate transgene-specific SNPs, was also tested and shown to be equally specific for the RB transgene. We propose extending the use of MAMA for the characterization of resistance transgenes. PMID:17177064

  7. Cross-kingdom amplification using Bacteria-specific primers: Complications for studies of coral microbial ecology

    USGS Publications Warehouse

    Galkiewicz, J.P.; Kellogg, C.A.

    2008-01-01

    PCR amplification of pure bacterial DNA is vital to the study of bacterial interactions with corals. Commonly used Bacteria-specific primers 8F and 27F paired with the universal primer 1492R amplify both eukaryotic and prokaryotic rRNA genes. An alternative primer set, 63F/1542R, is suggested to resolve this problem. Copyright ?? 2008, American Society for Microbiology. All Rights Reserved.

  8. New prediction model for probe specificity in an allele-specific extension reaction for haplotype-specific extraction (HSE) of Y chromosome mixtures.

    PubMed

    Rothe, Jessica; Watkins, Norman E; Nagy, Marion

    2012-01-01

    Allele-specific extension reactions (ASERs) use 3' terminus-specific primers for the selective extension of completely annealed matches by polymerase. The ability of the polymerase to extend non-specific 3' terminal mismatches leads to a failure of the reaction, a process that is only partly understood and predictable, and often requires time-consuming assay design. In our studies we investigated haplotype-specific extraction (HSE) for the separation of male DNA mixtures. HSE is an ASER and provides the ability to distinguish between diploid chromosomes from one or more individuals. Here, we show that the success of HSE and allele-specific extension depend strongly on the concentration difference between complete match and 3' terminal mismatch. Using the oligonucleotide-modeling platform Visual Omp, we demonstrated the dependency of the discrimination power of the polymerase on match- and mismatch-target hybridization between different probe lengths. Therefore, the probe specificity in HSE could be predicted by performing a relative comparison of different probe designs with their simulated differences between the duplex concentration of target-probe match and mismatches. We tested this new model for probe design in more than 300 HSE reactions with 137 different probes and obtained an accordance of 88%.

  9. DQB1*06:02 allele specific expression varies by allelic dosage, not narcolepsy status

    PubMed Central

    lachmi, Karin Weiner; Lin, Ling; Kornum, Birgitte Rahbek; Rico, Tom; Lo, Betty; Aran, Adi; Mignot, Emmanuel

    2012-01-01

    The association of narcolepsy-cataplexy, a sleep disorder caused by the loss of hypocretin/orexin neurons in the hypothalamus, with DQA1*01:02-DQB1*06:02 is one of the tightest known single allele HLA associations. In this study, we explored genome wide expression in peripheral white blood cells of 50 narcolepsy versus 47 controls (half of whom were DQB1*06:02 positive) and found the largest differences between the groups to be in the signal from HLA probes. Further studies of HLA-DQ expression (mRNA and protein in a subset) in 125 controls and 147 narcolepsy cases did not reveal any difference, a result we explain by the lack of proper control of allelic diversity in Affymetrix HLA probes. Rather, a clear effect of DQB1*06:02 allelic dosage on DQB1*06:02 mRNA levels (1.65 fold) and protein (1.59 fold) could be demonstrated independent of the disease status. These results indicate that allelic dosage is transmitted into changes in heterodimer availability, a phenomenon that may explain increased risk for narcolepsy in DQB1*06:02 homozygotes versus heterozygotes. PMID:22326585

  10. DQB1*06:02 allele-specific expression varies by allelic dosage, not narcolepsy status.

    PubMed

    Weiner Lachmi, Karin; Lin, Ling; Kornum, Birgitte Rahbek; Rico, Tom; Lo, Betty; Aran, Adi; Mignot, Emmanuel

    2012-04-01

    The association of narcolepsy-cataplexy, a sleep disorder caused by the loss of hypocretin/orexin neurons in the hypothalamus, with DQA1*01:02-DQB1*06:02 is one of the tightest known single-allele human leukocyte antigen (HLA) associations. In this study, we explored genome-wide expression in peripheral white blood cells of 50 narcolepsy versus 47 controls (half of whom were DQB1*06:02 positive) and observed the largest differences between the groups in the signal from HLA probes. Further studies of HLA-DQ expression (mRNA and protein in a subset) in 125 controls and 147 narcolepsy cases did not reveal any difference, a result we explain by the lack of proper control of allelic diversity in Affymetrix HLA probes. Rather, a clear effect of DQB1*06:02 allelic dosage on DQB1*06:02 mRNA levels (1.65-fold) and protein (1.59-fold) could be demonstrated independent of disease status. These results indicate that allelic dosage is transmitted into changes in heterodimer availability, a phenomenon that may explain the increased risk for narcolepsy in DQB1*06:02 homozygotes versus heterozygotes.

  11. MFEprimer-2.0: a fast thermodynamics-based program for checking PCR primer specificity.

    PubMed

    Qu, Wubin; Zhou, Yang; Zhang, Yanchun; Lu, Yiming; Wang, Xiaolei; Zhao, Dongsheng; Yang, Yi; Zhang, Chenggang

    2012-07-01

    Evaluating the specificity of polymerase chain reaction (PCR) primers is an essential step in PCR primer design. The MFEprimer-2.0 server allows users to check primer specificity against genomic DNA and messenger RNA/complementary DNA sequence databases quickly and easily. MFEprimer-2.0 uses a k-mer index algorithm to accelerate the search process for primer binding sites and uses thermodynamics to evaluate binding stability between each primer and its DNA template. Several important characteristics, such as the sequence, melting temperature and size of each amplicon, either specific or non-specific, are reported on the results page. Based on these characteristics and the user-friendly output, users can readily draw conclusions about the specificity of PCR primers. Analyses for degenerate primers and multiple PCR primers are also supported in MFEprimer-2.0. In addition, the databases supported by MFEprimer-2.0 are comprehensive, and custom databases can also be supported on request. The MFEprimer-2.0 server does not require a login and is freely available at http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0. More over, the MFEprimer-2.0 command-line version and local server version are open source and can be downloaded at https://github.com/quwubin/MFEprimer/wiki/Manual/.

  12. Allele-specific PCR for the beta-tubulin codon 200 TTC/TAC polymorphism using single adult and larval small strongyle (Cyathostominae) stages.

    PubMed

    von Samson-Himmelstjerna, G; Pape, M; von Witzendorff, C; Schnieder, T

    2002-04-01

    It has been shown that benzimidazole (BZ) resistance in sheep gastrointestinal nematodes is linked with an increase in beta-tubulin codon 200 tyrosine-expressing alleles in the resistant parasite populations. Here, an allele-specific PCR has been developed for the discrimination of the TAC/TTC polymorphism in the beta-tubulin 200 codon of small strongyles. One reverse primer was used in 2 separate amplifications with 1 of 2 forward primers that differed only in their final 3' nucleotide. The primers flank a facultative intron/exon. Therefore, the amplified fragments are either 251 or 308 bp in size, depending on the presence or absence of the intron in individual worms. Amplification of genomic DNA isolated from single adult small strongyles from a set of 7 species consistently generated allele-specific products. Three worms each of the following species were used: Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus elongatus, Cylicocyclus radiatus, Cyathostomum pateratum, Cyathostomum catinatum, and Cyathostomum coronatum. PCR with DNA isolated from single larvae also reproducibly generated specific fragments. This method might be applied for the future assessment of allele frequencies in susceptible and resistant populations to further investigate the mechanism of BZ-resistance in small strongyles. PMID:12053994

  13. Allele specific expression in worker reproduction genes in the bumblebee Bombus terrestris.

    PubMed

    Amarasinghe, Harindra E; Toghill, Bradley J; Nathanael, Despina; Mallon, Eamonn B

    2015-01-01

    Methylation has previously been associated with allele specific expression in ants. Recently, we found methylation is important in worker reproduction in the bumblebee Bombus terrestris. Here we searched for allele specific expression in twelve genes associated with worker reproduction in bees. We found allele specific expression in Ecdysone 20 monooxygenase and IMP-L2-like. Although we were unable to confirm a genetic or epigenetic cause for this allele specific expression, the expression patterns of the two genes match those predicted for imprinted genes.

  14. Construction of Specific Primers for Rapid Detection of South African Exportable Vegetable Macergens.

    PubMed

    Aremu, Bukola Rhoda; Babalola, Olubukola Oluranti

    2015-10-01

    Macergens are bacteria causing great damages to the parenchymatous tissues of vegetable both on the field and in transit. To effectively and rapidly investigate the diversity and distribution of these macergens, four specific primers were designed by retrieving 16S rDNA sequences of pectolytic bacteria from GenBank through the National Center for Biotechnology Information (NCBI). These were aligned using ClusterW via BioEdit and primers were designed using Primer3Plus platform. The size and primer location of each species and PCR product size were accurately defined. For specificity enhancement, DNA template of known macergens (Pectobacterium chrysanthermi) and fresh healthy vegetable were used. These primers yielded expected size of approximately 1100 bp product only when tested with known macergens and no amplicon with fresh healthy vegetable was detected. Rapid detection of macergens in rotten vegetable samples was then carried out using these primers. Nucleotide sequences of macergens identified were deposited into the GenBank and were assigned accession numbers. Hence, with these specific primers, macergens can be identified with minimal quantities of the vegetable tissues using molecular techniques, for future use of the quarantine section of the Agricultural Department of the country for quick and rapid detection of macergens before exportation. PMID:26437427

  15. Construction of Specific Primers for Rapid Detection of South African Exportable Vegetable Macergens

    PubMed Central

    Aremu, Bukola Rhoda; Babalola, Olubukola Oluranti

    2015-01-01

    Macergens are bacteria causing great damages to the parenchymatous tissues of vegetable both on the field and in transit. To effectively and rapidly investigate the diversity and distribution of these macergens, four specific primers were designed by retrieving 16S rDNA sequences of pectolytic bacteria from GenBank through the National Center for Biotechnology Information (NCBI). These were aligned using ClusterW via BioEdit and primers were designed using Primer3Plus platform. The size and primer location of each species and PCR product size were accurately defined. For specificity enhancement, DNA template of known macergens (Pectobacterium chrysanthermi) and fresh healthy vegetable were used. These primers yielded expected size of approximately 1100 bp product only when tested with known macergens and no amplicon with fresh healthy vegetable was detected. Rapid detection of macergens in rotten vegetable samples was then carried out using these primers. Nucleotide sequences of macergens identified were deposited into the GenBank and were assigned accession numbers. Hence, with these specific primers, macergens can be identified with minimal quantities of the vegetable tissues using molecular techniques, for future use of the quarantine section of the Agricultural Department of the country for quick and rapid detection of macergens before exportation. PMID:26437427

  16. Construction of Specific Primers for Rapid Detection of South African Exportable Vegetable Macergens.

    PubMed

    Aremu, Bukola Rhoda; Babalola, Olubukola Oluranti

    2015-09-30

    Macergens are bacteria causing great damages to the parenchymatous tissues of vegetable both on the field and in transit. To effectively and rapidly investigate the diversity and distribution of these macergens, four specific primers were designed by retrieving 16S rDNA sequences of pectolytic bacteria from GenBank through the National Center for Biotechnology Information (NCBI). These were aligned using ClusterW via BioEdit and primers were designed using Primer3Plus platform. The size and primer location of each species and PCR product size were accurately defined. For specificity enhancement, DNA template of known macergens (Pectobacterium chrysanthermi) and fresh healthy vegetable were used. These primers yielded expected size of approximately 1100 bp product only when tested with known macergens and no amplicon with fresh healthy vegetable was detected. Rapid detection of macergens in rotten vegetable samples was then carried out using these primers. Nucleotide sequences of macergens identified were deposited into the GenBank and were assigned accession numbers. Hence, with these specific primers, macergens can be identified with minimal quantities of the vegetable tissues using molecular techniques, for future use of the quarantine section of the Agricultural Department of the country for quick and rapid detection of macergens before exportation.

  17. Impriniting of human H19: Allele-specific CpG methylation, loss of the active allele in Wilms tumor, and potential for somatic allele switching

    SciTech Connect

    Zhang, Y.; Shields, T.; Crenshaw, T.; Hao, Y.; Moulton, T.; Tycko, B. )

    1993-07-01

    Genomic imprinting and monoallelic gene expression appear to play a role in human genetic disease and tumorigenesis. The human H19 gene, at chromosome 11p15, has previously been shown to be monoallelically expressed. Since CpG methylation has been implicated in imprinting, the authors analyzed methylation of H19 DNA. In fetal and adult organs the transcriptionally silent H19 allele was extensively hypermethylated through the entire gene and its promoter, and, consistent with a functional role for DNA methylation, expression of an H19 promoter-reporter construct was inhibited by in vitro methylation. Gynogenetic ovarian teratomas were found to contain only hypomethylated H19 DNA, suggesting that the expressed H19 allele might be maternal. This was confirmed by analysis of 11p15 polymorphisms in a patient with Wilms tumor. The tumor had lost the maternal 11p15, and H19 expression in the normal kidney was exclusively from this allele. Imprinting of human H19 appears to be susceptible to tissue-specific modulation in somatic development; in one individual, cerebellar cells were found to express only the otherwise silent allele. Implications of these findings for the role of DNA methylation in imprinting and for H19 as a candidate imprinted tumor-suppressor gene are discussed. 57 refs., 7 figs.

  18. primers4clades: a web server that uses phylogenetic trees to design lineage-specific PCR primers for metagenomic and diversity studies

    PubMed Central

    Contreras-Moreira, Bruno; Sachman-Ruiz, Bernardo; Figueroa-Palacios, Iraís; Vinuesa, Pablo

    2009-01-01

    Primers4clades is an easy-to-use web server that implements a fully automatic PCR primer design pipeline for cross-species amplification of novel sequences from metagenomic DNA, or from uncharacterized organisms, belonging to user-specified phylogenetic clades or taxa. The server takes a set of non-aligned protein coding genes, with or without introns, aligns them and computes a neighbor-joining tree, which is displayed on screen for easy selection of species or sequence clusters to design lineage-specific PCR primers. Primers4clades implements an extended CODEHOP primer design strategy based on both DNA and protein multiple sequence alignments. It evaluates several thermodynamic properties of the oligonucleotide pairs, and computes the phylogenetic information content of the predicted amplicon sets from Shimodaira–Hasegawa-like branch support values of maximum likelihood phylogenies. A non-redundant set of primer formulations is returned, ranked according to their thermodynamic properties. An amplicon distribution map provides a convenient overview of the coverage of the target locus. Altogether these features greatly help the user in making an informed choice between alternative primer pair formulations. Primers4clades is available at two mirror sites: http://maya.ccg.unam.mx/primers4clades/and http://floresta.eead.csic.es/primers4clades/. Three demo data sets and a comprehensive documentation/tutorial page are provided for easy testing of the server's capabilities and interface. PMID:19465390

  19. primers4clades: a web server that uses phylogenetic trees to design lineage-specific PCR primers for metagenomic and diversity studies.

    PubMed

    Contreras-Moreira, Bruno; Sachman-Ruiz, Bernardo; Figueroa-Palacios, Iraís; Vinuesa, Pablo

    2009-07-01

    Primers4clades is an easy-to-use web server that implements a fully automatic PCR primer design pipeline for cross-species amplification of novel sequences from metagenomic DNA, or from uncharacterized organisms, belonging to user-specified phylogenetic clades or taxa. The server takes a set of non-aligned protein coding genes, with or without introns, aligns them and computes a neighbor-joining tree, which is displayed on screen for easy selection of species or sequence clusters to design lineage-specific PCR primers. Primers4clades implements an extended CODEHOP primer design strategy based on both DNA and protein multiple sequence alignments. It evaluates several thermodynamic properties of the oligonucleotide pairs, and computes the phylogenetic information content of the predicted amplicon sets from Shimodaira-Hasegawa-like branch support values of maximum likelihood phylogenies. A non-redundant set of primer formulations is returned, ranked according to their thermodynamic properties. An amplicon distribution map provides a convenient overview of the coverage of the target locus. Altogether these features greatly help the user in making an informed choice between alternative primer pair formulations. Primers4clades is available at two mirror sites: http://maya.ccg.unam.mx/primers4clades/and http://floresta.eead.csic.es/primers4clades/. Three demo data sets and a comprehensive documentation/tutorial page are provided for easy testing of the server's capabilities and interface.

  20. An allele-specific PCR system for rapid detection and discrimination of the CYP2C19∗4A, ∗4B, and ∗17 alleles: implications for clopidogrel response testing.

    PubMed

    Scott, Stuart A; Tan, Qian; Baber, Usman; Yang, Yao; Martis, Suparna; Bander, Jeffrey; Kornreich, Ruth; Hulot, Jean-Sébastien; Desnick, Robert J

    2013-11-01

    CYP2C19 is involved in the metabolism of clinically relevant drugs, including the antiplatelet prodrug clopidogrel, which has prompted interest in clinical CYP2C19 genotyping. The CYP2C19∗4B allele is defined by both gain-of-function [c.-806C>T (∗17)] and loss-of-function [c.1A>G (∗4)] variants on the same haplotype; however, current genotyping and sequencing assays are unable to determine the phase of these variants. Thus, the aim of this study was to develop an assay that could rapidly detect and discriminate the related ∗4A, ∗4B, and ∗17 alleles. An allele-specific PCR assay, composed of four unique primer mixes that specifically interrogate the defining ∗17 and ∗4 variants, was developed by using samples (n = 20) with known genotypes, including the ∗4A, ∗4B, and/or ∗17 alleles. The assay was validated by testing 135 blinded samples, and the results were correlated with CYP2C19 genotyping and allele-specific cloning/sequencing. Importantly, among the six ∗4 carriers in the validation cohort, after allele-specific PCR testing both samples with a ∗1/∗4 genotype were reclassified to ∗1/∗4A, all three samples with a ∗4/∗17 genotype were reclassified to ∗1/∗4B, and a sample with a ∗4/∗17/∗17 genotype was reclassified to ∗4B/∗17. In conclusion, this rapid and robust allele-specific PCR assay can refine CYP2C19 genotyping and metabolizer phenotype classification by determining the phase of the defining ∗17 and ∗4 variants, which may have utility when testing CYP2C19 for clopidogrel response.

  1. Indexed PCR Primers Induce Template-Specific Bias in Large-Scale DNA Sequencing Studies

    PubMed Central

    O’Donnell, James L.; Kelly, Ryan P.; Lowell, Natalie C.; Port, Jesse A.

    2016-01-01

    Massively parallel sequencing is rapidly emerging as an efficient way to quantify biodiversity at all levels, from genetic variation and expression to ecological community assemblage. However, the number of reads produced per sequencing run far exceeds the number required per sample for many applications, compelling researchers to sequence multiple samples per run in order to maximize efficiency. For studies that include a PCR step, this can be accomplished using primers that include an index sequence allowing sample origin to be determined after sequencing. The use of indexed primers assumes they behave no differently than standard primers; however, we found that indexed primers cause substantial template sequence-specific bias, resulting in radically different profiles of the same environmental sample. Likely the outcome of differential amplification efficiency due to primer-template mismatch, two indexed primer sets spuriously change the inferred sequence abundance from the same DNA extraction by up to 77.1%. We demonstrate that a double PCR approach alleviates these effects in applications where indexed primers are necessary. PMID:26950069

  2. A computational workflow to identify allele-specific expression and epigenetic modification in maize.

    PubMed

    Wei, Xiaoxing; Wang, Xiangfeng

    2013-08-01

    Allele-specific expression refers to the preferential expression of one of the two alleles in a diploid genome, which has been thought largely attributable to the associated cis-element variation and allele-specific epigenetic modification patterns. Allele-specific expression may contribute to the heterosis (or hybrid vigor) effect in hybrid plants that are produced from crosses of closely-related species, subspecies and/or inbred lines. In this study, using Illumina high-throughput sequencing of maize transcriptomics, chromatic H3K27me3 histone modification and DNA methylation data, we developed a new computational framework to identify allele-specifically expressed genes by simultaneously tracking allele-specific gene expression patterns and the epigenetic modification landscape in the seedling tissues of hybrid maize. This approach relies on detecting nucleotide polymorphisms and any genomic structural variation between two parental genomes in order to distinguish paternally or maternally derived sequencing reads. This computational pipeline also incorporates a modified Chi-square test to statistically identify allele-specific gene expression and epigenetic modification based on the Poisson distribution.

  3. 5' and 3' untranslated regions contribute to the differential expression of specific HLA-A alleles.

    PubMed

    René, Céline; Lozano, Claire; Villalba, Martin; Eliaou, Jean-François

    2015-12-01

    In hematopoietic stem cell transplantation (HSCT), when no HLA full-matched donor is available, alternative donors could include one HLA-mismatched donor. Recently, the low expressed HLA-C alleles have been identified as permissive mismatches for the best donor choice. Concerning HLA-A, the degree of variability of expression is poorly understood. Here, we evaluated HLA-A expression in healthy individuals carrying HLA-A*02 allele in different genotypes using flow cytometry and allele-specific quantitative RT-PCR. While an interindividual variability of HLA-A*02 cell surface expression, not due to the allele associated, was observed, no difference of the mRNA expression level was shown, suggesting the involvement of the posttranscriptional regulation. The results of qRT-PCR analyses exhibit a differential expression of HLA-A alleles with HLA-A*02 as the strongest expressed allele independently of the second allele. The associated non-HLA-A*02 alleles were differentially expressed, particularly the HLA-A*31 and HLA-A*33 alleles (strong expression) and the HLA-A*29 (low expression). The presence of specific polymorphisms in the 5' and 3' untranslated regions of the HLA-A*31 and HLA-A*33 alleles could contribute to this high level of expression. As previously described for HLA-C, low-expressed HLA-A alleles, such as HLA-A*29, could be considered as a permissive mismatch, although this needs to be confirmed by clinical studies.

  4. Rapid deoxyribonucleic acid analysis by allele-specific polymerase chain reaction for detection of mutations in the steroid 21-hydroxylase gene

    SciTech Connect

    Wilson, R.C.; Wei, J.Q.; Cheng, K.C.

    1995-05-01

    Rapid DNA analysis based on allele-specific polymerase chain reaction (PCR) using mutation site-specific primers was developed to detect mutations in the CYP21 gene known to cause steroid 21-hydroxylase deficiency. In contrast to the previous method, in which PCR of genomic DNA was followed by dot blot analysis with radio active probes and multiple rounds of stripping and reprobing for each of the 8 most common mutation sites, the results using this new method were immediately visualized after the PCR run by ethidium bromide-stained agarose gel electrophoresis. Using allele-specific PCR, mutation(s) were identified on 148 affected chromosomes out of 160 tested. Although mutation(s) were identified on only one chromosome of 11 of these patients, their parents showed a consistent pattern on DNA analysis. The only exception was that in one family, in which the parents each had a detectable mutation, a mutation was detected on only one allele of the patient. Most likely there is a mutation in the patient`s other allele that could have arisen de novo or was inherited from the parent and was not evident in the transmitting parent`s phenotype. When compared with the dot blot procedure, allele-specific PCR is more rapid, less labor-intensive, and avoids the use of radioactivity. 26 refs., 3 figs., 2 tabs.

  5. A uniform survey of allele-specific binding and expression over 1000-Genomes-Project individuals.

    PubMed

    Chen, Jieming; Rozowsky, Joel; Galeev, Timur R; Harmanci, Arif; Kitchen, Robert; Bedford, Jason; Abyzov, Alexej; Kong, Yong; Regan, Lynne; Gerstein, Mark

    2016-04-18

    Large-scale sequencing in the 1000 Genomes Project has revealed multitudes of single nucleotide variants (SNVs). Here, we provide insights into the functional effect of these variants using allele-specific behaviour. This can be assessed for an individual by mapping ChIP-seq and RNA-seq reads to a personal genome, and then measuring 'allelic imbalances' between the numbers of reads mapped to the paternal and maternal chromosomes. We annotate variants associated with allele-specific binding and expression in 382 individuals by uniformly processing 1,263 functional genomics data sets, developing approaches to reduce the heterogeneity between data sets due to overdispersion and mapping bias. Since many allelic variants are rare, aggregation across multiple individuals is necessary to identify broadly applicable 'allelic elements'. We also found SNVs for which we can anticipate allelic imbalance from the disruption of a binding motif. Our results serve as an allele-specific annotation for the 1000 Genomes variant catalogue and are distributed as an online resource (alleledb.gersteinlab.org).

  6. A uniform survey of allele-specific binding and expression over 1000-Genomes-Project individuals

    PubMed Central

    Chen, Jieming; Rozowsky, Joel; Galeev, Timur R.; Harmanci, Arif; Kitchen, Robert; Bedford, Jason; Abyzov, Alexej; Kong, Yong; Regan, Lynne; Gerstein, Mark

    2016-01-01

    Large-scale sequencing in the 1000 Genomes Project has revealed multitudes of single nucleotide variants (SNVs). Here, we provide insights into the functional effect of these variants using allele-specific behaviour. This can be assessed for an individual by mapping ChIP-seq and RNA-seq reads to a personal genome, and then measuring ‘allelic imbalances' between the numbers of reads mapped to the paternal and maternal chromosomes. We annotate variants associated with allele-specific binding and expression in 382 individuals by uniformly processing 1,263 functional genomics data sets, developing approaches to reduce the heterogeneity between data sets due to overdispersion and mapping bias. Since many allelic variants are rare, aggregation across multiple individuals is necessary to identify broadly applicable ‘allelic elements'. We also found SNVs for which we can anticipate allelic imbalance from the disruption of a binding motif. Our results serve as an allele-specific annotation for the 1000 Genomes variant catalogue and are distributed as an online resource (alleledb.gersteinlab.org). PMID:27089393

  7. Allele-specific characterization of alanine: glyoxylate aminotransferase variants associated with primary hyperoxaluria.

    PubMed

    Lage, Melissa D; Pittman, Adrianne M C; Roncador, Alessandro; Cellini, Barbara; Tucker, Chandra L

    2014-01-01

    Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal recessive kidney stone disease caused by deficiency of the peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT), which is involved in glyoxylate detoxification. Over 75 different missense mutations in AGT have been found associated with PH1. While some of the mutations have been found to affect enzyme activity, stability, and/or localization, approximately half of these mutations are completely uncharacterized. In this study, we sought to systematically characterize AGT missense mutations associated with PH1. To facilitate analysis, we used two high-throughput yeast-based assays: one that assesses AGT specific activity, and one that assesses protein stability. Approximately 30% of PH1-associated missense mutations are found in conjunction with a minor allele polymorphic variant, which can interact to elicit complex effects on protein stability and trafficking. To better understand this allele interaction, we functionally characterized each of 34 mutants on both the major (wild-type) and minor allele backgrounds, identifying mutations that synergize with the minor allele. We classify these mutants into four distinct categories depending on activity/stability results in the different alleles. Twelve mutants were found to display reduced activity in combination with the minor allele, compared with the major allele background. When mapped on the AGT dimer structure, these mutants reveal localized regions of the protein that appear particularly sensitive to interactions with the minor allele variant. While the majority of the deleterious effects on activity in the minor allele can be attributed to synergistic interaction affecting protein stability, we identify one mutation, E274D, that appears to specifically affect activity when in combination with the minor allele.

  8. Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP).

    PubMed

    Bunce, M; O'Neill, C M; Barnardo, M C; Krausa, P; Browning, M J; Morris, P J; Welsh, K I

    1995-11-01

    We have developed a single DNA typing method which uses 144 sequence-specific primer (SSP) reactions to simultaneously detect all known HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 and DQB1 specificities in an allele specific or group specific manner using the same method, reagents, PCR parameters and protocols for all loci. The results from this integrated class I & II method can be visualized on a single photographic or electronic image and hence is described as "Phototyping". Phototyping has an overall resolution greater than or equivalent to good serology and results can be obtained in under 3 hours making the method suitable for genotyping potential cadaver donor peripheral blood without serological backup. This in turn produces the potential for reducing cold ischaemia times in renal transplantation as well as the application of prospective matching to cardiac and liver transplantation. The method has capacity to detect new alleles, for example, novel amplification patterns suggestive of 4 new HLA-B alleles have been detected. The Phototyping set has been used as the sole method of HLA typing for over 1010 individuals. Phototyping is not problem-free; deviations from the standard protocol, poor quality DNA and unsuitable PCR machines can result in individual PCR failures or in incorrect assignment of antigens. Approximately 5% of genotypes were repeated (either partially or fully) because of incomplete or equivocal results.

  9. Genus-Specific Primers for Study of Fusarium Communities in Field Samples.

    PubMed

    Karlsson, Ida; Edel-Hermann, Véronique; Gautheron, Nadine; Durling, Mikael Brandström; Kolseth, Anna-Karin; Steinberg, Christian; Persson, Paula; Friberg, Hanna

    2015-10-30

    Fusarium is a large and diverse genus of fungi of great agricultural and economic importance, containing many plant pathogens and mycotoxin producers. To date, high-throughput sequencing of Fusarium communities has been limited by the lack of genus-specific primers targeting regions with high discriminatory power at the species level. In the present study, we evaluated two Fusarium-specific primer pairs targeting translation elongation factor 1 (TEF1). We also present the new primer pair Fa+7/Ra+6. Mock Fusarium communities reflecting phylogenetic diversity were used to evaluate the accuracy of the primers in reflecting the relative abundance of the species. TEF1 amplicons were subjected to 454 high-throughput sequencing to characterize Fusarium communities. Field samples from soil and wheat kernels were included to test the method on more-complex material. For kernel samples, a single PCR was sufficient, while for soil samples, nested PCR was necessary. The newly developed primer pairs Fa+7/Ra+6 and Fa/Ra accurately reflected Fusarium species composition in mock DNA communities. In field samples, 47 Fusarium operational taxonomic units were identified, with the highest Fusarium diversity in soil. The Fusarium community in soil was dominated by members of the Fusarium incarnatum-Fusarium equiseti species complex, contradicting findings in previous studies. The method was successfully applied to analyze Fusarium communities in soil and plant material and can facilitate further studies of Fusarium ecology.

  10. Genus-Specific Primers for Study of Fusarium Communities in Field Samples

    PubMed Central

    Edel-Hermann, Véronique; Gautheron, Nadine; Durling, Mikael Brandström; Kolseth, Anna-Karin; Steinberg, Christian; Persson, Paula; Friberg, Hanna

    2015-01-01

    Fusarium is a large and diverse genus of fungi of great agricultural and economic importance, containing many plant pathogens and mycotoxin producers. To date, high-throughput sequencing of Fusarium communities has been limited by the lack of genus-specific primers targeting regions with high discriminatory power at the species level. In the present study, we evaluated two Fusarium-specific primer pairs targeting translation elongation factor 1 (TEF1). We also present the new primer pair Fa+7/Ra+6. Mock Fusarium communities reflecting phylogenetic diversity were used to evaluate the accuracy of the primers in reflecting the relative abundance of the species. TEF1 amplicons were subjected to 454 high-throughput sequencing to characterize Fusarium communities. Field samples from soil and wheat kernels were included to test the method on more-complex material. For kernel samples, a single PCR was sufficient, while for soil samples, nested PCR was necessary. The newly developed primer pairs Fa+7/Ra+6 and Fa/Ra accurately reflected Fusarium species composition in mock DNA communities. In field samples, 47 Fusarium operational taxonomic units were identified, with the highest Fusarium diversity in soil. The Fusarium community in soil was dominated by members of the Fusarium incarnatum-Fusarium equiseti species complex, contradicting findings in previous studies. The method was successfully applied to analyze Fusarium communities in soil and plant material and can facilitate further studies of Fusarium ecology. PMID:26519387

  11. Allele-Specific Interactions between CAST AWAY and NEVERSHED Control Abscission in Arabidopsis Flowers

    PubMed Central

    Groner, William D.; Christy, Megan E.; Kreiner, Catherine M.; Liljegren, Sarah J.

    2016-01-01

    An advantage of analyzing abscission in genetically tractable model plants is the ability to make use of classic genetic tools such as suppression analysis. We have investigated the regulation of organ abscission by carrying out suppression analysis in Arabidopsis flowers. Plants carrying mutations in the NEVERSHED (NEV) gene, which encodes an ADP-ribosylation factor GTPase-activating protein, retain their outer floral organs after fertilization. Mutant alleles of CAST AWAY (CST), which encodes a receptor-like cytoplasmic kinase, were found to restore organ abscission in nev flowers in an allele-specific manner. To further explore the basis of the interactions between CST and NEV, we tested whether the site of a nev mutation is predictive of its ability to be suppressed. Our results suggest instead that the strength of a nev allele influences whether organ abscission can be rescued by a specific allele of CST.

  12. Novel primers and PCR protocols for the specific detection and quantification of Sphingobium suberifaciens in situ

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pathogen causing corky root on lettuce, Sphingobium suberifaciens, is recalcitrant to standard epidemiological methods. Primers were selected from 16S rDNA sequences useful for the specific detection and quantification of S. suberifaciens. Conventional (PCR) and quantitative (qPCR) PCR protocols...

  13. GSP: a web-based platform for designing genome-specific primers in polyploids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The primary goal of this research was to develop a web-based platform named GSP for designing genome-specific primers to distinguish subgenome sequences in the polyploid genome background. GSP uses BLAST to extract homeologous sequences of the subgenomes in the existing databases, performed a multip...

  14. Development of Species-specific Primers for Rapid Detection of Phellinus linteus and P. baumii

    PubMed Central

    Kim, Mun-Ok; Kim, Gi-Young; Nam, Byung-Hyouk; Jin, Cheng-Yun; Lee, Ki-Won; Park, Jae-Min; Lee, Sang-Joon

    2005-01-01

    Genus Phellinus taxonomically belongs to Aphyllophorales and some species of this genus have been used as a medicinal ingredients and Indian folk medicines. Especially, P. linteus and morphological-related species are well-known medicinal fungi that have various biological activities such as humoral and cell-mediated, anti-mutagenic, and anti-cancer activities. However, little is known about the rapid detection for complex Phellinus species. Therefore, this study was carried out to develop specific primers for the rapid detection of P. linteus and other related species. Designing the species-specific primers was done based on internal transcribed spacer sequence data. Each primer set detected specifically P. linteus (PL2/PL5R) and P. baumii (PB1/PB4R). These primer sets could be useful for the rapid detection of specific-species among unidentified Phellinus species. Moreover, restriction fragment length polymorphism analysis of the ITS region with HaeIII was also useful for clarifying the relationship between each 5 Phellinus species. PMID:24049482

  15. Allele-Specific Deletions in Mouse Tumors Identify Fbxw7 as Germline Modifier of Tumor Susceptibility

    PubMed Central

    Perez-Losada, Jesus; Wu, Di; DelRosario, Reyno; Balmain, Allan; Mao, Jian-Hua

    2012-01-01

    Genome-wide association studies (GWAS) have been successful in finding associations between specific genetic variants and cancer susceptibility in human populations. These studies have identified a range of highly statistically significant associations between single nucleotide polymorphisms (SNPs) and susceptibility to development of a range of human tumors. However, the effect of each SNP in isolation is very small, and all of the SNPs combined only account for a relatively minor proportion of the total genetic risk (5–10%). There is therefore a major requirement for alternative routes to the discovery of genetic risk factors for cancer. We have previously shown using mouse models that chromosomal regions harboring susceptibility genes identified by linkage analysis frequently exhibit allele-specific genetic alterations in tumors. We demonstrate here that the Fbxw7 gene, a commonly mutated gene in a wide range of mouse and human cancers, shows allele-specific deletions in mouse lymphomas and skin tumors. Lymphomas from three different F1 hybrids show 100% allele-specificity in the patterns of allelic loss. Parental alleles from 129/Sv or Spretus/Gla mice are lost in tumors from F1 hybrids with C57BL/6 animals, due to the presence of a specific non-synonymous coding sequence polymorphism at the N-terminal portion of the gene. A specific genetic test of association between this SNP and lymphoma susceptibility in interspecific backcross mice showed a significant linkage (p = 0.001), but only in animals with a functional p53 gene. These data therefore identify Fbxw7 as a p53-dependent tumor susceptibility gene. Increased p53-dependent tumor susceptibility and allele-specific losses were also seen in a mouse skin model of skin tumor development. We propose that analysis of preferential allelic imbalances in tumors may provide an efficient means of uncovering genetic variants that affect mouse and human tumor susceptibility. PMID:22348067

  16. PrimerMapper: high throughput primer design and graphical assembly for PCR and SNP detection

    PubMed Central

    O’Halloran, Damien M.

    2016-01-01

    Primer design represents a widely employed gambit in diverse molecular applications including PCR, sequencing, and probe hybridization. Variations of PCR, including primer walking, allele-specific PCR, and nested PCR provide specialized validation and detection protocols for molecular analyses that often require screening large numbers of DNA fragments. In these cases, automated sequence retrieval and processing become important features, and furthermore, a graphic that provides the user with a visual guide to the distribution of designed primers across targets is most helpful in quickly ascertaining primer coverage. To this end, I describe here, PrimerMapper, which provides a comprehensive graphical user interface that designs robust primers from any number of inputted sequences while providing the user with both, graphical maps of primer distribution for each inputted sequence, and also a global assembled map of all inputted sequences with designed primers. PrimerMapper also enables the visualization of graphical maps within a browser and allows the user to draw new primers directly onto the webpage. Other features of PrimerMapper include allele-specific design features for SNP genotyping, a remote BLAST window to NCBI databases, and remote sequence retrieval from GenBank and dbSNP. PrimerMapper is hosted at GitHub and freely available without restriction. PMID:26853558

  17. PrimerMapper: high throughput primer design and graphical assembly for PCR and SNP detection.

    PubMed

    O'Halloran, Damien M

    2016-01-01

    Primer design represents a widely employed gambit in diverse molecular applications including PCR, sequencing, and probe hybridization. Variations of PCR, including primer walking, allele-specific PCR, and nested PCR provide specialized validation and detection protocols for molecular analyses that often require screening large numbers of DNA fragments. In these cases, automated sequence retrieval and processing become important features, and furthermore, a graphic that provides the user with a visual guide to the distribution of designed primers across targets is most helpful in quickly ascertaining primer coverage. To this end, I describe here, PrimerMapper, which provides a comprehensive graphical user interface that designs robust primers from any number of inputted sequences while providing the user with both, graphical maps of primer distribution for each inputted sequence, and also a global assembled map of all inputted sequences with designed primers. PrimerMapper also enables the visualization of graphical maps within a browser and allows the user to draw new primers directly onto the webpage. Other features of PrimerMapper include allele-specific design features for SNP genotyping, a remote BLAST window to NCBI databases, and remote sequence retrieval from GenBank and dbSNP. PrimerMapper is hosted at GitHub and freely available without restriction. PMID:26853558

  18. PrimerMapper: high throughput primer design and graphical assembly for PCR and SNP detection.

    PubMed

    O'Halloran, Damien M

    2016-01-01

    Primer design represents a widely employed gambit in diverse molecular applications including PCR, sequencing, and probe hybridization. Variations of PCR, including primer walking, allele-specific PCR, and nested PCR provide specialized validation and detection protocols for molecular analyses that often require screening large numbers of DNA fragments. In these cases, automated sequence retrieval and processing become important features, and furthermore, a graphic that provides the user with a visual guide to the distribution of designed primers across targets is most helpful in quickly ascertaining primer coverage. To this end, I describe here, PrimerMapper, which provides a comprehensive graphical user interface that designs robust primers from any number of inputted sequences while providing the user with both, graphical maps of primer distribution for each inputted sequence, and also a global assembled map of all inputted sequences with designed primers. PrimerMapper also enables the visualization of graphical maps within a browser and allows the user to draw new primers directly onto the webpage. Other features of PrimerMapper include allele-specific design features for SNP genotyping, a remote BLAST window to NCBI databases, and remote sequence retrieval from GenBank and dbSNP. PrimerMapper is hosted at GitHub and freely available without restriction.

  19. Improved group-specific primers based on the full SILVA 16S rRNA gene reference database.

    PubMed

    Pfeiffer, Stefan; Pastar, Milica; Mitter, Birgit; Lippert, Kathrin; Hackl, Evelyn; Lojan, Paul; Oswald, Andreas; Sessitsch, Angela

    2014-08-01

    Quantitative PCR (qPCR) and community fingerprinting methods, such as the Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis,are well-suited techniques for the examination of microbial community structures. The use of phylum and class-specific primers can provide enhanced sensitivity and phylogenetic resolution as compared with domain-specific primers. To date, several phylum- and class-specific primers targeting the 16S ribosomal RNA gene have been published. However, many of these primers exhibit low discriminatory power against non-target bacteria in PCR. In this study, we evaluated the precision of certain published primers in silico and via specific PCR. We designed new qPCR and T-RFLP primer pairs (for the classes Alphaproteobacteria and Betaproteobacteria, and the phyla Bacteroidetes, Firmicutes and Actinobacteria) by combining the sequence information from a public dataset (SILVA SSU Ref 102 NR) with manual primer design. We evaluated the primer pairs via PCR using isolates of the above-mentioned groups and via screening of clone libraries from environmental soil samples and human faecal samples. As observed through theoretical and practical evaluation, the primers developed in this study showed a higher level of precision than previously published primers, thus allowing a deeper insight into microbial community dynamics. PMID:25229098

  20. Allele-specific copy-number discovery from whole-genome and whole-exome sequencing.

    PubMed

    Wang, WeiBo; Wang, Wei; Sun, Wei; Crowley, James J; Szatkiewicz, Jin P

    2015-08-18

    Copy-number variants (CNVs) are a major form of genetic variation and a risk factor for various human diseases, so it is crucial to accurately detect and characterize them. It is conceivable that allele-specific reads from high-throughput sequencing data could be leveraged to both enhance CNV detection and produce allele-specific copy number (ASCN) calls. Although statistical methods have been developed to detect CNVs using whole-genome sequence (WGS) and/or whole-exome sequence (WES) data, information from allele-specific read counts has not yet been adequately exploited. In this paper, we develop an integrated method, called AS-GENSENG, which incorporates allele-specific read counts in CNV detection and estimates ASCN using either WGS or WES data. To evaluate the performance of AS-GENSENG, we conducted extensive simulations, generated empirical data using existing WGS and WES data sets and validated predicted CNVs using an independent methodology. We conclude that AS-GENSENG not only predicts accurate ASCN calls but also improves the accuracy of total copy number calls, owing to its unique ability to exploit information from both total and allele-specific read counts while accounting for various experimental biases in sequence data. Our novel, user-friendly and computationally efficient method and a complete analytic protocol is freely available at https://sourceforge.net/projects/asgenseng/. PMID:25883151

  1. Genome destabilizing mutator alleles drive specific mutational trajectories in Saccharomyces cerevisiae.

    PubMed

    Stirling, Peter C; Shen, Yaoqing; Corbett, Richard; Jones, Steven J M; Hieter, Philip

    2014-02-01

    In addition to environmental factors and intrinsic variations in base substitution rates, specific genome-destabilizing mutations can shape the mutational trajectory of genomes. How specific alleles influence the nature and position of accumulated mutations in a genomic context is largely unknown. Understanding the impact of genome-destabilizing alleles is particularly relevant to cancer genomes where biased mutational signatures are identifiable. We first created a more complete picture of cellular pathways that impact mutation rate using a primary screen to identify essential Saccharomyces cerevisiae gene mutations that cause mutator phenotypes. Drawing primarily on new alleles identified in this resource, we measure the impact of diverse mutator alleles on mutation patterns directly by whole-genome sequencing of 68 mutation-accumulation strains derived from wild-type and 11 parental mutator genotypes. The accumulated mutations differ across mutator strains, displaying base-substitution biases, allele-specific mutation hotspots, and break-associated mutation clustering. For example, in mutants of POLα and the Cdc13-Stn1-Ten1 complex, we find a distinct subtelomeric bias for mutations that we show is independent of the target sequence. Together our data suggest that specific genome-instability mutations are sufficient to drive discrete mutational signatures, some of which share properties with mutation patterns seen in tumors. Thus, in a population of cells, genome-instability mutations could influence clonal evolution by establishing discrete mutational trajectories for genomes.

  2. Genome Destabilizing Mutator Alleles Drive Specific Mutational Trajectories in Saccharomyces cerevisiae

    PubMed Central

    Stirling, Peter C.; Shen, Yaoqing; Corbett, Richard; Jones, Steven J. M.; Hieter, Philip

    2014-01-01

    In addition to environmental factors and intrinsic variations in base substitution rates, specific genome-destabilizing mutations can shape the mutational trajectory of genomes. How specific alleles influence the nature and position of accumulated mutations in a genomic context is largely unknown. Understanding the impact of genome-destabilizing alleles is particularly relevant to cancer genomes where biased mutational signatures are identifiable. We first created a more complete picture of cellular pathways that impact mutation rate using a primary screen to identify essential Saccharomyces cerevisiae gene mutations that cause mutator phenotypes. Drawing primarily on new alleles identified in this resource, we measure the impact of diverse mutator alleles on mutation patterns directly by whole-genome sequencing of 68 mutation-accumulation strains derived from wild-type and 11 parental mutator genotypes. The accumulated mutations differ across mutator strains, displaying base-substitution biases, allele-specific mutation hotspots, and break-associated mutation clustering. For example, in mutants of POLα and the Cdc13–Stn1–Ten1 complex, we find a distinct subtelomeric bias for mutations that we show is independent of the target sequence. Together our data suggest that specific genome-instability mutations are sufficient to drive discrete mutational signatures, some of which share properties with mutation patterns seen in tumors. Thus, in a population of cells, genome-instability mutations could influence clonal evolution by establishing discrete mutational trajectories for genomes. PMID:24336748

  3. ACNE: a summarization method to estimate allele-specific copy numbers for Affymetrix SNP arrays

    PubMed Central

    Ortiz-Estevez, Maria; Bengtsson, Henrik; Rubio, Angel

    2010-01-01

    Motivation: Current algorithms for estimating DNA copy numbers (CNs) borrow concepts from gene expression analysis methods. However, single nucleotide polymorphism (SNP) arrays have special characteristics that, if taken into account, can improve the overall performance. For example, cross hybridization between alleles occurs in SNP probe pairs. In addition, most of the current CN methods are focused on total CNs, while it has been shown that allele-specific CNs are of paramount importance for some studies. Therefore, we have developed a summarization method that estimates high-quality allele-specific CNs. Results: The proposed method estimates the allele-specific DNA CNs for all Affymetrix SNP arrays dealing directly with the cross hybridization between probes within SNP probesets. This algorithm outperforms (or at least it performs as well as) other state-of-the-art algorithms for computing DNA CNs. It better discerns an aberration from a normal state and it also gives more precise allele-specific CNs. Availability: The method is available in the open-source R package ACNE, which also includes an add on to the aroma.affymetrix framework (http://www.aroma-project.org/). Contact: arubio@ceit.es Supplementaruy information: Supplementary data are available at Bioinformatics online. PMID:20529889

  4. Specificity of Methylation Assays in Cancer Research: A Guideline for Designing Primers and Probes

    PubMed Central

    Barekati, Zeinab; Radpour, Ramin; Kohler, Corina; Zhong, Xiao Yan

    2010-01-01

    DNA methylation is an epigenetic regulation mechanism of genomic function, and aberrant methylation pattern has been found to be a common event in many diseases and human cancers. A large number of cancer studies have been focused on identification of methylation changes as biomarkers (i.e., breast cancer). However, still clinical use of them is very limited because of lack of specificity and sensitivity for diagnostic test. This highlights the critical need for specific primer and probe design to avoid false-positive detection of methylation profiling. The guideline and online web tools that are introduced in this paper might help to perform a successful experiment and to develop specific diagnosis biomarkers by designing right primer pair and probe prior to experimental step. PMID:20798774

  5. SNPsplit: Allele-specific splitting of alignments between genomes with known SNP genotypes

    PubMed Central

    Krueger, Felix; Andrews, Simon R.

    2016-01-01

    Sequencing reads overlapping polymorphic sites in diploid mammalian genomes may be assigned to one allele or the other. This holds the potential to detect gene expression, chromatin modifications, DNA methylation or nuclear interactions in an allele-specific fashion. SNPsplit is an allele-specific alignment sorter designed to read files in SAM/BAM format and determine the allelic origin of reads or read-pairs that cover known single nucleotide polymorphic (SNP) positions. For this to work libraries must have been aligned to a genome in which all known SNP positions were masked with the ambiguity base 'N' and aligned using a suitable mapping program such as Bowtie2, TopHat, STAR, HISAT2, HiCUP or Bismark. SNPsplit also provides an automated solution to generate N-masked reference genomes for hybrid mouse strains based on the variant call information provided by the Mouse Genomes Project. The unique ability of SNPsplit to work with various different kinds of sequencing data including RNA-Seq, ChIP-Seq, Bisulfite-Seq or Hi-C opens new avenues for the integrative exploration of allele-specific data. PMID:27429743

  6. Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection

    PubMed Central

    KC, R.; Srivastava, A.; Wilkowski, J. M.; Richter, C. E.; Shavit, J. A.; Burke, D. T.; Bielas, S. L.

    2016-01-01

    CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies. PMID:27557703

  7. SNPsplit: Allele-specific splitting of alignments between genomes with known SNP genotypes.

    PubMed

    Krueger, Felix; Andrews, Simon R

    2016-01-01

    Sequencing reads overlapping polymorphic sites in diploid mammalian genomes may be assigned to one allele or the other. This holds the potential to detect gene expression, chromatin modifications, DNA methylation or nuclear interactions in an allele-specific fashion. SNPsplit is an allele-specific alignment sorter designed to read files in SAM/BAM format and determine the allelic origin of reads or read-pairs that cover known single nucleotide polymorphic (SNP) positions. For this to work libraries must have been aligned to a genome in which all known SNP positions were masked with the ambiguity base 'N' and aligned using a suitable mapping program such as Bowtie2, TopHat, STAR, HISAT2, HiCUP or Bismark. SNPsplit also provides an automated solution to generate N-masked reference genomes for hybrid mouse strains based on the variant call information provided by the Mouse Genomes Project. The unique ability of SNPsplit to work with various different kinds of sequencing data including RNA-Seq, ChIP-Seq, Bisulfite-Seq or Hi-C opens new avenues for the integrative exploration of allele-specific data. PMID:27429743

  8. Epidemiological survey of Theileria parasite infection of cattle in Northeast China by allele-specific PCR.

    PubMed

    Yu, Longzheng; Zhang, Shoufa; Liang, Wanfeng; Jin, Chunmei; Jia, Lijun; Luo, Yuzi; Li, Yan; Cao, Shinuo; Yamagishi, Junya; Nishikawa, Yoshifumi; Kawano, Suguru; Fujisaki, Kozo; Xuan, Xuenan

    2011-11-01

    An epidemiological survey on a Theileria parasite infection of cattle in Northeast China was carried out using allele-specific PCR and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. The results showed that 14 of 104 blood samples were positive for Theileria by PCR. Among the positive cases, co-infection with various combinations of C- and I-type parasites was detected in 12 samples; no B- and Thai-type parasites were detected by allele-specific PCR. Phylogenetic analysis based on the MPSP gene sequences revealed that Theileria parasites with the MPSP types 1, 2, and 4 were distributed in Northeast China.

  9. SP-Designer: a user-friendly program for designing species-specific primer pairs from DNA sequence alignments.

    PubMed

    Villard, Pierre; Malausa, Thibaut

    2013-07-01

    SP-Designer is an open-source program providing a user-friendly tool for the design of specific PCR primer pairs from a DNA sequence alignment containing sequences from various taxa. SP-Designer selects PCR primer pairs for the amplification of DNA from a target species on the basis of several criteria: (i) primer specificity, as assessed by interspecific sequence polymorphism in the annealing regions, (ii) the biochemical characteristics of the primers and (iii) the intended PCR conditions. SP-Designer generates tables, detailing the primer pair and PCR characteristics, and a FASTA file locating the primer sequences in the original sequence alignment. SP-Designer is Windows-compatible and freely available from http://www2.sophia.inra.fr/urih/sophia_mart/sp_designer/info_sp_designer.php.

  10. Development of species-specific primers for rapid identification of Debaryomyces hansenii.

    PubMed

    Wrent, Petra; Rivas, Eva-María; Gil de Prado, Elena; Peinado, José M; de Silóniz, María-Isabel

    2015-01-16

    In this work, we developed a specific PCR assay for Debaryomyces hansenii strains that uses a putative homologous PAD1 region (729 bp) present in this yeast species as a target. The amplification of this sequence with the D. hansenii specific primer pair (DhPADF/DhPADR) was found to be a rapid, specific and an affordable method enabling identification of D. hansenii from other yeast strains. Primers were tested in almost 100 strains, 49 strains from Type Culture Collection belonging to the genus Debaryomyces and to other yeast species commonly found in foods or related genera. These primers were able to discriminate between closely related species of Debaryomyces, such as Debaryomyces fabryi and Debaryomyces subglobosus, with a 100% detection rate for D. hansenii. Also, the method was tested in 45 strains from different foods. Results confirmed the specificity of the PCR method and detected two earlier misidentifications of D. hansenii strains obtained by RFLP analysis of the 5.8S ITS rDNA region. Subsequently we confirmed by sequencing the D1/D2 domain of 26S rDNA that these strains belonged to D. fabryi. We call attention in this work to the fact that the RFLPs of the 5.8S ITS rDNA profiles of D. hansenii, D. fabryi and D. subglobosus are the same and this technique will thus lead to incorrect identifications. PMID:25462930

  11. Reliable and fast allele-specific extension of 3'-LNA modified oligonucleotides covalently immobilized on a plastic base, combined with biotin-dUTP mediated optical detection.

    PubMed

    Michikawa, Yuichi; Fujimoto, Kentaro; Kinoshita, Kenji; Kawai, Seiko; Sugahara, Keisuke; Suga, Tomo; Otsuka, Yoshimi; Fujiwara, Kazuhiko; Iwakawa, Mayumi; Imai, Takashi

    2006-12-01

    In the present work, a convenient microarray SNP typing system has been developed using a plastic base that covalently immobilizes amino-modified oligonucleotides. Reliable SNP allele discrimination was achieved by using allelic specificity-enhanced enzymatic extension of immobilized oligonucleotide primer, with a locked nucleic acid (LNA) modification at the SNP-discriminating 3'-end nucleotide. Incorporation of multiple biotin-dUTP molecules during primer extension, followed by binding of alkaline phosphatase-conjugated streptavidin, allowed optical detection of the genotyping results through precipitation of colored alkaline phosphatase substrates onto the surface of the plastic base. Notably, rapid primer extension was demonstrated without a preliminary annealing step of double-stranded template DNA, allowing overall processes to be performed within a couple of hours. Simultaneous evaluation of three SNPs in the genes TGFB1, SOD2 and APEX1, previously investigated for association with radiation sensitivity, in 25 individuals has shown perfect assignment with data obtained by another established technique (MassARRAY system).

  12. Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

    PubMed Central

    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni

    2012-01-01

    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes. PMID:22558339

  13. Allele specific locked nucleic acid quantitative PCR (ASLNAqPCR): an accurate and cost-effective assay to diagnose and quantify KRAS and BRAF mutation.

    PubMed

    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni

    2012-01-01

    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes.

  14. Specific Polymerase Chain Reaction Primers for the Detection of Plasmodiophora brassicae in Soil and Water.

    PubMed

    Faggian, R; Bulman, S R; Lawrie, A C; Porter, I J

    1999-05-01

    ABSTRACT The development of specific oligonucleotide primers for Plasmodiophora brassicae has led to a nested polymerase chain reaction (PCR) detection method for P. brassicae in soil and water. Initially, the PCR was used to amplify a section of the rDNA repeat. The PCR products were sequenced and the data used to design primers that were directed at the ribosomal RNA genes and internal transcribed spacer regions. Specificity was tested against more than 40 common soil organisms, host plants, and spore suspension contaminants, as well as P. brassicae isolates from around Australia and the world. Sensitivity was determined to be 0.1 fentograms (fg; 10(-15) g) for pure template and as low as 1,000 spores per g of potting mix. In soil, P. brassicae was detected in all soils where the inoculum was sufficient to result in clubroot symptoms. Also outlined is a simple method of DNA extraction from soil. PMID:18944752

  15. Specific oligonucleotide primers for detection of lecithinase-positive Bacillus spp. by PCR.

    PubMed Central

    Schraft, H; Griffiths, M W

    1995-01-01

    An assay based on the PCR has been developed to facilitate detection and identification of Bacillus cereus in foods. Three primers for the PCR have been designed within the sequence for cereolysin AB, a cytolytic determinant that encodes lecithin-hydrolyzing and hemolytic activities of B. cereus. With the PCR and hybridization, the specificity of the primers was tested with 39 isolates of the B. cereus group, with 17 other Bacillus spp., and with 21 non-Bacillus strains. Results demonstrate a high specificity of the three oligonucleotides for isolates of the B. cereus group. With a combined PCR-hybridization assay, the detection limit for B. cereus in artificially contaminated milk was 1 CFU/ml of milk. PMID:7887632

  16. Specific HLA-DQB and HLA-DRB1 alleles confer susceptibility to pemphigus vulgaris.

    PubMed Central

    Scharf, S J; Freidmann, A; Steinman, L; Brautbar, C; Erlich, H A

    1989-01-01

    The autoimmune dermatologic disease pemphigus vulgaris (PV) is associated with the serotypes HLA-DR4 and HLA-DRw6. Based on nucleotide sequence and oligonucleotide probe analysis of enzymatically amplified DNA encoding HLA-DR beta chain (HLA-DRB) and HLA-DQ beta chain (HLA-DQB; henceforth HLA is omitted from designations), we showed previously that the DR4 susceptibility was associated with the Dw10 DRB1 allele [encoding the mixed lymphocyte culture (MLC)-defined Dw10 specificity]. The DRw6 susceptibility similarly was shown to be associated with a rare DQB allele (DQB1.3), which differed from another nonsusceptible allele by only a valine-to-aspartic acid substitution at position 57. Given the linkage disequilibrium that characterizes HLA haplotypes, it is difficult to assign disease susceptibility to a specific locus rather than to a closely linked gene(s) on the same haplotype. To address this problem, we have analyzed all of the polymorphic loci of the class II HLA region (DRB1, DRB3, DQA, DQB, and DPB) on the DRw6 haplotypes in patients and controls. In 22 PV patients, 4 different DRw6 haplotypes were found that encode the same DQ beta chain (DQB1.3) but contained silent nucleotide differences at the DQB locus as well as coding sequence differences in the DQA and DRB loci. These results, obtained by using a method for allele-specific polymerase chain reaction amplification, strongly support the hypothesis that the allele DQB1.3 confers susceptibility. This DQB allele is correlated with the MLC-defined Dw9 specificity and is associated with two different DRB1 alleles (the common "6A" associated with DRw13 and the rare "6B" associated with DRw14). Since 86% (19 of 22) of DRw6+ patients contain the DQB1.3 allele (vs. 3% of controls), whereas 64% (14 of 22) contain the DRB1 allele 6B (vs. 6% of the controls), we conclude that most of the DRw6 susceptibility to PV can be accounted for by the DQ beta chain. Images PMID:2503828

  17. Allele-Specific Reprogramming of Cancer Metabolism by the Long Non-coding RNA CCAT2.

    PubMed

    Redis, Roxana S; Vela, Luz E; Lu, Weiqin; Ferreira de Oliveira, Juliana; Ivan, Cristina; Rodriguez-Aguayo, Cristian; Adamoski, Douglas; Pasculli, Barbara; Taguchi, Ayumu; Chen, Yunyun; Fernandez, Agustin F; Valledor, Luis; Van Roosbroeck, Katrien; Chang, Samuel; Shah, Maitri; Kinnebrew, Garrett; Han, Leng; Atlasi, Yaser; Cheung, Lawrence H; Huang, Gilbert Y; Monroig, Paloma; Ramirez, Marc S; Catela Ivkovic, Tina; Van, Long; Ling, Hui; Gafà, Roberta; Kapitanovic, Sanja; Lanza, Giovanni; Bankson, James A; Huang, Peng; Lai, Stephen Y; Bast, Robert C; Rosenblum, Michael G; Radovich, Milan; Ivan, Mircea; Bartholomeusz, Geoffrey; Liang, Han; Fraga, Mario F; Widger, William R; Hanash, Samir; Berindan-Neagoe, Ioana; Lopez-Berestein, Gabriel; Ambrosio, Andre L B; Gomes Dias, Sandra M; Calin, George A

    2016-02-18

    Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA. PMID:26853146

  18. Loss of RNA expression and allele-specific expression associated with congenital heart disease

    PubMed Central

    McKean, David M.; Homsy, Jason; Wakimoto, Hiroko; Patel, Neil; Gorham, Joshua; DePalma, Steven R.; Ware, James S.; Zaidi, Samir; Ma, Wenji; Patel, Nihir; Lifton, Richard P.; Chung, Wendy K.; Kim, Richard; Shen, Yufeng; Brueckner, Martina; Goldmuntz, Elizabeth; Sharp, Andrew J.; Seidman, Christine E.; Gelb, Bruce D.; Seidman, J. G.

    2016-01-01

    Congenital heart disease (CHD), a prevalent birth defect occurring in 1% of newborns, likely results from aberrant expression of cardiac developmental genes. Mutations in a variety of cardiac transcription factors, developmental signalling molecules and molecules that modify chromatin cause at least 20% of disease, but most CHD remains unexplained. We employ RNAseq analyses to assess allele-specific expression (ASE) and biallelic loss-of-expression (LOE) in 172 tissue samples from 144 surgically repaired CHD subjects. Here we show that only 5% of known imprinted genes with paternal allele silencing are monoallelic versus 56% with paternal allele expression—this cardiac-specific phenomenon seems unrelated to CHD. Further, compared with control subjects, CHD subjects have a significant burden of both LOE genes and ASE events associated with altered gene expression. These studies identify FGFBP2, LBH, RBFOX2, SGSM1 and ZBTB16 as candidate CHD genes because of significantly altered transcriptional expression. PMID:27670201

  19. In silico vs in vitro analysis of primer specificity for the detection of Gardnerella vaginalis, Atopobium vaginae and Lactobacillus spp.

    PubMed Central

    2012-01-01

    Background Bacterial vaginosis (BV) is a common pathology of women in reproductive age that can lead to serious health complications, and is associated with shifts in the normal microflora from predominance of Lactobacillus spp. to a proliferation of other anaerobes such as G. vaginalis and A vaginae, which can be detected by PCR. The optimal PCR pathogen detection assay relies mainly on the specificity and sensitivity of the primers used. Findings Here we demonstrate that in silico analytical testing of primer specificity is not a synonym to in vitro analytical specificity by testing a range of published and newly designed primers with both techniques for the detection of BV-associated microorganisms. Conclusions By testing primer in vitro specificity with a sufficient range of bacterial strains, we were able to design primers with higher specificity and sensitivity. Also by comparing the results obtained for the newly designed primers with other previously published primers, we confirmed that in silico analysis is not sufficient to predict in vitro specificity. As such care must be taken when choosing the primers for a detection assay. PMID:23153093

  20. Authentication of official Da-huang by sequencing and multiplex allele-specific PCR of a short maturase K gene.

    PubMed

    Xu, Guojie; Wang, Xueyong; Liu, Chunsheng; Li, Weidong; Wei, Shengli; Liu, Ying; Cheng, Xiaoli; Liu, Juan

    2013-02-01

    Rhubarb (official Da-huang) is an important medicinal herb in Asia. Many adulterants of official Da-huang have been discovered in Chinese markets in recent years, which has resulted in adverse effects in medicinal treatment. Here, novel molecular markers based on a short maturase K (matK) gene were developed for authenticating official Da-huang. This study showed that all the species from official Da-huang were clustered together in one clade in the polygenetic trees based on short matK. Two highly conserved single nucleotide polymorphisms of short matK were mined in the species from official Da-huang. Based on these polymophisms, four improved specific primers of official Da-huang were successfully developed that generated reproducible specific bands. These results suggest that the short matK sequence can be considered as a favorable candidate for distinguishing official Da-huang from its adulterants. The established multiplex allele-specific PCR was determined to be simple and accurate and may serve as a preferable tool for authentication of official Da-huang. In addition, we suggest that short-sized specific bands be developed to authenticate materials used in traditional Chinese medicine.

  1. Direct micro-haplotyping by multiple double PCR amplifications of specific alleles (MD-PASA)

    PubMed Central

    Eitan, Yuval; Kashi, Yechezkel

    2002-01-01

    Analysis of haplotypes is an important tool in population genetics, familial heredity and gene mapping. Determination of haplotypes of multiple single nucleotide polymorphisms (SNPs) or other simple mutations is time consuming and expensive when analyzing large populations, and often requires the help of computational and statistical procedures. Based on double PCR amplification of specific alleles, described previously, we have developed a simple, rapid and low-cost method for direct haplotyping of multiple SNPs and simple mutations found within relatively short specific regions or genes (micro-haplotypes). Using this method, it is possible to directly determine the physical linkage of multiple heterozygous alleles, by conducting a series of double allele-specific PCR amplification sets with simple analysis by gel electrophoresis. Application of the method requires prior information as to the sequence of the segment to be haplotyped, including the polymorphic sites. We applied the method to haplotyping of nine sites in the chicken HSP108 gene. One of the haplotypes in the population apparently arose by recombination between two existing haplotypes, and we were able to locate the point of recombination within a segment of 19 bp. We anticipate rapidly growing needs for SNP haplotyping in human (medical and pharmacogenetics), animal and plant genetics; in this context, the multiple double PCR amplifications of specific alleles (MD-PASA) method offers a useful haplotyping tool. PMID:12060700

  2. Isolation of Fungal Pathogens to an Edible Mushroom, Pleurotus eryngii, and Development of Specific ITS Primers.

    PubMed

    Kim, Sang-Woo; Kim, Sinil; Lee, Hyun-Jun; Park, Ju-Wan; Ro, Hyeon-Su

    2013-12-01

    Fungal pathogens have caused severe damage to the commercial production of Pleurotus eryngii, the king oyster mushroom, by reducing production yield, causing deterioration of commercial value, and shortening shelf-life. Four strains of pathogenic fungi, including Trichoderma koningiopsis DC3, Phomopsis sp. MP4, Mucor circinelloides MP5, and Cladosporium bruhnei MP6, were isolated from the bottle culture of diseased P. eryngii. A species-specific primer set was designed for each fungus from the ITS1-5.8S rDNA-ITS2 sequences. PCR using the ITS primer set yielded a unique DNA band for each fungus without any cross-reaction, proving the validity of our method in detection of mushroom fungal pathogens. PMID:24493949

  3. Sex-specific allelic transmission bias suggests sexual conflict at MC1R.

    PubMed

    Ducret, Valérie; Gaigher, Arnaud; Simon, Céline; Goudet, Jérôme; Roulin, Alexandre

    2016-09-01

    Sexual conflict arises when selection in one sex causes the displacement of the other sex from its phenotypic optimum, leading to an inevitable tension within the genome - called intralocus sexual conflict. Although the autosomal melanocortin-1-receptor gene (MC1R) can generate colour variation in sexually dichromatic species, most previous studies have not considered the possibility that MC1R may be subject to sexual conflict. In the barn owl (Tyto alba), the allele MC1RWHITE is associated with whitish plumage coloration, typical of males, and the allele MC1RRUFOUS is associated with dark rufous coloration, typical of females, although each sex can express any phenotype. Because each colour variant is adapted to specific environmental conditions, the allele MC1RWHITE may be more strongly selected in males and the allele MC1RRUFOUS in females. We therefore investigated whether MC1R genotypes are in excess or deficit in male and female fledglings compared with the expected Hardy-Weinberg proportions. Our results show an overall deficit of 7.5% in the proportion of heterozygotes in males and of 12.9% in females. In males, interannual variation in assortative pairing with respect to MC1R explained the year-specific deviations from Hardy-Weinberg proportions, whereas in females, the deficit was better explained by the interannual variation in the probability of inheriting the MC1RWHITE or MC1RRUFOUS allele. Additionally, we observed that sons inherit the MC1RRUFOUS allele from their fathers on average slightly less often than expected under the first Mendelian law. Transmission ratio distortion may be adaptive in this sexually dichromatic species if males and females are, respectively, selected to display white and rufous plumages. PMID:27480981

  4. Advancing allele group-specific amplification of the complete HLA-C gene--isolation of novel alleles from three allele groups (C*04, C*07 and C*08).

    PubMed

    Cisneros, E; Martínez-Pomar, N; Vilches, M; Martín, P; de Pablo, R; Nuñez Del Prado, N; Nieto, A; Matamoros, N; Moraru, M; Vilches, C

    2013-10-01

    A variety of strategies have been designed for sequence-based HLA typing (SBT) and for the isolation of new human leucocyte antigen (HLA) alleles, but unambiguous characterization of complete genomic sequences remains a challenge. We recently reported a simple method for the group-specific amplification (GSA) and sequencing of a full-length C*04 genomic sequence in isolation from the accompanying allele. Here we build on this strategy and present homologous methods that enable the isolation of HLA-C alleles belonging to another two allele groups. Using this approach, which can be applied to sequence-based typing in some clinical settings, we have successfully characterized three novel HLA-C alleles (C*04:128, C*07:01:01:02, and C*08:62).

  5. Human-specific derived alleles of CD33 and other genes protect against postreproductive cognitive decline

    PubMed Central

    Schwarz, Flavio; Springer, Stevan A.; Altheide, Tasha K.; Varki, Nissi M.; Gagneux, Pascal; Varki, Ajit

    2016-01-01

    The individuals of most vertebrate species die when they can no longer reproduce. Humans are a rare exception, having evolved a prolonged postreproductive lifespan. Elders contribute to cooperative offspring care, assist in foraging, and communicate important ecological and cultural knowledge, increasing the survival of younger individuals. Age-related deterioration of cognitive capacity in humans compromises these benefits and also burdens the group with socially costly members. We investigated the contribution of the immunoregulatory receptor CD33 to a uniquely human postreproductive disease, Alzheimer’s dementia. Surprisingly, even though selection at advanced age is expected to be weak, a CD33 allele protective against Alzheimer’s disease is derived and unique to humans and favors a functional molecular state of CD33 resembling that of the chimpanzee. Thus, derived alleles may be compensatory and restore interactions altered as a consequence of human-specific brain evolution. We found several other examples of derived alleles at other human loci that protect against age-related cognitive deterioration arising from neurodegenerative disease or cerebrovascular insufficiency. Selection by inclusive fitness may be strong enough to favor alleles protecting specifically against cognitive decline in postreproductive humans. Such selection would operate by maximizing the contributions of postreproductive individuals to the fitness of younger kin. PMID:26621708

  6. Human-specific derived alleles of CD33 and other genes protect against postreproductive cognitive decline.

    PubMed

    Schwarz, Flavio; Springer, Stevan A; Altheide, Tasha K; Varki, Nissi M; Gagneux, Pascal; Varki, Ajit

    2016-01-01

    The individuals of most vertebrate species die when they can no longer reproduce. Humans are a rare exception, having evolved a prolonged postreproductive lifespan. Elders contribute to cooperative offspring care, assist in foraging, and communicate important ecological and cultural knowledge, increasing the survival of younger individuals. Age-related deterioration of cognitive capacity in humans compromises these benefits and also burdens the group with socially costly members. We investigated the contribution of the immunoregulatory receptor CD33 to a uniquely human postreproductive disease, Alzheimer's dementia. Surprisingly, even though selection at advanced age is expected to be weak, a CD33 allele protective against Alzheimer's disease is derived and unique to humans and favors a functional molecular state of CD33 resembling that of the chimpanzee. Thus, derived alleles may be compensatory and restore interactions altered as a consequence of human-specific brain evolution. We found several other examples of derived alleles at other human loci that protect against age-related cognitive deterioration arising from neurodegenerative disease or cerebrovascular insufficiency. Selection by inclusive fitness may be strong enough to favor alleles protecting specifically against cognitive decline in postreproductive humans. Such selection would operate by maximizing the contributions of postreproductive individuals to the fitness of younger kin. PMID:26621708

  7. Extracellular Superoxide Dismutase Polymorphism in Mice: Allele- Specific Effects on Phenotype

    PubMed Central

    Jun, Sujung; Pierce, Anson; Dory, Ladislav

    2010-01-01

    Extracellular superoxide dismutase (ecSOD) protects the extracellular matrix (ECM) from oxidative stress. We previously reported a new allele for ecSOD, expressed in 129P3/J mice (129), which differs from the wild-type (wt), expressed in C57BL/6J and other strains, by two amino acid substitutions and a 10 bp deletion in the 3' UTR of the mRNA [1]. The newly discovered allele is associated with a phenotype of significantly increased circulating and heparin-releasable enzyme activities and levels. In order to examine the properties of the two forms of ecSOD in an identical environment we generated, by extensive backcrossing of ecSOD heterozygous progeny to C57BL/6J females, a congenic C57 strain with the 129 (or wt) allele of ecSOD. These mice are homozygous for nearly 5,000 SNPs across all chromosomes, as determined by Affymetrix Parallele Mouse 5K SNP panel. The present study describes the generation of the congenic mice (genetically >99.8 % identical) and their ecSOD phenotype. The congenic mice plasma ecSOD activities before and after heparin administration recapitulate the differences reported in the founder mice. Tissue enzyme distribution is similar in both congenic groups, although the 129 allele is associated with higher levels of enzyme expression despite lower levels of enzyme mRNA. In these characteristics the phenotype is also allele driven, with little impact by the rest of the genome. The congenic mice carrying the 129 allele have mRNA levels that are in between those found in the founder 129P3/J and C57BL/6J strains. We conclude that the ecSOD phenotype in most aspects of enzyme expression is allele- driven, with the exception of tissue mRNA levels, where a significant contribution by the surrounding (host) genome is observed. These results also suggest potential allele-specific differences in the regulation of ecSOD synthesis and intracellular processing/secretion of ecSOD, independent of the genotype context. Most importantly, the congenic mice

  8. Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR

    PubMed Central

    Orue, Andrea; Rieber, Manuel

    2016-01-01

    Establishing the KRAS mutational status of tumor samples is essential to manage patients with colorectal or lung cancer, since these mutations preclude treatment with monoclonal anti-epidermal growth factor receptor (EGFR) antibodies. We report an inexpensive, rapid multiplex allele-specific qPCR method detecting the 7 most clinically relevant KRAS somatic mutations with concomitant amplification of non-mutated KRAS in tumor cells and tissues from CRC patients. Positive samples evidenced in the multiplex assay were further subjected to individual allele-specific analysis, to define the specific mutation. Reference human cancer DNA harbouring either G12A, G12C, G12D, G12R, G12S, G12V and G13D confirmed assay specificity with ≤1% sensitivity of mutant alleles. KRAS multiplex mutation analysis usefulness was also demonstrated with formalin-fixed paraffin embedded (FFPE) from CRC biopsies. Conclusion. Co-amplification of non-mutated DNA avoided false negatives from degraded samples. Moreover, this cost effective assay is compatible with mutation detection by DNA sequencing in FFPE tissues, but with a greater sensitivity when mutant DNA concentrations are limiting. PMID:27632281

  9. Correction of Hair Shaft Defects through Allele-Specific Silencing of Mutant Krt75.

    PubMed

    Liu, Ying; Snedecor, Elizabeth R; Zhang, Xu; Xu, Yanfeng; Huang, Lan; Jones, Evan C; Zhang, Lianfeng; Clark, Richard A; Roop, Dennis R; Qin, Chuan; Chen, Jiang

    2016-01-01

    Dominant mutations in keratin genes can cause a number of inheritable skin disorders characterized by intraepidermal blistering, epidermal hyperkeratosis, or abnormalities in skin appendages, such as nail plate dystrophy and structural defects in hair. Allele-specific silencing of mutant keratins through RNA interference is a promising therapeutic approach for suppressing the expression of mutant keratins and related phenotypes in the epidermis. However, its effectiveness on skin appendages remains to be confirmed in vivo. In this study, we developed allele-specific small interfering RNAs capable of selectively suppressing the expression of a mutant Krt75, which causes hair shaft structural defects characterized by the development of blebs along the hair shaft in mice. Hair regenerated from epidermal keratinocyte progenitor cells isolated from mutant Krt75 mouse models reproduced the blebbing phenotype when grafted in vivo. In contrast, mutant cells manipulated with a lentiviral vector expressing mutant Krt75-specific short hairpin RNA (shRNA) persistently suppressed this phenotype. The phenotypic correction was associated with a significant reduction of mutant Krt75 mRNA in the skin grafts. Thus, data obtained from this study demonstrated the feasibility of utilizing RNA interference to achieve durable correction of hair structural phenotypes through allele-specific silencing of mutant keratin genes. PMID:26763422

  10. Extensive allele-specific translational regulation in hybrid mice

    PubMed Central

    Hou, Jingyi; Wang, Xi; McShane, Erik; Zauber, Henrik; Sun, Wei; Selbach, Matthias; Chen, Wei

    2015-01-01

    Translational regulation is mediated through the interaction between diffusible trans-factors and cis-elements residing within mRNA transcripts. In contrast to extensively studied transcriptional regulation, cis-regulation on translation remains underexplored. Using deep sequencing-based transcriptome and polysome profiling, we globally profiled allele-specific translational efficiency for the first time in an F1 hybrid mouse. Out of 7,156 genes with reliable quantification of both alleles, we found 1,008 (14.1%) exhibiting significant allelic divergence in translational efficiency. Systematic analysis of sequence features of the genes with biased allelic translation revealed that local RNA secondary structure surrounding the start codon and proximal out-of-frame upstream AUGs could affect translational efficiency. Finally, we observed that the cis-effect was quantitatively comparable between transcriptional and translational regulation. Such effects in the two regulatory processes were more frequently compensatory, suggesting that the regulation at the two levels could be coordinated in maintaining robustness of protein expression. PMID:26253569

  11. Detection of mutation by allele-specific loop-mediated isothermal amplification (AS-LAMP).

    PubMed

    Aonuma, Hiroka; Badolo, Athanase; Okado, Kiyoshi; Kanuka, Hirotaka

    2013-01-01

    For effective control of pathogen-transmitting mosquitoes, precise surveillance data of mosquito distribution are essential. Recently, an increase of insecticide resistance due to the kdr mutation in Anopheles gambiae, a mosquito that transmits the malaria parasite, has been reported. With the aim of developing a simple and effective method for surveying resistant mosquitoes, LAMP was applied to the allele-specific detection of the kdr gene in An. gambiae. Allele-specific LAMP (AS-LAMP) method successfully distinguished the kdr homozygote from the heterozygote and the wild type. The robustness of AS-LAMP suggests its usefulness for routine identification of insects, not only mosquitoes but also other vectors and agricultural pests. Here we describe the method of AS-LAMP to detect mutation in Anopheles mosquitoes. PMID:24026691

  12. Genome-wide survey of allele-specific splicing in humans

    PubMed Central

    Nembaware, Victoria; Lupindo, Bukiwe; Schouest, Katherine; Spillane, Charles; Scheffler, Konrad; Seoighe, Cathal

    2008-01-01

    Background Accurate mRNA splicing depends on multiple regulatory signals encoded in the transcribed RNA sequence. Many examples of mutations within human splice regulatory regions that alter splicing qualitatively or quantitatively have been reported and allelic differences in mRNA splicing are likely to be a common and important source of phenotypic diversity at the molecular level, in addition to their contribution to genetic disease susceptibility. However, because the effect of a mutation on the efficiency of mRNA splicing is often difficult to predict, many mutations that cause disease through an effect on splicing are likely to remain undiscovered. Results We have combined a genome-wide scan for sequence polymorphisms likely to affect mRNA splicing with analysis of publicly available Expressed Sequence Tag (EST) and exon array data. The genome-wide scan uses published tools and identified 30,977 SNPs located within donor and acceptor splice sites, branch points and exonic splicing enhancer elements. For 1,185 candidate splicing polymorphisms the difference in splicing between alternative alleles was corroborated by publicly available exon array data from 166 lymphoblastoid cell lines. We developed a novel probabilistic method to infer allele-specific splicing from EST data. The method uses SNPs and alternative mRNA isoforms mapped to EST sequences and models both regulated alternative splicing as well as allele-specific splicing. We have also estimated heritability of splicing and report that a greater proportion of genes show evidence of splicing heritability than show heritability of overall gene expression level. Our results provide an extensive resource that can be used to assess the possible effect on splicing of human polymorphisms in putative splice-regulatory sites. Conclusion We report a set of genes showing evidence of allele-specific splicing from an integrated analysis of genomic polymorphisms, EST data and exon array data, including several

  13. High-speed droplet-allele-specific polymerase chain reaction for genotyping of single nucleotide polymorphisms.

    PubMed

    Matsuda, Kazuyuki; Honda, Takayuki

    2015-01-01

    Single nucleotide alternations such as single nucleotide polymorphisms (SNPs) or single nucleotide mutations are useful genetic markers for molecular diagnosis, prognosis, drug response, and predisposition to diseases. Rapid identification of SNPs or mutations is clinically important, especially for determining drug responses and selection of molecular-targeted therapy. Here, we describe a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) by using our droplet-PCR machine (droplet-AS-PCR).

  14. Design of phylum-specific hybrid primers for DNA barcoding: addressing the need for efficient COI amplification in the Echinodermata.

    PubMed

    Hoareau, T B; Boissin, E

    2010-11-01

    Recent research has shown the usefulness of the Folmer region of the cytochrome oxidase I (COI) as a genetic barcode to assist in species delimitation of echinoderms. However, amplification of COI is often challenging in echinoderms (low success or pseudogenes). We present a method that allows the design of phylum-specific hybrid primers, and use this to develop COI primers for the Echinodermata. We aligned COI sequences from 310 echinoderm species and designed all possible primers along the consensus sequence with two methods (standard degenerate and hybrid). We found much lower degeneracy for hybrid primers (4-fold degeneracy) than for standard degenerate primers (≥48-fold degeneracy). We then designed the most conserved hybrid primers to amplify a >500-bp region within COI. These primers successfully amplified this gene region in all tested taxa (123 species across all echinoderm classes). Sequencing of 30 species among these confirmed both the quality of the sequences (>500 bp, no pseudogenes) and their utility as a DNA barcode. This method should be useful for developing primers for other mitochondrial genes and other phyla. The method will also be of interest for the development of future projects involving both community-based genetic assessments on macroorganisms and biodiversity assessment of environmental samples using high-throughput sequencing.

  15. PCR amplification of the hrcV gene through specific primers for detecting Pseudomonas syringae pathovars.

    PubMed

    Vaseghi, Akbar; Bakhshinejad, Babak; Safaie, Naser; Parchin, Reza Ashrafi; Sadeghizadeh, Majid

    2014-02-01

    Pseudomonas syringae pathovars are important pathogens among phytopathogenic bacteria causing a variety of diseases in plants. These pathogens can rapidly disseminate in a large area leading to infection and destruction of plants. To prevent the incidence of the bacteria, appropriate detection methods should be employed. Routinely serological tests, being time-consuming and costly, are exploited to detect these pathogens in plants, soil, water and other resources. Over the recent years, DNA-based detection approaches which are stable, rapid, specific and reliable have been developed and sequence analysis of various genes are widely utilized to identify different strains of P. syringe. However, the greatest limitation of these genes is inability to detect numerous pathovars of P. syringae. Herein, by using bioinformatic analysis, we found the hrcV gene located at pathogenicity islands of bacterial genome with the potential of being used as a new marker for phylogenetic detection of numerous pathovars of P. syringae. Following design of specific primers to hrcV, we amplified a 440 bp fragment. Of 13 assayed pathovars, 11 were detected. Also, through experimental procedures and bioinformatic analysis it was revealed that the designed primers have the capacity to detect 19 pathovars. Our findings suggest that hrcV could be used as a gene with the merit of detecting more pathovars of P. syringae in comparison with other genes used frequently for detection purposes.

  16. Exploration of Deinococcus-Thermus molecular diversity by novel group-specific PCR primers.

    PubMed

    Theodorakopoulos, Nicolas; Bachar, Dipankar; Christen, Richard; Alain, Karine; Chapon, Virginie

    2013-10-01

    The deeply branching Deinococcus-Thermus lineage is recognized as one of the most extremophilic phylum of bacteria. In previous studies, the presence of Deinococcus-related bacteria in the hot arid Tunisian desert of Tataouine was demonstrated through combined molecular and culture-based approaches. Similarly, Thermus-related bacteria have been detected in Tunisian geothermal springs. The present work was conducted to explore the molecular diversity within the Deinococcus-Thermus phylum in these extreme environments. A set of specific primers was designed in silico on the basis of 16S rRNA gene sequences, validated for the specific detection of reference strains, and used for the polymerase chain reaction (PCR) amplification of metagenomic DNA retrieved from the Tataouine desert sand and Tunisian hot spring water samples. These analyses have revealed the presence of previously undescribed Deinococcus-Thermus bacterial sequences within these extreme environments. The primers designed in this study thus represent a powerful tool for the rapid detection of Deinococcus-Thermus in environmental samples and could also be applicable to clarify the biogeography of the Deinococcus-Thermus phylum.

  17. Exploration of Deinococcus-Thermus molecular diversity by novel group-specific PCR primers

    PubMed Central

    Theodorakopoulos, Nicolas; Bachar, Dipankar; Christen, Richard; Alain, Karine; Chapon, Virginie

    2013-01-01

    The deeply branching Deinococcus-Thermus lineage is recognized as one of the most extremophilic phylum of bacteria. In previous studies, the presence of Deinococcus-related bacteria in the hot arid Tunisian desert of Tataouine was demonstrated through combined molecular and culture-based approaches. Similarly, Thermus-related bacteria have been detected in Tunisian geothermal springs. The present work was conducted to explore the molecular diversity within the Deinococcus-Thermus phylum in these extreme environments. A set of specific primers was designed in silico on the basis of 16S rRNA gene sequences, validated for the specific detection of reference strains, and used for the polymerase chain reaction (PCR) amplification of metagenomic DNA retrieved from the Tataouine desert sand and Tunisian hot spring water samples. These analyses have revealed the presence of previously undescribed Deinococcus-Thermus bacterial sequences within these extreme environments. The primers designed in this study thus represent a powerful tool for the rapid detection of Deinococcus-Thermus in environmental samples and could also be applicable to clarify the biogeography of the Deinococcus-Thermus phylum. PMID:23996915

  18. Mining of novel species-specific primers for PCR detection of Listeria monocytogenes based on genomic approach.

    PubMed

    Tao, Tingting; Chen, Qiming; Bie, Xiaomei; Lu, Fengxia; Lu, Zhaoxin

    2015-12-01

    Listeria monocytogenes in contaminated food is considered as a serious health threat for consumers due to its high mortality rate. The objective of this study was to obtain novel species-specific target-genes and primers for the molecular detection of L. monocytogenes using a comparative genomic approach. By comparative analysis of L. monocytogenes and non-L. monocytogenes genome sequences in the GenBank database with BLAST program, 26 specific target sequences were used as candidates and the primers were designed for L. monocytogenes species-specificity verification by using PCR assay. Finally, the three genes LMOf2365_0970, LMOf2365_2721 and mpl were identified to have L. monocytogenes species-specificity and be unique as detection targets for diagnostic application. The species-specific primer Lm8 of gene LMOf2365_0970, Lm13 of gene LMOf2365_2721 and Lm20 of gene mpl showed better specificity and sensitivity than the primers described previously. The PCR detection limits of the three specific primer sets were 430, 43, 4.3 fg/μL for genomic DNA, and 5 × 10(3), 50, 5 cfu/mL for pure culture of L. monocytogenes. There was no interference in specificity of detecting L. monocytogenes by co-culture with other foodborne pathogens in high concentration. Moreover, after 6-8 h of enrichment, L. monocytogenes in the artificially contaminated milk samples at an inoculum dose of 38 cfu/10 mL milk could be detected successfully with the studied three primers. Therefore, the three specific genes and primers can be applied to establish a novel rapid and accurate method for detecting L. monocytogenes in food materials. PMID:26354019

  19. Survey of benign Theileria parasites of cattle and buffaloes in Thailand using allele-specific polymerase chain reaction of major piroplasm surface protein gene.

    PubMed

    Sarataphan, Nopporn; Kakuda, Tsutomu; Chansiri, Kosum; Onuma, Misao

    2003-01-01

    During a year from 1999 to 2000, a total of 247 blood samples were collected from 214 cattle and 33 water buffaloes in 16 distinct geographical locations of Thailand and analyzed by allele-specific PCR amplification of major piroplasm surface protein (MPSP) genes of benign Theileria parasites. Four allelic MPSP gene types were determined namely C-type, I-type, B-type and Thai-type, which were originally designated from Japanese Theileria orientalis (Chitose, Ikeda), Australian T. buffeli (Warwick) and Thai T. sp. (Kamphaeng Saen), respectively. Only two allelic MPSP gene types were successively amplified from 204 (82.6%) blood samples. Among positive cases, 138 (67.6%) and 17 (8.3%) samples contained either Thai-type or C-type parasites, respectively, while 49 (24%) samples contained both types. However, nucleotide sequences of MPSP genes of Thai T. sp. amplified by C-type specific primers revealed higher (96.3%) similarity to Indonesian T. sp. rather than (87.8% similarity) to Japanese T. orientalis (Chitose) designated as C-type.

  20. Regulation of the imprinted Dlk1-Dio3 locus by allele-specific enhancer activity.

    PubMed

    Luo, Zhuojuan; Lin, Chengqi; Woodfin, Ashley R; Bartom, Elizabeth T; Gao, Xin; Smith, Edwin R; Shilatifard, Ali

    2016-01-01

    Genomic imprinting is a critical developmental process characteristic of parent of origin-specific gene expression. It is well accepted that differentially DNA-methylated regions (DMRs) and enhancers are two major classes of cis-elements determining parent of origin-specific gene expression, with each recruiting different sets of transcription factors. Previously, we identified the AF4/FMR2 (AFF) family protein AFF3 within the transcription elongation complex SEC-L3. Here, we report that AFF3 can specifically bind both gametic DMRs (gDMRs) and enhancers within imprinted loci in an allele-specific manner. We identify the molecular regulators involved in the recruitment of AFF3 to gDMRs and provide mechanistic insight into the requirement of AFF3 at an enhancer for the expression of an ∼200-kb polycistronic transcript within the imprinted Dlk1-Dio3 locus. Our data suggest that the heterochromatic environment at the gDMR reinforces silencing of its related enhancer by controlling the binding and activity of AFF3 in an allele-specific manner. In summary, this study provides molecular details about the regulation of dosage-critical imprinted gene expression through the regulated binding of the transcription elongation factor AFF3 between a DMR and an enhancer. PMID:26728555

  1. Regulation of the imprinted Dlk1-Dio3 locus by allele-specific enhancer activity

    PubMed Central

    Luo, Zhuojuan; Lin, Chengqi; Woodfin, Ashley R.; Bartom, Elizabeth T.; Gao, Xin; Smith, Edwin R.; Shilatifard, Ali

    2016-01-01

    Genomic imprinting is a critical developmental process characteristic of parent of origin-specific gene expression. It is well accepted that differentially DNA-methylated regions (DMRs) and enhancers are two major classes of cis-elements determining parent of origin-specific gene expression, with each recruiting different sets of transcription factors. Previously, we identified the AF4/FMR2 (AFF) family protein AFF3 within the transcription elongation complex SEC-L3. Here, we report that AFF3 can specifically bind both gametic DMRs (gDMRs) and enhancers within imprinted loci in an allele-specific manner. We identify the molecular regulators involved in the recruitment of AFF3 to gDMRs and provide mechanistic insight into the requirement of AFF3 at an enhancer for the expression of an ∼200-kb polycistronic transcript within the imprinted Dlk1-Dio3 locus. Our data suggest that the heterochromatic environment at the gDMR reinforces silencing of its related enhancer by controlling the binding and activity of AFF3 in an allele-specific manner. In summary, this study provides molecular details about the regulation of dosage-critical imprinted gene expression through the regulated binding of the transcription elongation factor AFF3 between a DMR and an enhancer. PMID:26728555

  2. Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus

    PubMed Central

    Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

    2016-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) system has been adopted for a wide range of biological applications including genome editing. In some cases, dissection of genome functions requires allele-specific genome editing, but the use of CRISPR for this purpose has not been studied in detail. In this study, using the p16INK4a gene in HCT116 as a model locus, we investigated whether chromatin states, such as CpG methylation, or a single-nucleotide gap form in a target site can be exploited for allele-specific locus binding and genome editing by CRISPR in vivo. First, we showed that allele-specific locus binding and genome editing could be achieved by targeting allele-specific CpG-methylated regions, which was successful for one, but not all guide RNAs. In this regard, molecular basis underlying the success remains elusive at this stage. Next, we demonstrated that an allele-specific single-nucleotide gap form could be employed for allele-specific locus binding and genome editing by CRISPR, although it was important to avoid CRISPR tolerance of a single nucleotide mismatch brought about by mismatched base skipping. Our results provide information that might be useful for applications of CRISPR in studies of allele-specific functions in the genomes. PMID:27465215

  3. Allele-specific deposition of macroH2A1 in Imprinting Control Regions

    SciTech Connect

    Choo, J H; Kim, J D; Chung, J H; Stubbs, L; Kim, J

    2006-01-13

    In the current study, we analyzed the deposition patterns of macroH2A1 at a number of different genomic loci located in X chromosome and autosomes. MacroH2A1 is preferentially deposited at methylated CpG CpG-rich regions located close to promoters. The macroH2A1 deposition patterns at the methylated CpG islands of several imprinted domains, including the Imprinting Control Regions (ICRs) of Xist, Peg3, H19/Igf2 Igf2, Gtl2/Dlk1, and Gnas domains, show consistent allele-specificity towards inactive, methylated alleles. The macroH2A1 deposition levels at the ICRs and other Differentially Methylated Regions (DMRs) of these domains are also either higher or comparable to those observed at the inactive X chromosome of female mammals. Overall, our results indicate that besides DNA methylation macroH2A1 is another epigenetic component in the chromatin of ICRs displaying differential association with two parental alleles.

  4. Efficient and allele-specific genome editing of disease loci in human iPSCs.

    PubMed

    Smith, Cory; Abalde-Atristain, Leire; He, Chaoxia; Brodsky, Brett R; Braunstein, Evan M; Chaudhari, Pooja; Jang, Yoon-Young; Cheng, Linzhao; Ye, Zhaohui

    2015-03-01

    Efficient and precise genome editing is crucial for realizing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucleases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed targets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloproliferative neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) deficiency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleotide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption.

  5. Identification of Mycobacterium bovis in bovine clinical samples by PCR species-specific primers.

    PubMed Central

    Romero, R E; Garzón, D L; Mejía, G A; Monroy, W; Patarroyo, M E; Murillo, L A

    1999-01-01

    Tuberculosis, caused by Mycobacterium bovis is emerging as the most important disease affecting cattle. Furthermore, it results in a major public health problem when transmitted to humans. Due to its difficult and non-specific diagnosis, M. bovis has been declared to be one of the etiologic agents causing significant economic loss in the cattle industry. Our group evaluated a more rapid and specific method, based on a new polymerase chain reaction species-specific primers, which amplifies a 470-base pair fragment of the M. bovis genome. A total of 275 milk-producing cows were studied by intradermal tuberculin test (ITT) which gave 184 positive and 91 negative cases. From them, 50 animals were taken from a cattle ranch free of tuberculosis. Three different samples were collected from each animal (blood, nasal mucus, and milk). Positive results were obtained from 26 animals by PCR (11.4%), 1 by bacteriological culturing (0.4%) and 1 by bacilloscopy (0.4%). This finding suggests, as in previous reports, that ITT, normally used for bovine tuberculosis detection, has the inconvenience of having a broad range of specificity and sensitivity, and the PCR technique is a more specific and sensitive test to detect infection associated with M. bovis. Therefore, we propose this PCR assay as a useful tool in the epidemiological characterization of infected animals in areas considered to be at high risk of transmission. Images Figure 1. PMID:10369566

  6. A multiplex allele-specific real-time polymerase chain reaction assay for HLA-B*13:01 genotyping in four Chinese populations.

    PubMed

    Liu, Z; Chen, G; Kang, X; Han, M; Chen, R; Chen, C; Wang, H

    2016-10-01

    Human leukocyte antigen HLA-B*13:01 is identified currently as a marker of individual susceptibility to drug-induced hypersensitivity reaction, such as dapsone-induced hypersensitivity reactions (DIHRs) and trichloroethylene-induced dermatitis. Therefore, screening for the HLA-B*13:01 allele can assist clinics in identifying patients at risk of developing DIHRs. By combining the allele-specific primers with TaqMan probes, we established a single tube, triplex real-time PCR to detect HLA-B*13:01. The reliability of this assay was validated by the comparison of genotyping results with those by sequence-based typing (SBT). With this assay, the distribution of HLA-B*13:01 in a total of 350 blood samples from four ethnic groups: Han, Tibetan, Uighur, and Buyei were determined. A 100% concordance was observed between the results with the established real-time PCR and SBT in 100 samples. The detection limit of this assay was 0.016 ng genomic DNA. The prevalence of HLA-B*13:01 carriers were 11%, 8%, 1%, and 2% in the Buyei (n = 100), Northern Han (n = 100), Tibetan (n = 100), and Uighur (n = 50) populations, respectively. The multiplex real-time PCR assay provided a fast and reliable method for accurate detection of HLA-B*13:01 allele prior to dapsone administration in clinical practice and onset of the reaction after exposure to trichloroethylene.

  7. Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts

    PubMed Central

    Scholefield, Janine; Watson, Lauren; Smith, Danielle; Greenberg, Jacquie; Wood, Matthew JA

    2014-01-01

    Polyglutamine (polyQ) disorders are inherited neurodegenerative conditions defined by a common pathogenic CAG repeat expansion leading to a toxic gain-of-function of the mutant protein. Consequences of this toxicity include activation of heat-shock proteins (HSPs), impairment of the ubiquitin-proteasome pathway and transcriptional dysregulation. Several studies in animal models have shown that reducing levels of toxic protein using small RNAs would be an ideal therapeutic approach for such disorders, including spinocerebellar ataxia-7 (SCA7). However, testing such RNA interference (RNAi) effectors in genetically appropriate patient cell lines with a disease-relevant phenotype has yet to be explored. Here, we have used primary adult dermal fibroblasts from SCA7 patients and controls to assess the endogenous allele-specific silencing of ataxin-7 by two distinct siRNAs. We further identified altered expression of two disease-relevant transcripts in SCA7 patient cells: a twofold increase in levels of the HSP DNAJA1 and a twofold decrease in levels of the de-ubiquitinating enzyme, UCHL1. After siRNA treatment, the expression of both genes was restored towards normal levels. To our knowledge, this is the first time that allele-specific silencing of mutant ataxin-7, targeting a common SNP, has been demonstrated in patient cells. These findings highlight the advantage of an allele-specific RNAi-based therapeutic approach, and indicate the value of primary patient-derived cells as useful models for mechanistic studies and for measuring efficacy of RNAi effectors on a patient-to-patient basis in the polyQ diseases. PMID:24667781

  8. Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts.

    PubMed

    Scholefield, Janine; Watson, Lauren; Smith, Danielle; Greenberg, Jacquie; Wood, Matthew J A

    2014-12-01

    Polyglutamine (polyQ) disorders are inherited neurodegenerative conditions defined by a common pathogenic CAG repeat expansion leading to a toxic gain-of-function of the mutant protein. Consequences of this toxicity include activation of heat-shock proteins (HSPs), impairment of the ubiquitin-proteasome pathway and transcriptional dysregulation. Several studies in animal models have shown that reducing levels of toxic protein using small RNAs would be an ideal therapeutic approach for such disorders, including spinocerebellar ataxia-7 (SCA7). However, testing such RNA interference (RNAi) effectors in genetically appropriate patient cell lines with a disease-relevant phenotype has yet to be explored. Here, we have used primary adult dermal fibroblasts from SCA7 patients and controls to assess the endogenous allele-specific silencing of ataxin-7 by two distinct siRNAs. We further identified altered expression of two disease-relevant transcripts in SCA7 patient cells: a twofold increase in levels of the HSP DNAJA1 and a twofold decrease in levels of the de-ubiquitinating enzyme, UCHL1. After siRNA treatment, the expression of both genes was restored towards normal levels. To our knowledge, this is the first time that allele-specific silencing of mutant ataxin-7, targeting a common SNP, has been demonstrated in patient cells. These findings highlight the advantage of an allele-specific RNAi-based therapeutic approach, and indicate the value of primary patient-derived cells as useful models for mechanistic studies and for measuring efficacy of RNAi effectors on a patient-to-patient basis in the polyQ diseases.

  9. Development of Species-Specific Primers for Agronomical Thrips and Multiplex Assay for Quarantine Identification of Western Flower Thrips.

    PubMed

    Yeh, W B; Tseng, M J; Chang, N T; Wu, S Y; Tsai, Y S

    2014-10-01

    While morphological identification of thrips species has been difficult because of their minute size and a lack of easily recognizable characteristics, molecular identification based on the development of specific molecular markers can be easily and reliably carried out. Among the known molecular markers, the nuclear internal transcribed spacer (ITS) exhibits distinguishable variations among thrips species. In this study, sequences of ITS2 region of 10 agriculturally important thrips were established to design species-specific primers for polymerase chain reaction (PCR). ITS2 sequence variations within these species were far less than those among species, indicating the suitability of this marker for species-specific primers design. These primers, though with one or two sporadically variable positions, showed a good efficacy within species. The specificity of these primers, examined on thrips species belonging to 15 genera, proved satisfactory. Furthermore, a multiplex PCR was used successfully for identifying Frankliniella occidentalis (Pergande), an insect pest monitored for quarantine purpose, and three additional thrips also commonly found in imported agricultural products and field samples, i.e., Thrips tabaci Lindeman, Thrips hawaiiensis (Morgan), and Frankliniella intonsa (Trybom). This study has demonstrated that specific primers and multiplex PCR based on ITS2 are reliable, convenient, and diagnostic tool to discriminate thrips species of quarantine and agricultural importance.

  10. Development of Prevotella nigrescens-specific PCR primers based on the nucleotide sequence of a Pn23 DNA probe.

    PubMed

    Kim, Min Jung; Lee, Young-Seok; Park, Jae-Yoon; Kook, Joong-Ki

    2011-02-01

    A previous study reported the cloning of a putative Prevotella nigrescens-specific DNA probe, Pn23, using random shotgun method. The present study evaluated the species-specificity of Pn23 for P. nigrescens using the clinical strains of Prevotella intermedia and P. nigrescens to develop P. nigrescens-specific polymerase chain reaction (PCR) primers. Southern blot analysis showed that the DNA probe, Pn23, detected only the genomic DNA of P. nigrescens strains. PCR showed that the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, had species-specificity for P. nigrescens. Interestingly, the two sets of PCR primers, Pn23-F6/Pn23-R6 and Pn23-F7/Pn23-R7, had strain-specificity for P. nigrescens ATCC 33563. The detection limits of the four primer sets were 40 or 4 pg of the purified genomic DNA of P. nigrescens ATCC 33563. These results suggest that the DNA probe, Pn23, and the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, can be useful for the detection of P. nigrescens in the molecular epidemiological studies of oral infectious diseases. PMID:21184839

  11. Multiple Avirulence Loci and Allele-Specific Effector Recognition Control the Pm3 Race-Specific Resistance of Wheat to Powdery Mildew[OPEN

    PubMed Central

    Roffler, Stefan; Stirnweis, Daniel; Treier, Georges; Herren, Gerhard; Korol, Abraham B.; Wicker, Thomas

    2015-01-01

    In cereals, several mildew resistance genes occur as large allelic series; for example, in wheat (Triticum aestivum and Triticum turgidum), 17 functional Pm3 alleles confer agronomically important race-specific resistance to powdery mildew (Blumeria graminis). The molecular basis of race specificity has been characterized in wheat, but little is known about the corresponding avirulence genes in powdery mildew. Here, we dissected the genetics of avirulence for six Pm3 alleles and found that three major Avr loci affect avirulence, with a common locus_1 involved in all AvrPm3-Pm3 interactions. We cloned the effector gene AvrPm3a2/f2 from locus_2, which is recognized by the Pm3a and Pm3f alleles. Induction of a Pm3 allele-dependent hypersensitive response in transient assays in Nicotiana benthamiana and in wheat demonstrated specificity. Gene expression analysis of Bcg1 (encoded by locus_1) and AvrPm3 a2/f2 revealed significant differences between isolates, indicating that in addition to protein polymorphisms, expression levels play a role in avirulence. We propose a model for race specificity involving three components: an allele-specific avirulence effector, a resistance gene allele, and a pathogen-encoded suppressor of avirulence. Thus, whereas a genetically simple allelic series controls specificity in the plant host, recognition on the pathogen side is more complex, allowing flexible evolutionary responses and adaptation to resistance genes. PMID:26452600

  12. Comparative analysis of type 2 diabetes-associated SNP alleles identifies allele-specific DNA-binding proteins for the KCNQ1 locus.

    PubMed

    Hiramoto, Masaki; Udagawa, Haruhide; Watanabe, Atsushi; Miyazawa, Keisuke; Ishibashi, Naoko; Kawaguchi, Miho; Uebanso, Takashi; Nishimura, Wataru; Nammo, Takao; Yasuda, Kazuki

    2015-07-01

    Although recent genome-wide association studies (GWAS) have been extremely successful, it remains a big challenge to functionally annotate disease‑associated single nucleotide polymorphisms (SNPs), as the majority of these SNPs are located in non‑coding regions of the genome. In this study, we described a novel strategy for identifying the proteins that bind to the SNP‑containing locus in an allele‑specific manner and successfully applied this method to SNPs in the type 2 diabetes mellitus susceptibility gene, potassium voltage‑gated channel, KQT‑like subfamily Q, member 1 (KCNQ1). DNA fragments encompassing SNPs, and risk or non‑risk alleles were immobilized onto the novel nanobeads and DNA‑binding proteins were purified from the nuclear extracts of pancreatic β cells using these DNA‑immobilized nanobeads. Comparative analysis of the allele-specific DNA-binding proteins indicated that the affinities of several proteins for the examined SNPs differed between the alleles. Nuclear transcription factor Y (NF‑Y) specifically bound the non‑risk allele of the SNP rs2074196 region and stimulated the transcriptional activity of an artificial promoter containing SNP rs2074196 in an allele‑specific manner. These results suggest that SNP rs2074196 modulates the affinity of the locus for NF‑Y and possibly induces subsequent changes in gene expression. The findings of this study indicate that our comparative method using novel nanobeads is effective for the identification of allele‑specific DNA‑binding proteins, which may provide important clues for the functional impact of disease‑associated non‑coding SNPs.

  13. A species-specific primer pair for distinguishing between Paramisgurnus dabryanus and Misgurnus anguillicaudatus based on mitochondrial DNA polymorphisms.

    PubMed

    Liu, Yongfu; Hou, Jilun; Wang, Guixing; Zhang, Xiaoyan; Liu, Haijin

    2016-07-01

    Paramisgurnus dabryanus and Misgurnus anguillicaudatus (family Cobitidae) are loaches with high morphological similarity. In this study, we designed primers to distinguish between Paramisgurnus dabryanus and Misgurnus anguillicaudatus based on the length differences in the mitochondrial COXII to tRNA(Lys) gene region. Samples of P. dabryanus and M. anguillicaudatus from different geographical locations were collected and amplified to verify primer specificity. The results of electrophoresis revealed the successful amplification of all P. dabryanus and M. anguillicaudatus DNA samples, which had distinct, specific-specific sizes (214 bp for P. dabryanus and 285 bp for M. anguillicaudatus). In conclusion, the new primers provide fast, reliable, and accurate identification between P. dabryanus and M. anguillicaudatus.

  14. In Vivo Evaluation of Candidate Allele-specific Mutant Huntingtin Gene Silencing Antisense Oligonucleotides

    PubMed Central

    Southwell, Amber L; Skotte, Niels H; Kordasiewicz, Holly B; Østergaard, Michael E; Watt, Andrew T; Carroll, Jeffrey B; Doty, Crystal N; Villanueva, Erika B; Petoukhov, Eugenia; Vaid, Kuljeet; Xie, Yuanyun; Freier, Susan M; Swayze, Eric E; Seth, Punit P; Bennett, Clarence Frank; Hayden, Michael R

    2014-01-01

    Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than nonselective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. Here we evaluate ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 16 weeks or more after a single intracerebroventricular (ICV) injection. With further validation, these ASOs could provide a therapeutic option for individuals afflicted with HD. PMID:25101598

  15. Allele-specific RNAi Mitigates Phenotypic Progression in a Transgenic Model of Alzheimer's Disease

    PubMed Central

    Rodríguez-Lebrón, Edgardo; Gouvion, Cynthia M; Moore, Steven A; Davidson, Beverly L; Paulson, Henry L

    2009-01-01

    Despite recent advances suggesting new therapeutic targets, Alzheimer's disease (AD) remains incurable. Aberrant production and accumulation of the Aβ peptide resulting from altered processing of the amyloid precursor protein (APP) is central to the pathogenesis of disease, particularly in dominantly inherited forms of AD. Thus, modulating the production of APP is a potential route to effective AD therapy. Here, we describe the successful use of an allele-specific RNA interference (RNAi) approach targeting the Swedish variant of APP (APPsw) in a transgenic mouse model of AD. Using recombinant adeno-associated virus (rAAV), we delivered an anti-APPsw short-hairpin RNA (shRNA) to the hippocampus of AD transgenic mice (APP/PS1). In short- and long-term transduction experiments, reduced levels of APPsw transprotein were observed throughout targeted regions of the hippocampus while levels of wild-type murine APP remained unaltered. Moreover, intracellular production of transfer RNA (tRNA)-valine promoter–driven shRNAs did not lead to detectable neuronal toxicity. Finally, long-term bilateral hippocampal expression of anti-APPsw shRNA mitigated abnormal behaviors in this mouse model of AD. The difference in phenotype progression was associated with reduced levels of soluble Aβ but not with a reduced number of amyloid plaques. Our results support the development of allele-specific RNAi strategies to treat familial AD and other dominantly inherited neurodegenerative diseases. PMID:19532137

  16. Rapid plant identification using species- and group-specific primers targeting chloroplast DNA.

    PubMed

    Wallinger, Corinna; Juen, Anita; Staudacher, Karin; Schallhart, Nikolaus; Mitterrutzner, Evi; Steiner, Eva-Maria; Thalinger, Bettina; Traugott, Michael

    2012-01-01

    Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae), the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory. PMID:22253728

  17. Rapid Plant Identification Using Species- and Group-Specific Primers Targeting Chloroplast DNA

    PubMed Central

    Staudacher, Karin; Schallhart, Nikolaus; Mitterrutzner, Evi; Steiner, Eva-Maria; Thalinger, Bettina; Traugott, Michael

    2012-01-01

    Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae), the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory. PMID:22253728

  18. Characterization of the 18S rRNA gene for designing universal eukaryote specific primers.

    PubMed

    Hadziavdic, Kenan; Lekang, Katrine; Lanzen, Anders; Jonassen, Inge; Thompson, Eric M; Troedsson, Christofer

    2014-01-01

    High throughput sequencing technology has great promise for biodiversity studies. However, an underlying assumption is that the primers used in these studies are universal for the prokaryotic or eukaryotic groups of interest. Full primer universality is difficult or impossible to achieve and studies using different primer sets make biodiversity comparisons problematic. The aim of this study was to design and optimize universal eukaryotic primers that could be used as a standard in future biodiversity studies. Using the alignment of all eukaryotic sequences from the publicly available SILVA database, we generated a full characterization of variable versus conserved regions in the 18S rRNA gene. All variable regions within this gene were analyzed and our results suggested that the V2, V4 and V9 regions were best suited for biodiversity assessments. Previously published universal eukaryotic primers as well as a number of self-designed primers were mapped to the alignment. Primer selection will depend on sequencing technology used, and this study focused on the 454 pyrosequencing GS FLX Titanium platform. The results generated a primer pair yielding theoretical matches to 80% of the eukaryotic and 0% of the prokaryotic sequences in the SILVA database. An empirical test of marine sediments using the AmpliconNoise pipeline for analysis of the high throughput sequencing data yielded amplification of sequences for 71% of all eukaryotic phyla with no isolation of prokaryotic sequences. To our knowledge this is the first characterization of the complete 18S rRNA gene using all eukaryotes present in the SILVA database, providing a robust test for universal eukaryotic primers. Since both in silico and empirical tests using high throughput sequencing retained high inclusion of eukaryotic phyla and exclusion of prokaryotes, we conclude that these primers are well suited for assessing eukaryote diversity, and can be used as a standard in biodiversity studies.

  19. Characterization of the 18S rRNA Gene for Designing Universal Eukaryote Specific Primers

    PubMed Central

    Hadziavdic, Kenan; Lekang, Katrine; Lanzen, Anders; Jonassen, Inge; Thompson, Eric M.; Troedsson, Christofer

    2014-01-01

    High throughput sequencing technology has great promise for biodiversity studies. However, an underlying assumption is that the primers used in these studies are universal for the prokaryotic or eukaryotic groups of interest. Full primer universality is difficult or impossible to achieve and studies using different primer sets make biodiversity comparisons problematic. The aim of this study was to design and optimize universal eukaryotic primers that could be used as a standard in future biodiversity studies. Using the alignment of all eukaryotic sequences from the publicly available SILVA database, we generated a full characterization of variable versus conserved regions in the 18S rRNA gene. All variable regions within this gene were analyzed and our results suggested that the V2, V4 and V9 regions were best suited for biodiversity assessments. Previously published universal eukaryotic primers as well as a number of self-designed primers were mapped to the alignment. Primer selection will depend on sequencing technology used, and this study focused on the 454 pyrosequencing GS FLX Titanium platform. The results generated a primer pair yielding theoretical matches to 80% of the eukaryotic and 0% of the prokaryotic sequences in the SILVA database. An empirical test of marine sediments using the AmpliconNoise pipeline for analysis of the high throughput sequencing data yielded amplification of sequences for 71% of all eukaryotic phyla with no isolation of prokaryotic sequences. To our knowledge this is the first characterization of the complete 18S rRNA gene using all eukaryotes present in the SILVA database, providing a robust test for universal eukaryotic primers. Since both in silico and empirical tests using high throughput sequencing retained high inclusion of eukaryotic phyla and exclusion of prokaryotes, we conclude that these primers are well suited for assessing eukaryote diversity, and can be used as a standard in biodiversity studies. PMID:24516555

  20. Geographically Distinct and Domain-Specific Sequence Variations in the Alleles of Rice Blast Resistance Gene Pib

    PubMed Central

    Vasudevan, Kumar; Vera Cruz, Casiana M.; Gruissem, Wilhelm; Bhullar, Navreet K.

    2016-01-01

    Rice blast is caused by Magnaporthe oryzae, which is the most destructive fungal pathogen affecting rice growing regions worldwide. The rice blast resistance gene Pib confers broad-spectrum resistance against Southeast Asian M. oryzae races. We investigated the allelic diversity of Pib in rice germplasm originating from 12 major rice growing countries. Twenty-five new Pib alleles were identified that have unique single nucleotide polymorphisms (SNPs), insertions and/or deletions, in addition to the polymorphic nucleotides that are shared between the different alleles. These partially or completely shared polymorphic nucleotides indicate frequent sequence exchange events between the Pib alleles. In some of the new Pib alleles, nucleotide diversity is high in the LRR domain, whereas, in others it is distributed among the NB-ARC and LRR domains. Most of the polymorphic amino acids in LRR and NB-ARC2 domains are predicted as solvent-exposed. Several of the alleles and the unique SNPs are country specific, suggesting a diversifying selection of alleles in various geographical locations in response to the locally prevalent M. oryzae population. Together, the new Pib alleles are an important genetic resource for rice blast resistance breeding programs and provide new information on rice-M. oryzae interactions at the molecular level. PMID:27446145

  1. Geographically Distinct and Domain-Specific Sequence Variations in the Alleles of Rice Blast Resistance Gene Pib.

    PubMed

    Vasudevan, Kumar; Vera Cruz, Casiana M; Gruissem, Wilhelm; Bhullar, Navreet K

    2016-01-01

    Rice blast is caused by Magnaporthe oryzae, which is the most destructive fungal pathogen affecting rice growing regions worldwide. The rice blast resistance gene Pib confers broad-spectrum resistance against Southeast Asian M. oryzae races. We investigated the allelic diversity of Pib in rice germplasm originating from 12 major rice growing countries. Twenty-five new Pib alleles were identified that have unique single nucleotide polymorphisms (SNPs), insertions and/or deletions, in addition to the polymorphic nucleotides that are shared between the different alleles. These partially or completely shared polymorphic nucleotides indicate frequent sequence exchange events between the Pib alleles. In some of the new Pib alleles, nucleotide diversity is high in the LRR domain, whereas, in others it is distributed among the NB-ARC and LRR domains. Most of the polymorphic amino acids in LRR and NB-ARC2 domains are predicted as solvent-exposed. Several of the alleles and the unique SNPs are country specific, suggesting a diversifying selection of alleles in various geographical locations in response to the locally prevalent M. oryzae population. Together, the new Pib alleles are an important genetic resource for rice blast resistance breeding programs and provide new information on rice-M. oryzae interactions at the molecular level. PMID:27446145

  2. Microarrays for Genotyping Human Group A Rotavirus by Multiplex Capture and Type-Specific Primer Extension

    PubMed Central

    Lovmar, Lovisa; Fock, Caroline; Espinoza, Felix; Bucardo, Filemon; Syvänen, Ann-Christine; Bondeson, Kåre

    2003-01-01

    Human group A rotavirus (HRV) is the major cause of severe gastroenteritis in infants worldwide. HRV shares the feature of a high degree of genetic diversity with many other RNA viruses, and therefore, genotyping of this organism is more complicated than genotyping of more stable DNA viruses. We describe a novel microarray-based method that allows high-throughput genotyping of RNA viruses with a high degree of polymorphism by multiplex capture and type-specific extension on microarrays. Denatured reverse transcription (RT)-PCR products derived from two outer capsid genes of clinical isolates of HRV were hybridized to immobilized capture oligonucleotides representing the most commonly occurring P and G genotypes on a microarray. Specific primer extension of the type-specific capture oligonucleotides was applied to incorporate the fluorescent nucleotide analogue cyanine 5-labeled dUTP as a detectable label. Laser scanning and fluorescence detection of the microarrays was followed by visual or computer-assisted interpretation of the fluorescence patterns generated on the microarrays. Initially, the method detected HRV in all 40 samples and correctly determined both the G and the P genotypes of 35 of the 40 strains analyzed. After modification by inclusion of additional capture oligonucleotides specific for the initially unassigned genotypes, all genotypes could be correctly defined. The results of genotyping with the microarray fully agreed with the results obtained by nucleotide sequence analysis and sequence-specific multiplex RT-PCR. Owing to its robustness, simplicity, and general utility, the microarray-based method may gain wide applicability for the genotyping of microorganisms, including highly variable RNA and DNA viruses. PMID:14605152

  3. Microarrays for genotyping human group a rotavirus by multiplex capture and type-specific primer extension.

    PubMed

    Lovmar, Lovisa; Fock, Caroline; Espinoza, Felix; Bucardo, Filemon; Syvänen, Ann-Christine; Bondeson, Kåre

    2003-11-01

    Human group A rotavirus (HRV) is the major cause of severe gastroenteritis in infants worldwide. HRV shares the feature of a high degree of genetic diversity with many other RNA viruses, and therefore, genotyping of this organism is more complicated than genotyping of more stable DNA viruses. We describe a novel microarray-based method that allows high-throughput genotyping of RNA viruses with a high degree of polymorphism by multiplex capture and type-specific extension on microarrays. Denatured reverse transcription (RT)-PCR products derived from two outer capsid genes of clinical isolates of HRV were hybridized to immobilized capture oligonucleotides representing the most commonly occurring P and G genotypes on a microarray. Specific primer extension of the type-specific capture oligonucleotides was applied to incorporate the fluorescent nucleotide analogue cyanine 5-labeled dUTP as a detectable label. Laser scanning and fluorescence detection of the microarrays was followed by visual or computer-assisted interpretation of the fluorescence patterns generated on the microarrays. Initially, the method detected HRV in all 40 samples and correctly determined both the G and the P genotypes of 35 of the 40 strains analyzed. After modification by inclusion of additional capture oligonucleotides specific for the initially unassigned genotypes, all genotypes could be correctly defined. The results of genotyping with the microarray fully agreed with the results obtained by nucleotide sequence analysis and sequence-specific multiplex RT-PCR. Owing to its robustness, simplicity, and general utility, the microarray-based method may gain wide applicability for the genotyping of microorganisms, including highly variable RNA and DNA viruses.

  4. Robust Detection of Rare Species Using Environmental DNA: The Importance of Primer Specificity

    PubMed Central

    Wilcox, Taylor M.; McKelvey, Kevin S.; Young, Michael K.; Jane, Stephen F.; Lowe, Winsor H.; Whiteley, Andrew R.; Schwartz, Michael K.

    2013-01-01

    Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probe-based chemistries may represent a particularly powerful tool because of the method’s sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence the specificity and sensitivity of TaqMan MGB assays using co-occurring, closely related brook trout (Salvelinus fontinalis) and bull trout (S. confluentus) as a case study. We found qPCR to be substantially more sensitive than traditional PCR, with a high probability of detection at concentrations as low as 0.5 target copies/µl. We also found that number and placement of base pair mismatches between the Taqman MGB assay and non-target templates was important to target specificity, and that specificity was most influenced by base pair mismatches in the primers, rather than in the probe. We found that insufficient specificity can result in both false positive and false negative results, particularly in the presence of abundant related species. Our results highlight the utility of qPCR as a highly sensitive eDNA tool, and underscore the importance of careful assay design. PMID:23555689

  5. Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay

    PubMed Central

    Zhang, Guoqing; Cai, Fangping; Zhou, Zhiyong; DeVos, Joshua; Wagar, Nick; Diallo, Karidia; Zulu, Isaac; Wadonda-Kabondo, Nellie; Stringer, Jeffrey S. A.; Weidle, Paul J.; Ndongmo, Clement B.; Sikazwe, Izukanji; Sarr, Abdoulaye; Kagoli, Matthew; Nkengasong, John

    2013-01-01

    High-throughput, sensitive, and cost-effective HIV drug resistance (HIVDR) detection assays are needed for large-scale monitoring of the emergence and transmission of HIVDR in resource-limited settings. Using suspension array technology, we have developed a multiplex allele-specific (MAS) assay that can simultaneously detect major HIVDR mutations at 20 loci. Forty-five allele-specific primers tagged with unique 24-base oligonucleotides at the 5′ end were designed to detect wild-type and mutant alleles at the 20 loci of HIV-1 subtype C. The MAS assay was first established and optimized with three plasmid templates (C-wt, C-mut1, and C-mut2) and then evaluated using 148 plasma specimens from HIV-1 subtype C-infected individuals. All the wild-type and mutant alleles were unequivocally distinguished with plasmid templates, and the limits of detection were 1.56% for K219Q and K219E, 3.13% for L76V, 6.25% for K65R, K70R, L74V, L100I, K103N, K103R, Q151M, Y181C, and I47V, and 12.5% for M41L, K101P, K101E, V106A, V106M, Y115F, M184V, Y188L, G190A, V32I, I47A, I84V, and L90M. Analyses of 148 plasma specimens revealed that the MAS assay gave 100% concordance with conventional sequencing at eight loci and >95% (range, 95.21% to 99.32%) concordance at the remaining 12 loci. The differences observed were caused mainly by 24 additional low-abundance alleles detected by the MAS assay. Ultradeep sequencing analysis confirmed 15 of the 16 low-abundance alleles. This multiplex, sensitive, and straightforward result-reporting assay represents a new efficient genotyping tool for HIVDR surveillance and monitoring. PMID:23985909

  6. Development of Prevotella intermedia-specific PCR primers based on the nucleotide sequences of a DNA probe Pig27.

    PubMed

    Kim, Min Jung; Hwang, Kyung Hwan; Lee, Young-Seok; Park, Jae-Yoon; Kook, Joong-Ki

    2011-03-01

    The aim of this study was to develop Prevotella intermedia-specific PCR primers based on the P. intermedia-specific DNA probe. The P. intermedia-specific DNA probe was screened by inverted dot blot hybridization and confirmed by Southern blot hybridization. The nucleotide sequences of the species-specific DNA probes were determined using a chain termination method. Southern blot analysis showed that the DNA probe, Pig27, detected only the genomic DNA of P. intermedia strains. PCR showed that the PCR primers, Pin-F1/Pin-R1, had species-specificity for P. intermedia. The detection limits of the PCR primer sets were 0.4pg of the purified genomic DNA of P. intermedia ATCC 49046. These results suggest that the PCR primers, Pin-F1/Pin-R1, could be useful in the detection of P. intermedia as well as in the development of a PCR kit in epidemiological studies related to periodontal diseases. PMID:21192988

  7. A novel PCR technique using Alu-specific primers to identify unknown flanking sequences from the human genome

    SciTech Connect

    Minami, M.; Poussin, K.; Brechot, C.; Paterlini, P.

    1995-09-20

    The rapid and reproducible identification of new cellular DNA sequences is difficult to achieve with the currently available procedures. Here we describe a novel approach based on the polymerase chain reaction (PCR) using a primer specific to the known sequence and another directed to a human Alu repeat. To avoid undesirable amplifications between Alu sequences, primers are constructed with dUTPs and destroyed by uracil DNA glycosylase treatment after 10 initial cycles of amplification. Only desirable fragments are then further amplified with specific primers to the known region and to a tag sequence introduced in the Alu-specific primer. Using this protocol, we have successfully indentified cellular sequences flanking integrated hepatitis B virus DNA from the human genome of three hepatoma tissues. The method enables a direct specific amplification without any ligation or nonspecific annealing steps as required by previous PCR-based protocols. This rapid and straightforward approach will be a powerful tool for the study of viral integration sites, but is also widely applicable to other studies of the human genome. 39 refs., 4 figs.

  8. Metabarcoding Analysis of Phytophthora Diversity Using Genus-Specific Primers and 454 Pyrosequencing.

    PubMed

    Prigigallo, Maria I; Abdelfattah, Ahmed; Cacciola, Santa O; Faedda, Roberto; Sanzani, Simona M; Cooke, David E L; Schena, L

    2016-03-01

    A metabarcoding method based on genus-specific primers and 454 pyrosequencing was utilized to investigate the genetic diversity of Phytophthora spp. in soil and root samples of potted plants, from eight nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in seven different clades and provided a much higher resolution than a corresponding cloning/Sanger sequencing approach. Eleven of these phylotypes, including P. cactorum, P. citricola s.str., P. palmivora, P. palmivora-like, P. megasperma or P. gonapodyides, P. ramorum, and five putative new Phytophthora species phylogenetically related to clades 1, 2, 4, 6, and 7 were detected only with the 454 pyrosequencing approach. We also found an additional 18 novel records of a phylotype in a particular nursery that were not detected with cloning/Sanger sequencing. Several aspects confirmed the reliability of the method: (i) many identical sequence types were identified independently in different nurseries, (ii) most sequence types identified with 454 pyrosequencing were identical to those from the cloning/Sanger sequencing approach and/or perfectly matched GenBank deposited sequences, and (iii) the divergence noted between sequence types of putative new Phytophthora species and all other detected sequences was sufficient to rule out sequencing errors. The proposed method represents a powerful tool to study Phytophthora diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types.

  9. Metabarcoding Analysis of Phytophthora Diversity Using Genus-Specific Primers and 454 Pyrosequencing.

    PubMed

    Prigigallo, Maria I; Abdelfattah, Ahmed; Cacciola, Santa O; Faedda, Roberto; Sanzani, Simona M; Cooke, David E L; Schena, L

    2016-03-01

    A metabarcoding method based on genus-specific primers and 454 pyrosequencing was utilized to investigate the genetic diversity of Phytophthora spp. in soil and root samples of potted plants, from eight nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in seven different clades and provided a much higher resolution than a corresponding cloning/Sanger sequencing approach. Eleven of these phylotypes, including P. cactorum, P. citricola s.str., P. palmivora, P. palmivora-like, P. megasperma or P. gonapodyides, P. ramorum, and five putative new Phytophthora species phylogenetically related to clades 1, 2, 4, 6, and 7 were detected only with the 454 pyrosequencing approach. We also found an additional 18 novel records of a phylotype in a particular nursery that were not detected with cloning/Sanger sequencing. Several aspects confirmed the reliability of the method: (i) many identical sequence types were identified independently in different nurseries, (ii) most sequence types identified with 454 pyrosequencing were identical to those from the cloning/Sanger sequencing approach and/or perfectly matched GenBank deposited sequences, and (iii) the divergence noted between sequence types of putative new Phytophthora species and all other detected sequences was sufficient to rule out sequencing errors. The proposed method represents a powerful tool to study Phytophthora diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types. PMID:26574783

  10. FUNGAL-SPECIFIC PCR PRIMERS DEVELOPED FOR ANALYSIS OF THE ITS REGION OF ENVIRONMENTAL DNA EXTRACTS

    EPA Science Inventory

    Background The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmenta...

  11. Utilizing ethnic-specific differences in minor allele frequency to recategorize reported pathogenic deafness variants.

    PubMed

    Shearer, A Eliot; Eppsteiner, Robert W; Booth, Kevin T; Ephraim, Sean S; Gurrola, José; Simpson, Allen; Black-Ziegelbein, E Ann; Joshi, Swati; Ravi, Harini; Giuffre, Angelica C; Happe, Scott; Hildebrand, Michael S; Azaiez, Hela; Bayazit, Yildirim A; Erdal, Mehmet Emin; Lopez-Escamez, Jose A; Gazquez, Irene; Tamayo, Marta L; Gelvez, Nancy Y; Leal, Greizy Lopez; Jalas, Chaim; Ekstein, Josef; Yang, Tao; Usami, Shin-ichi; Kahrizi, Kimia; Bazazzadegan, Niloofar; Najmabadi, Hossein; Scheetz, Todd E; Braun, Terry A; Casavant, Thomas L; LeProust, Emily M; Smith, Richard J H

    2014-10-01

    Ethnic-specific differences in minor allele frequency impact variant categorization for genetic screening of nonsyndromic hearing loss (NSHL) and other genetic disorders. We sought to evaluate all previously reported pathogenic NSHL variants in the context of a large number of controls from ethnically distinct populations sequenced with orthogonal massively parallel sequencing methods. We used HGMD, ClinVar, and dbSNP to generate a comprehensive list of reported pathogenic NSHL variants and re-evaluated these variants in the context of 8,595 individuals from 12 populations and 6 ethnically distinct major human evolutionary phylogenetic groups from three sources (Exome Variant Server, 1000 Genomes project, and a control set of individuals created for this study, the OtoDB). Of the 2,197 reported pathogenic deafness variants, 325 (14.8%) were present in at least one of the 8,595 controls, indicating a minor allele frequency (MAF) > 0.00006. MAFs ranged as high as 0.72, a level incompatible with pathogenicity for a fully penetrant disease like NSHL. Based on these data, we established MAF thresholds of 0.005 for autosomal-recessive variants (excluding specific variants in GJB2) and 0.0005 for autosomal-dominant variants. Using these thresholds, we recategorized 93 (4.2%) of reported pathogenic variants as benign. Our data show that evaluation of reported pathogenic deafness variants using variant MAFs from multiple distinct ethnicities and sequenced by orthogonal methods provides a powerful filter for determining pathogenicity. The proposed MAF thresholds will facilitate clinical interpretation of variants identified in genetic testing for NSHL. All data are publicly available to facilitate interpretation of genetic variants causing deafness. PMID:25262649

  12. HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA.

    PubMed Central

    Barat, C; Lullien, V; Schatz, O; Keith, G; Nugeyre, M T; Grüninger-Leitch, F; Barré-Sinoussi, F; LeGrice, S F; Darlix, J L

    1989-01-01

    The virion cores of the replication competent type 1 human immunodeficiency virus (HIV-1), a retrovirus, contain and RNA genome associated with nucleocapsid (NC) and reverse transcriptase (RT p66/p51) molecules. In vitro reconstructions of these complexes with purified components show that NC is required for efficient annealing of the primer tRNALys,3. In the absence of NC, HIV-1 RT is unable to retrotranscribe the viral RNA template from the tRNA primer. We demonstrate that the HIV-1 RT p66/p51 specifically binds to its cognate primer tRNALys,3 even in the presence of a 100-fold molar excess of other tRNAs. Cross-linking analysis of this interaction locates the contact site to a region within the heavily modified anti-codon domain of tRNALys,3. Images PMID:2479543

  13. Spelt-specific alleles in HMW glutenin genes from modern and historical European spelt ( Triticum spelta L.).

    PubMed

    Blatter, Robert H. E.; Jacomet, Stefanie; Schlumbaum, Angela

    2002-02-01

    A partial promoter region of the high-molecular weight (HMW) glutenin genes was studied in two wheat specimens, a 300 year-old spelt ( Triticum spelta L.) and an approximately 250 year-old bread wheat ( Triticum aestivum L.) from Switzerland. Sequences were compared to a recent Swiss landrace T. spelta'Oberkulmer.' The alleles from the historical bread wheat were most similar to those of modern T. aestivumcultivars, whereas in the historical and the recent spelt specific alleles were detected. Pairwise genetic distances up to 0.03 within 200 bp from the HMW Glu-A1-2, Glu-B1-1 and Glu-B1-2 alleles in spelt to the most-similar alleles from bread wheat suggest a polyphyletic origin. The spelt Glu-B1-1 allele, which was unlike the corresponding alleles in bread wheat, was closer related to an allele found in tetraploid wheat cultivars. The results are discussed in context of the origin of European spelt.

  14. A new PCR method: one primer amplification of PCR-CTPP products.

    PubMed

    Yin, Guang; Mitsuda, Yoko; Ezaki, Takayuki; Hamajima, Nobuyuki

    2012-10-01

    Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is a convenient method for genotyping single nucleotide polymorphisms, saving time, and costs. It uses four primers for PCR; F1 and R1 for one allele, and F2 and R2 for the other allele, by which three different sizes of DNA are amplified; between F1 and R1, between F2 and R2, and between F1 and R2. To date, we have applied PCR-CTPP successfully for genotyping more than 60 polymorphisms. However, it is not rare that PCR does not produce balanced amplification of allele specific bands. Accordingly, the method was modified by attaching a common sequence at the 5' end of two-pair primers and adding another primer with the common sequence in PCR, in total five different primers in a tube for PCR. The modification allowed one primer amplification for the products of initial PCR with confronting two-pair primers, named as one primer amplification of PCR-CTPP products (OPA-CTPP). This article demonstrates an example for an A/G polymorphism of paraoxonase 1 (PON1) Gln192Arg (rs662). PCR-CTPP failed clear genotyping for the polymorphism, while OPA-CTPP successfully produced PCR products corresponding to the allele. The present example indicated that the OPA-CTPP would be useful in the case that PCR-CTPP failed to produce balanced PCR products specific to each allele.

  15. Development of a Novel Allele-Specific PCR Method for Rapid Assessment of Nervous Necrosis Virus Genotypes.

    PubMed

    Toubanaki, Dimitra K; Margaroni, Maritsa; Karagouni, Evdokia

    2015-11-01

    Viral nervous necrosis infections are causing severe problems on aquaculture industry due to ecological and economic impacts. Their causal agent is nervous necrosis virus or nodavirus, which has been classified into four genotypes. Different genotypes correlate with differences in viral pathogenicity. Therefore, rational development of effective vaccines and diagnostic reagents requires analysis of the genetic variation. The development and validation of a polymerase chain reaction amplification (PCR)-based methodology for nodavirus genotype assessment in a simple and robust format is described. Degenerate external primers and two genotype-specific internal primers were utilized for simultaneous amplification of nodavirus products in a single PCR. A first set of cycles produced a long PCR product, defined by the outer primers, and the internal primers amplified short DNA fragments specific for each genotype in lower annealing temperature. Detection was based on the size of the short products. Nodavirus infected and healthy samples were analyzed and none of the non-infected samples showed any bands, while all infected samples were positive. The proposed method can be performed within 4 h and consumes standard PCR and electrophoresis reagents, with costs lower than 2€ per sample. Tetra-primer PCR is a suitable alternative for virus sequencing in medium scale research laboratories and farming facilities. PMID:26210900

  16. Development of a Novel Allele-Specific PCR Method for Rapid Assessment of Nervous Necrosis Virus Genotypes.

    PubMed

    Toubanaki, Dimitra K; Margaroni, Maritsa; Karagouni, Evdokia

    2015-11-01

    Viral nervous necrosis infections are causing severe problems on aquaculture industry due to ecological and economic impacts. Their causal agent is nervous necrosis virus or nodavirus, which has been classified into four genotypes. Different genotypes correlate with differences in viral pathogenicity. Therefore, rational development of effective vaccines and diagnostic reagents requires analysis of the genetic variation. The development and validation of a polymerase chain reaction amplification (PCR)-based methodology for nodavirus genotype assessment in a simple and robust format is described. Degenerate external primers and two genotype-specific internal primers were utilized for simultaneous amplification of nodavirus products in a single PCR. A first set of cycles produced a long PCR product, defined by the outer primers, and the internal primers amplified short DNA fragments specific for each genotype in lower annealing temperature. Detection was based on the size of the short products. Nodavirus infected and healthy samples were analyzed and none of the non-infected samples showed any bands, while all infected samples were positive. The proposed method can be performed within 4 h and consumes standard PCR and electrophoresis reagents, with costs lower than 2€ per sample. Tetra-primer PCR is a suitable alternative for virus sequencing in medium scale research laboratories and farming facilities.

  17. Evaluation of PCR primer selectivity and phylogenetic specificity by using amplification of 16S rRNA genes from betaproteobacterial ammonia-oxidizing bacteria in environmental samples.

    PubMed

    Junier, Pilar; Kim, Ok-Sun; Hadas, Ora; Imhoff, Johannes F; Witzel, Karl-Paul

    2008-08-01

    The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (betaAOB) was evaluated. betaAOB represent a group of phylogenetically related organisms for which the 16S rRNA gene approach is especially suitable. We used experimental comparisons of primer performance with water samples, together with an in silico analysis of published sequences and a literature review of clone libraries made with four specific PCR primers for the betaAOB 16S rRNA gene. With four aquatic samples, the primers NitA/NitB produced the highest frequency of ammonia-oxidizing-bacterium-like sequences compared to clone libraries with products amplified with the primer combinations betaAMOf/betaAMOr, betaAMOf/Nso1255g, and NitA/Nso1225g. Both the experimental examination of ammonia-oxidizing-bacterium-specific 16S rRNA gene primers and the literature search showed that neither specificity nor sensitivity of primer combinations can be evaluated reliably only by sequence comparison. Apparently, the combination of sequence comparison and experimental data is the best approach to detect possible biases of PCR primers. Although this study focused on betaAOB, the results presented here more generally exemplify the importance of primer selection and potential primer bias when analyzing microbial communities in environmental samples.

  18. A specific HLA-DP beta allele is associated with pauciarticular juvenile rheumatoid arthritis but not adult rheumatoid arthritis.

    PubMed Central

    Begovich, A B; Bugawan, T L; Nepom, B S; Klitz, W; Nepom, G T; Erlich, H A

    1989-01-01

    Nonradioactive sequence-specific oligonucleotide probes specific for the HLA-DP beta locus have been used in a simple dot-blot format to type samples amplified by the polymerase chain reaction from 44 patients with pauciarticular juvenile rheumatoid arthritis, 32 patients with adult rheumatoid arthritis, and 50 random controls. The sequences of four new DP beta alleles derived from these patients and controls are reported, bringing the total number of alleles identified thus far to 19. The DPB2.1 allele is significantly increased in juvenile rheumatoid arthritis patients over controls; this allele is not increased in patients with adult rheumatoid arthritis. The association of juvenile rheumatoid arthritis with the DPB2.1 allele is independent of linkage with previously defined HLA-D region markers of disease. Analysis of the DPB2.1 sequence shows that it differs from the nonsusceptible DPB4.2 allele by only 1 amino acid at position 69 in the beta 1 domain. PMID:2512583

  19. A hypervariable STR polymorphism in the CFI gene: southern origin of East Asian-specific group H alleles.

    PubMed

    Yuasa, Isao; Jin, Feng; Harihara, Shinji; Matsusue, Aya; Fujihara, Junko; Takeshita, Haruo; Akane, Atsushi; Umetsu, Kazuo; Saitou, Naruya; Chattopadhyay, Prasanta K

    2013-09-01

    Previous studies of four populations revealed that a hypervariable short tandem repeat (iSTR) in intron 7 of the human complement factor I (CFI) gene on chromosome 4q was unique, with 17 possible East Asian-specific group H alleles observed at relatively high frequencies. To develop a deeper anthropological and forensic understanding of iSTR, 1161 additional individuals from 11 Asian populations were investigated. Group H alleles of iSTR and c.1217A allele of a SNP in exon 11 of the CFI gene were associated with each other and were almost entirely confined to East Asian populations. Han Chinese in Changsha, southern China, showed the highest frequency for East Asian-specific group H alleles (0.201) among 15 populations. Group H alleles were observed to decrease gradually from south to north in 11 East Asian populations. This expansion of group H alleles provides evidence that southern China and Southeast Asia are a hotspot of Asian diversity and a genetic reservoir of Asians after they entered East Asia. The expected heterozygosity values of iSTR ranged from 0.927 in Thais to 0.874 in Oroqens, higher than those of an STR in the fibrinogen alpha chain (FGA) gene on chromosome 4q. Thus, iSTR is a useful marker for anthropological and forensic genetics.

  20. Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi

    PubMed Central

    Kaulich, Manuel; Lee, Yeon J.; Lönn, Peter; Springer, Aaron D.; Meade, Bryan R.; Dowdy, Steven F.

    2015-01-01

    Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms. PMID:25586224

  1. Diversity of Methane-Cycling Archaea in Hydrothermal Sediment Investigated by General and Group-Specific PCR Primers

    PubMed Central

    Teske, Andreas P.

    2014-01-01

    The zonation of anaerobic methane-cycling Archaea in hydrothermal sediment of Guaymas Basin was studied by general primer pairs (mcrI, ME1/ME2, mcrIRD) targeting the alpha subunit of methyl coenzyme M reductase gene (mcrA) and by new group-specific mcrA and 16S rRNA gene primer pairs. The mcrIRD primer pair outperformed the other general mcrA primer pairs in detection sensitivity and phylogenetic coverage. Methanotrophic ANME-1 Archaea were the only group detected with group-specific primers only. The detection of 14 mcrA lineages surpasses the diversity previously found in this location. Most phylotypes have high sequence similarities to hydrogenotrophs, methylotrophs, and anaerobic methanotrophs previously detected at Guaymas Basin or at hydrothermal vents, cold seeps, and oil reservoirs worldwide. Additionally, five mcrA phylotypes belonging to newly defined lineages are detected. Two of these belong to deeply branching new orders, while the others are new species or genera of Methanopyraceae and Methermicoccaceae. Downcore diversity decreases from all groups detected in the upper 6 cm (∼2 to 40°C, sulfate measurable to 4 cm) to only two groups below 6 cm (>40°C). Despite the presence of hyperthermophilic genera (Methanopyrus, Methanocaldococcus) in cooler surface strata, no genes were detected below 10 cm (≥60°C). While mcrA-based and 16S rRNA gene-based community compositions are generally congruent, the deeply branching mcrA cannot be assigned to specific 16S rRNA gene lineages. Our study indicates that even among well-studied metabolic groups and in previously characterized model environments, major evolutionary branches are overlooked. Detecting these groups by improved molecular biological methods is a crucial first step toward understanding their roles in nature. PMID:25527539

  2. Searching for Beta-Haemolysin hlb Gene in Staphylococcus pseudintermedius with Species-Specific Primers.

    PubMed

    Kmieciak, Wioletta; Szewczyk, Eligia M; Ciszewski, Marcin

    2016-07-01

    The paper presents an analysis of 51 Staphylococcus pseudintermedius clinically isolated strains from humans and from animals. Staphylococcus pseudintermedius strains' ability to produce β-haemolysin was evaluated with phenotypic methods (hot-cold effect, reverse CAMP test). In order to determine the hlb gene presence (coding for β-haemolysin) in a genomic DNA, PCR reactions were conducted with two different pairs of primers: one described in the literature for Staphylococcus aureus and recommended for analysing SIG group staphylococci and newly designed one in CLC Main Workbench software. Only reactions with newly designed primers resulted in product amplification, the presence of which was fully compatible with the results of phenotypic β-haemolysin test. Negative results for S. aureus and S. intermedius reference ATCC strains suggest that after further analysis the fragment of hlb gene amplified with primers described in this study might be included in the process of S. pseudintermedius strains identification.

  3. Searching for Beta-Haemolysin hlb Gene in Staphylococcus pseudintermedius with Species-Specific Primers.

    PubMed

    Kmieciak, Wioletta; Szewczyk, Eligia M; Ciszewski, Marcin

    2016-07-01

    The paper presents an analysis of 51 Staphylococcus pseudintermedius clinically isolated strains from humans and from animals. Staphylococcus pseudintermedius strains' ability to produce β-haemolysin was evaluated with phenotypic methods (hot-cold effect, reverse CAMP test). In order to determine the hlb gene presence (coding for β-haemolysin) in a genomic DNA, PCR reactions were conducted with two different pairs of primers: one described in the literature for Staphylococcus aureus and recommended for analysing SIG group staphylococci and newly designed one in CLC Main Workbench software. Only reactions with newly designed primers resulted in product amplification, the presence of which was fully compatible with the results of phenotypic β-haemolysin test. Negative results for S. aureus and S. intermedius reference ATCC strains suggest that after further analysis the fragment of hlb gene amplified with primers described in this study might be included in the process of S. pseudintermedius strains identification. PMID:27086303

  4. Alleles of the maize P gene with distinct tissue specificities encode Myb-homologous proteins with C-terminal replacements.

    PubMed Central

    Chopra, S; Athma, P; Peterson, T

    1996-01-01

    The maize P gene is a transcriptional regulator of genes encoding enzymes for flavonoid biosynthesis in the pathway leading to the production of a red phlobaphene pigment. Multiple alleles of the P gene confer distinct patterns of pigmentation to specific floral organs, such as the kernel pericarp and cob tissues. To determine the basis of allele-specific pigmentation, we have characterized the gene products and transcript accumulation patterns of the P-wr allele, which specifies colorless pericarps and red cob tissues. RNA transcripts of P-wr are present in colorless pericarps as well as in the colored cob tissues; however, the expression of P-wr in pericarp does not induce the accumulation of transcripts from the C2 and A1 genes, which encode enzymes for flavonoid pigment biosynthesis. The coding sequences of P-wr were compared with the P-rr allele, which specifies red pericarp and red cob. The P-wr and P-rr cDNA sequences are very similar in their 5' regions. There are only two nucleotide changes that result in amino acid differences; both are outside of the Myb-homologous DNA binding domain. In contrast, the 3' coding region of P-rr is replaced by a unique 210-bp sequence in P-wr. The predicted P-wr protein has a C-terminal sequence resembling a cysteine-containing metal binding domain that is not present in the P-rr protein. These results indicate that the differential pericarp pigmentation specified by the P-rr and P-wr alleles does not result from an absence of P-wr transcripts in pericarps. Rather, the allele-specific patterns of P-rr and P-wr pigmentation may be associated with structural differences in the proteins encoded by each allele. PMID:8768374

  5. Allele-specific analysis of DNA replication origins in mammalian cells.

    PubMed

    Bartholdy, Boris; Mukhopadhyay, Rituparna; Lajugie, Julien; Aladjem, Mirit I; Bouhassira, Eric E

    2015-05-19

    The mechanisms that control the location and timing of firing of replication origins are poorly understood. Using a novel functional genomic approach based on the analysis of SNPs and indels in phased human genomes, we observe that replication asynchrony is associated with small cumulative variations in the initiation efficiency of multiple origins between the chromosome homologues, rather than with the activation of dormant origins. Allele-specific measurements demonstrate that the presence of G-quadruplex-forming sequences does not correlate with the efficiency of initiation. Sequence analysis reveals that the origins are highly enriched in sequences with profoundly asymmetric G/C and A/T nucleotide distributions and are almost completely depleted of antiparallel triplex-forming sequences. We therefore propose that although G4-forming sequences are abundant in replication origins, an asymmetry in nucleotide distribution, which increases the propensity of origins to unwind and adopt non-B DNA structure, rather than the ability to form G4, is directly associated with origin activity.

  6. Allele-Specific Methylation Occurs at Genetic Variants Associated with Complex Disease

    PubMed Central

    Hutchinson, John N.; Raj, Towfique; Fagerness, Jes; Stahl, Eli; Viloria, Fernando T.; Gimelbrant, Alexander; Seddon, Johanna; Daly, Mark; Chess, Andrew; Plenge, Robert

    2014-01-01

    We hypothesize that the phenomenon of allele-specific methylation (ASM) may underlie the phenotypic effects of multiple variants identified by Genome-Wide Association studies (GWAS). We evaluate ASM in a human population and document its genome-wide patterns in an initial screen at up to 380,678 sites within the genome, or up to 5% of the total genomic CpGs. We show that while substantial inter-individual variation exists, 5% of assessed sites show evidence of ASM in at least six samples; the majority of these events (81%) are under genetic influence. Many of these cis-regulated ASM variants are also eQTLs in peripheral blood mononuclear cells and monocytes and/or in high linkage-disequilibrium with variants linked to complex disease. Finally, focusing on autoimmune phenotypes, we extend this initial screen to confirm the association of cis-regulated ASM with multiple complex disease-associated variants in an independent population using next-generation bisulfite sequencing. These four variants are implicated in complex phenotypes such as ulcerative colitis and AIDS progression disease (rs10491434), Celiac disease (rs2762051), Crohn's disease, IgA nephropathy and early-onset inflammatory bowel disease (rs713875) and height (rs6569648). Our results suggest cis-regulated ASM may provide a mechanistic link between the non-coding genetic changes and phenotypic variation observed in these diseases and further suggests a route to integrating DNA methylation status with GWAS results. PMID:24911414

  7. Allele-specific methylation occurs at genetic variants associated with complex disease.

    PubMed

    Hutchinson, John N; Raj, Towfique; Fagerness, Jes; Stahl, Eli; Viloria, Fernando T; Gimelbrant, Alexander; Seddon, Johanna; Daly, Mark; Chess, Andrew; Plenge, Robert

    2014-01-01

    We hypothesize that the phenomenon of allele-specific methylation (ASM) may underlie the phenotypic effects of multiple variants identified by Genome-Wide Association studies (GWAS). We evaluate ASM in a human population and document its genome-wide patterns in an initial screen at up to 380,678 sites within the genome, or up to 5% of the total genomic CpGs. We show that while substantial inter-individual variation exists, 5% of assessed sites show evidence of ASM in at least six samples; the majority of these events (81%) are under genetic influence. Many of these cis-regulated ASM variants are also eQTLs in peripheral blood mononuclear cells and monocytes and/or in high linkage-disequilibrium with variants linked to complex disease. Finally, focusing on autoimmune phenotypes, we extend this initial screen to confirm the association of cis-regulated ASM with multiple complex disease-associated variants in an independent population using next-generation bisulfite sequencing. These four variants are implicated in complex phenotypes such as ulcerative colitis and AIDS progression disease (rs10491434), Celiac disease (rs2762051), Crohn's disease, IgA nephropathy and early-onset inflammatory bowel disease (rs713875) and height (rs6569648). Our results suggest cis-regulated ASM may provide a mechanistic link between the non-coding genetic changes and phenotypic variation observed in these diseases and further suggests a route to integrating DNA methylation status with GWAS results.

  8. The allele-specific suppressor sup-39 alters use of cryptic splice sites in Caenorhabditis elegans.

    PubMed Central

    Roller, A B; Hoffman, D C; Zahler, A M

    2000-01-01

    Mutations in the Caenorhabditis elegans sup-39 gene cause allele-specific suppression of the uncoordination defect of unc-73(e936). e936 is a point mutation that changes the canonical G at the 5' end of intron 16 to a U. This mutation activates three splice donors, two of which define introns beginning with the canonical GU. Use of these two cryptic splice sites causes loss of reading frame; interestingly these messages are not substrates for nonsense-mediated decay. The third splice donor, used in 10% of steady-state e936 messages, is the mutated splice donor at the wild-type position, which defines an intron beginning with UU. In the presence of a sup-39 mutation, these same three splice donors are used, but the ratio of messages produced by splicing at these sites changes. The percentage of unc-73(e936) messages containing the wild-type splice junction is increased to 33% with a corresponding increase in the level of UNC-73 protein. This sup-39-induced change was also observed when the e936 mutant intron region was inserted into a heterologous splicing reporter construct transfected into worms. Experiments with splicing reporter constructs showed that the degree of 5' splice site match to the splicing consensus sequence can strongly influence cryptic splice site choice. We propose that mutant SUP-39 is a new type of informational suppressor that alters the use of weak splice donors. PMID:10757761

  9. Analysis of common mitochondrial DNA mutations by allele-specific oligonucleotide and Southern blot hybridization.

    PubMed

    Tang, Sha; Halberg, Michelle C; Floyd, Kristen C; Wang, Jing

    2012-01-01

    Mitochondrial disorders are clinically and genetically heterogeneous. There are a set of recurrent point mutations in the mitochondrial DNA (mtDNA) that are responsible for common mitochondrial diseases, including MELAS (mitochondrial encephalopathy, lactic acidosis, stroke-like episodes), MERRF (myoclonic epilepsy and ragged red fibers), LHON (Leber's hereditary optic neuropathy), NARP (neuropathy, ataxia, retinitis pigmentosa), and Leigh syndrome. Most of the pathogenic mtDNA point mutations are present in the heteroplasmic state, meaning that the wild-type and mutant-containing mtDNA molecules are coexisting. Clinical heterogeneity may be due to the degree of mutant load (heteroplasmy) and distribution of heteroplasmic mutations in affected tissues. Additionally, Kearns-Sayre syndrome and Pearson syndrome are caused by large mtDNA deletions. In this chapter, we describe a multiplex PCR/allele-specific oligonucleotide (ASO) hybridization method for the screening of 13 common point mutations. This method allows the detection of low percentage of mutant heteroplasmy. In addition, a nonradioactive Southern blot hybridization protocol for the analysis of mtDNA large deletions is also described. PMID:22215554

  10. Assessing allele-specific expression across multiple tissues from RNA-seq read data

    PubMed Central

    Pirinen, Matti; Lappalainen, Tuuli; Zaitlen, Noah A.; Dermitzakis, Emmanouil T.; Donnelly, Peter; McCarthy, Mark I.; Rivas, Manuel A.

    2015-01-01

    Motivation: RNA sequencing enables allele-specific expression (ASE) studies that complement standard genotype expression studies for common variants and, importantly, also allow measuring the regulatory impact of rare variants. The Genotype-Tissue Expression (GTEx) project is collecting RNA-seq data on multiple tissues of a same set of individuals and novel methods are required for the analysis of these data. Results: We present a statistical method to compare different patterns of ASE across tissues and to classify genetic variants according to their impact on the tissue-wide expression profile. We focus on strong ASE effects that we are expecting to see for protein-truncating variants, but our method can also be adjusted for other types of ASE effects. We illustrate the method with a real data example on a tissue-wide expression profile of a variant causal for lipoid proteinosis, and with a simulation study to assess our method more generally. Availability and implementation: http://www.well.ox.ac.uk/~rivas/mamba/. R-sources and data examples http://www.iki.fi/mpirinen/ Contact: matti.pirinen@helsinki.fi or rivas@well.ox.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25819081

  11. Allele Specific Expression of MICA Variants in Human Fibroblasts Suggests a Pathogenic Mechanism.

    PubMed

    Shi, Chunhua; Li, Hongye; Couturier, Jacob P; Yang, Karen; Guo, Xinjian; He, Dongyi; Lewis, Dorothy E; Zhou, Xiaodong

    2015-01-01

    The major histocompatibility complex class I chain-related gene A (MICA) is involved in immune responses of both nature killer (NK) cells and subsets of T cells with its receptor NKG2D. MICA is highly polymorphic in sequence which leads to MICA protein variants with distinct features. Specific polymorphisms of MICA have been associated with inflammatory diseases, including ankylosing spondylitis (AS), ulcerative colitis (UC) and Behçet's disease. Studies herein characterize expression features of three MICA variants including MICA*008, a common variant in general population, and *MICA*007 and *019, which are associated with susceptibility to inflammatory diseases. MICA*019 was highly expressed on the surface of fibroblasts whereas expression of MICA*007 was the lowest in the culture supernatant. MICA*008 had low cell surface expression but was the only MICA allele in which exosomal material was detected. Surface or membrane-bound MICA activates NKG2D-mediated cytotoxicity, whereas soluble and exosomal MICAs down-regulate NKG2D. Therefore, comparisons of these three MICA variants in fibroblasts provides insight into understanding how MICA associated immune responses could be regulated to influence levels of inflammation.

  12. Allele-specific FKBP5 DNA demethylation mediates gene-childhood trauma interactions.

    PubMed

    Klengel, Torsten; Mehta, Divya; Anacker, Christoph; Rex-Haffner, Monika; Pruessner, Jens C; Pariante, Carmine M; Pace, Thaddeus W W; Mercer, Kristina B; Mayberg, Helen S; Bradley, Bekh; Nemeroff, Charles B; Holsboer, Florian; Heim, Christine M; Ressler, Kerry J; Rein, Theo; Binder, Elisabeth B

    2013-01-01

    Although the fact that genetic predisposition and environmental exposures interact to shape development and function of the human brain and, ultimately, the risk of psychiatric disorders has drawn wide interest, the corresponding molecular mechanisms have not yet been elucidated. We found that a functional polymorphism altering chromatin interaction between the transcription start site and long-range enhancers in the FK506 binding protein 5 (FKBP5) gene, an important regulator of the stress hormone system, increased the risk of developing stress-related psychiatric disorders in adulthood by allele-specific, childhood trauma-dependent DNA demethylation in functional glucocorticoid response elements of FKBP5. This demethylation was linked to increased stress-dependent gene transcription followed by a long-term dysregulation of the stress hormone system and a global effect on the function of immune cells and brain areas associated with stress regulation. This identification of molecular mechanisms of genotype-directed long-term environmental reactivity will be useful for designing more effective treatment strategies for stress-related disorders. PMID:23201972

  13. Allele-specific expression at the RET locus in blood and gut tissue of individuals carrying risk alleles for Hirschsprung disease.

    PubMed

    Matera, Ivana; Musso, Marco; Griseri, Paola; Rusmini, Marta; Di Duca, Marco; So, Man-Ting; Mavilio, Domenico; Miao, Xiaoping; Tam, Paul Hk; Ravazzolo, Roberto; Ceccherini, Isabella; Garcia-Barcelo, Merce

    2013-05-01

    RET common variants are associated with Hirschsprung disease (HSCR; colon aganglionosis), a congenital defect of the enteric nervous system. We analyzed a well-known HSCR-associated RET haplotype that encompasses linked alleles in coding and noncoding/regulatory sequences. This risk haplotype correlates with reduced level of RET expression when compared with the wild-type counterpart. As allele-specific expression (ASE) contributes to phenotypic variability in health and disease, we investigated whether RET ASE could contribute to the overall reduction of RET mRNA detected in carriers. We tested heterozygous neuroblastoma cell lines, ganglionic gut tissues (18 HSCR and 14 non-HSCR individuals) and peripheral blood mononuclear cells (PBMCs; 16 HSCR and 14 non-HSCR individuals). Analysis of the data generated by SNaPshot and Pyrosequencing revealed that the RET risk haplotype is significantly more expressed in gut than in PBMCs (P = 0.0045). No ASE difference was detected between patients and controls, irrespective of the sample type. Comparison of total RET expression levels between gut samples with and without ASE, correlated reduced RET expression with preferential transcription from the RET risk haplotype. Nonrandom RET ASE occurs in ganglionic gut regardless of the disease status. RET ASE should not be excluded as a disease mechanism acting during development.

  14. Primer pairs for the specific environmental detection and T-RFLP analysis of the ubiquitous flagellate taxa Chrysophyceae and Kinetoplastea.

    PubMed

    Glaser, Karin; Kuppardt, Anke; Krohn, Sandra; Heidtmann, Anett; Harms, Hauke; Chatzinotas, Antonis

    2014-05-01

    Bacterivorous protists play a key role in microbial soil food webs, however due to the lack of specific PCR protocols targeting selected protist taxa, knowledge on the diversity and dynamics of these groups is scarce. We developed specific PCR primers in combination with a T-RFLP protocol for the cultivation-independent analysis of two important taxa of bacterivorous flagellates, the Chrysophyceae and Kinetoplastea, in soil samples. Sequence analysis of clone libraries originating from two soils in temperate regions demonstrated the specificity of the respective primer pairs. Clone sequences affiliating to the Chrysophyceae mainly clustered within the clade C2, which has been known so far for its presence mainly in cold climatic regions, whereas Kinetoplastea sequences were mainly related to the Neobodonid clade. Based on an in silico restriction analysis of database sequence entries, suitable restriction enzymes for a T-RFLP approach were selected. This in silico approach revealed the necessity to use a combination of two restriction enzymes for T-RFLP analysis of the Chrysophyceae. Soil T-RFLP profiles reflected all T-RFs of the clone library sequences obtained from the same soils and allowed to distinguish flagellate communities from different sites. We propose to use these primer pairs for PCR detection and rapid fingerprint screening in environmental samples and envisage their use also for quantitative PCR or next generation sequencing approaches.

  15. Characterization and machine learning prediction of allele-specific DNA methylation.

    PubMed

    He, Jianlin; Sun, Ming-an; Wang, Zhong; Wang, Qianfei; Li, Qing; Xie, Hehuang

    2015-12-01

    A large collection of Single Nucleotide Polymorphisms (SNPs) has been identified in the human genome. Currently, the epigenetic influences of SNPs on their neighboring CpG sites remain elusive. A growing body of evidence suggests that locus-specific information, including genomic features and local epigenetic state, may play important roles in the epigenetic readout of SNPs. In this study, we made use of mouse methylomes with known SNPs to develop statistical models for the prediction of SNP associated allele-specific DNA methylation (ASM). ASM has been classified into parent-of-origin dependent ASM (P-ASM) and sequence-dependent ASM (S-ASM), which comprises scattered-S-ASM (sS-ASM) and clustered-S-ASM (cS-ASM). We found that P-ASM and cS-ASM CpG sites are both enriched in CpG rich regions, promoters and exons, while sS-ASM CpG sites are enriched in simple repeat and regions with high frequent SNP occurrence. Using Lasso-grouped Logistic Regression (LGLR), we selected 21 out of 282 genomic and methylation related features that are powerful in distinguishing cS-ASM CpG sites and trained the classifiers with machine learning techniques. Based on 5-fold cross-validation, the logistic regression classifier was found to be the best for cS-ASM prediction with an ACC of 0.77, an AUC of 0.84 and an MCC of 0.54. Lastly, we applied the logistic regression classifier on human brain methylome and predicted 608 genes associated with cS-ASM. Gene ontology term enrichment analysis indicated that these cS-ASM associated genes are significantly enriched in the category coding for transcripts with alternative splicing forms. In summary, this study provided an analytical procedure for cS-ASM prediction and shed new light on the understanding of different types of ASM events.

  16. Allele-specific transcription factor binding to common and rare variants associated with disease and gene expression.

    PubMed

    Cavalli, Marco; Pan, Gang; Nord, Helena; Wallerman, Ola; Wallén Arzt, Emelie; Berggren, Olof; Elvers, Ingegerd; Eloranta, Maija-Leena; Rönnblom, Lars; Lindblad Toh, Kerstin; Wadelius, Claes

    2016-05-01

    Genome-wide association studies (GWAS) have identified a large number of disease-associated SNPs, but in few cases the functional variant and the gene it controls have been identified. To systematically identify candidate regulatory variants, we sequenced ENCODE cell lines and used public ChIP-seq data to look for transcription factors binding preferentially to one allele. We found 9962 candidate regulatory SNPs, of which 16 % were rare and showed evidence of larger functional effect than common ones. Functionally rare variants may explain divergent GWAS results between populations and are candidates for a partial explanation of the missing heritability. The majority of allele-specific variants (96 %) were specific to a cell type. Furthermore, by examining GWAS loci we found >400 allele-specific candidate SNPs, 141 of which were highly relevant in our cell types. Functionally validated SNPs support identification of an SNP in SYNGR1 which may expose to the risk of rheumatoid arthritis and primary biliary cirrhosis, as well as an SNP in the last intron of COG6 exposing to the risk of psoriasis. We propose that by repeating the ChIP-seq experiments of 20 selected transcription factors in three to ten people, the most common polymorphisms can be interrogated for allele-specific binding. Our strategy may help to remove the current bottleneck in functional annotation of the genome. PMID:26993500

  17. Allele-specific rpoB PCR assays for detection of rifampin-resistant Mycobacterium tuberculosis in sputum smears.

    PubMed

    Mokrousov, Igor; Otten, Tatiana; Vyshnevskiy, Boris; Narvskaya, Olga

    2003-07-01

    We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M. tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB). Three different allele-specific PCRs are carried out either directly with purified DNA (single-step multiplex allele-specific PCR), or with preamplified rpoB fragment (nested allele-specific PCR [NAS-PCR]). The method was optimized and validated following analysis of 36 strains with known rpoB sequence. A retrospective analysis of the 287 DNA preparations from epidemiologically unlinked RIF-resistant clinical strains from Russia, collected from 1996 to 2002, revealed that 247 (86.1%) of them harbored a mutation in one of the targeted rpoB codons. A prospective study of microscopy-positive consecutive sputum samples from new and chronic TB patients validated the method for direct analysis of DNA extracted from sputum smears. The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of these samples. The developed rpoB-PCR assay can be used in clinical laboratories to detect RIF-resistant and hence MDR M. tuberculosis in the regions with high burdens of the MDR-TB. PMID:12821473

  18. Diversity of methane-cycling archaea in hydrothermal sediment investigated by general and group-specific PCR primers.

    PubMed

    Lever, Mark A; Teske, Andreas P

    2015-02-01

    The zonation of anaerobic methane-cycling Archaea in hydrothermal sediment of Guaymas Basin was studied by general primerpairs (mcrI, ME1/ME2, mcrIRD) targeting the alpha subunit of methyl coenzyme M reductase gene (mcrA) and by new group specific mcrA and 16S rRNA gene primer pairs. The mcrIRD primer pair outperformed the other general mcrA primer pairs indetection sensitivity and phylogenetic coverage. Methanotrophic ANME-1 Archaea were the only group detected with group specific primers only. The detection of 14 mcrA lineages surpasses the diversity previously found in this location. Most phylotypes have high sequence similarities to hydrogenotrophs, methylotrophs, and anaerobic methanotrophs previously detected at Guaymas Basin or at hydrothermal vents, cold seeps, and oil reservoirs worldwide. Additionally, five mcrA phylotypes belonging to newly defined lineages are detected. Two of these belong to deeply branching new orders, while the others are new species or genera of Methanopyraceae and Methermicoccaceae. Downcore diversity decreases from all groups detected in the upper 6 cm(2 to 40 °C, sulfate measurable to 4 cm) to only two groups below 6 cm (>40 °C). Despite the presence of hyperthermophilic genera (Methanopyrus, Methanocaldococcus) in cooler surface strata, no genes were detected below 10 cm (>60 °C). While mcrAbased and 16S rRNA gene-based community compositions are generally congruent, the deeply branching mcrA cannot be assigned to specific 16S rRNA gene lineages. Our study indicates that even among well-studied metabolic groups and in previously characterized model environments, major evolutionary branches are overlooked. Detecting these groups by improved molecular biological methods is a crucial first step toward understanding their roles in nature.

  19. Identification of self-incompatibility genotypes of apricot (Prunus armeniaca L.) by S-allele-specific PCR analysis.

    PubMed

    Jie, Qi; Shupeng, Gai; Jixiang, Zhang; Manru, Gu; Huairui, Shu

    2005-08-01

    A cDNA of 417 bp encoding an S-RNase gene, named PA S3, was isolated from apricot, Prunus aremeniaca. Nine S-alleles, S1-S9, were recognized by S-allele-specific PCR and confirmed by Southern blot analysis using PA S3 as probe. The S-genotypes of the six cultivars were determined and the results of self- and cross-pollination tests among the six cultivars were consistent with the predicted S-haplotypes by PCR analysis.

  20. Nonsyntenic Genes Drive Tissue-Specific Dynamics of Differential, Nonadditive, and Allelic Expression Patterns in Maize Hybrids1[OPEN

    PubMed Central

    2016-01-01

    Distantly related maize (Zea mays) inbred lines display an exceptional degree of genomic diversity. F1 progeny of such inbred lines are often more vigorous than their parents, a phenomenon known as heterosis. In this study, we investigated how the genetic divergence of the maize inbred lines B73 and Mo17 and their F1 hybrid progeny is reflected in differential, nonadditive, and allelic expression patterns in primary root tissues. In pairwise comparisons of the four genotypes, the number of differentially expressed genes between the two parental inbred lines significantly exceeded those of parent versus hybrid comparisons in all four tissues under analysis. No differentially expressed genes were detected between reciprocal hybrids, which share the same nuclear genome. Moreover, hundreds of nonadditive and allelic expression ratios that were different from the expression ratios of the parents were observed in the reciprocal hybrids. The overlap of both nonadditive and allelic expression patterns in the reciprocal hybrids significantly exceeded the expected values. For all studied types of expression - differential, nonadditive, and allelic - substantial tissue-specific plasticity was observed. Significantly, nonsyntenic genes that evolved after the last whole genome duplication of a maize progenitor from genes with synteny to sorghum (Sorghum bicolor) were highly overrepresented among differential, nonadditive, and allelic expression patterns compared with the fraction of these genes among all expressed genes. This observation underscores the role of nonsyntenic genes in shaping the transcriptomic landscape of maize hybrids during the early developmental manifestation of heterosis in root tissues of maize hybrids. PMID:27208302

  1. Paternal-specific S-allele transmission in sweet cherry (Prunus avium L.): the potential for sexual selection.

    PubMed

    Hedhly, A; Wünsch, A; Kartal, Ö; Herrero, M; Hormaza, J I

    2016-03-01

    Homomorphic self-incompatibility is a well-studied example of a physiological process that is thought to increase population diversity and reduce the expression of inbreeding depression. Whereas theoretical models predict the presence of a large number of S-haplotypes with equal frequencies at equilibrium, unequal allele frequencies have been repeatedly reported and attributed to sampling effects, population structure, demographic perturbation, sheltered deleterious mutations or selection pressure on linked genes. However, it is unclear to what extent unequal segregations are the results of gametophytic or sexual selection. Although these two forces are difficult to disentangle, testing S-alleles in the offspring of controlled crosses provides an opportunity to separate these two phenomena. In this work, segregation and transmission of S-alleles have been characterized in progenies of mixed donors and fully compatible pollinations under field conditions in Prunus avium. Seed set patterns and pollen performance have also been characterized. The results reveal paternal-specific distorted transmission of S-alleles in most of the crosses. Interestingly, S-allele segregation within any given paternal or maternal S-locus was random. Observations on pollen germination, pollen tube growth rate, pollen tube cohort size, seed set dynamics and transmission patterns strongly suggest post-pollination, prezygotic sexual selection, with male-male competition as the most likely mechanism. According to these results, post-pollination sexual selection takes precedence over frequency-dependent selection in explaining unequal S-haplotype frequencies.

  2. Specific expression of apomixis-linked alleles revealed by comparative transcriptomic analysis of sexual and apomictic Paspalum simplex Morong flowers.

    PubMed

    Polegri, Livia; Calderini, Ornella; Arcioni, Sergio; Pupilli, Fulvio

    2010-06-01

    Apomixis is defined as clonal reproduction by seed. A comparative transcriptomic analysis was undertaken between apomictic and sexual genotypes of Paspalum simplex Morong to identify apomixis-related polymorphisms at the level of mRNA. cDNA-AFLP (amplified fragment length polymorphism) profiling of apomictic and sexual flowers at several stages of development yielded 202 amplicons that showed several kinds of expression specificities. Among these, the large majority consisted of amplicons that were present only in specific stages of development of the apomictic flowers. Ten percent of polymorphic amplicons were present with almost identical intensity in all stages of the apomictic flowers and never in the sexual flowers. Reverse transcription-PCR (RT-PCR) and Southern analyses of these amplicons showed that they belong to constitutively expressed alleles that are specifically present on the apomixis-controlling locus of P. simplex. The most frequent biological functions inferred from the sequence homology of the apomixis-linked alleles were related to signal transduction and nucleic acid/protein-binding activities. Most of these apomixis-linked alleles showed nonsense and frameshift mutations, revealing their probable pseudogene nature. None of the amplicons that were present only in specific stages of development of the apomictic flowers co-segregated with apomixis, indicating they did not originate from additional apomictic alleles but more probably from differential regulation of the same allele in apomictic and sexual flowers. The molecular functions inferred from sequence analysis of these latter amplicons were related to seed storage protein and regulatory genes of various types. The results are discussed regarding the possible role in apomictic reproduction of the differentially expressed genes in relation to their specificity of expression and inferred molecular functions.

  3. High-throughput analysis of candidate imprinted genes and allele-specific gene expression in the human term placenta

    PubMed Central

    2010-01-01

    Background Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE) is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue. Results Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA) and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes) remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs) were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%). Conclusions Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes in the human term

  4. Development of species-specific PCR primers and polyphasic characterization of Lactobacillus sanfranciscensis isolated from Korean sourdough.

    PubMed

    Lee, Hyeongrho; Baek, Hyunwook; Lim, Sae Bom; Hur, Jin Soo; Shim, Sangmin; Shin, So-Yeon; Han, Nam Soo; Seo, Jin-Ho

    2015-05-01

    Lactobacillus sanfranciscensis is a bacterium used in sourdough that provides desirable properties such as better flavor and texture to the sourdough bread. Here, the intra-species diversity of L. sanfranciscensis strains isolated from Korean sourdough was studied using genotypic (multiplex-RAPD-PCR: multiplex-Randomly Amplified Polymorphic DNA-polymerase chain reaction) and phenotypic (VITEK2 Compact system) analyses. For this, a novel species-specific set of PCR primers was developed to identify L. sanfranciscensis using the recently published genome database. The primers were able to detect L. sanfranciscensis isolated from Korean sourdough with 100% accuracy. Genotyping and phenotyping analyses at the strain level demonstrated that Korean sourdough possesses various biotypes of L. sanfranciscensis strains. These strains were clustered into 5 subtypes (genotyping) or 7 subtypes (phenotyping). In summary, this strategy to construct novel primers reduced the chance of cross amplification and was able to identify the desired strain. The various strains isolated in this study can be used to develop a sourdough starter after the analysis of their fermentation characteristics.

  5. A multiplex set of species-specific primers for rapid identification of members of the genus Saccharomyces.

    PubMed

    Muir, Alastair; Harrison, Elizabeth; Wheals, Alan

    2011-11-01

    The Saccharomyces genus (previously Saccharomyces sensu stricto) formally comprises Saccharomyces arboricola, Saccharomyces bayanus, Saccharomyces cariocanus, Saccharomyces cerevisiae, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces paradoxus and Saccharomyces pastorianus. Species-specific primer pairs that produce a single band of known and different product size have been developed for each member of the clade with the exception of S. pastorianus, which is a polyphyletic allopolyploid hybrid only found in lager breweries, and for which signature sequences could not be reliably created. Saccharomyces cariocanus is now regarded as an American variant of S. paradoxus, and accordingly a single primer pair that recognizes both species was developed. A different orthologous and essential housekeeping gene was used to detect each species, potentially avoiding competition between PCR primers and overlap between amplicons. In multiplex format, two or more different species could be identified in a single reaction; double and triple hybrids could not always be correctly identified. Forty-two unidentified yeasts from sugar cane juice fermentations were correctly identified as S. cerevisiae. A colony PCR method was developed that is rapid, robust, inexpensive and capable of automation, requires no mycological expertise on the part of the user and is thus useful for large-scale preliminary screens.

  6. A multiplex set of species-specific primers for rapid identification of members of the genus Saccharomyces.

    PubMed

    Muir, Alastair; Harrison, Elizabeth; Wheals, Alan

    2011-11-01

    The Saccharomyces genus (previously Saccharomyces sensu stricto) formally comprises Saccharomyces arboricola, Saccharomyces bayanus, Saccharomyces cariocanus, Saccharomyces cerevisiae, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces paradoxus and Saccharomyces pastorianus. Species-specific primer pairs that produce a single band of known and different product size have been developed for each member of the clade with the exception of S. pastorianus, which is a polyphyletic allopolyploid hybrid only found in lager breweries, and for which signature sequences could not be reliably created. Saccharomyces cariocanus is now regarded as an American variant of S. paradoxus, and accordingly a single primer pair that recognizes both species was developed. A different orthologous and essential housekeeping gene was used to detect each species, potentially avoiding competition between PCR primers and overlap between amplicons. In multiplex format, two or more different species could be identified in a single reaction; double and triple hybrids could not always be correctly identified. Forty-two unidentified yeasts from sugar cane juice fermentations were correctly identified as S. cerevisiae. A colony PCR method was developed that is rapid, robust, inexpensive and capable of automation, requires no mycological expertise on the part of the user and is thus useful for large-scale preliminary screens. PMID:22093682

  7. Trans-specific evolution of opsin alleles and the maintenance of trichromatic colour vision in Callitrichine primates.

    PubMed

    Surridge, Alison K; Mundy, Nicholas I

    2002-10-01

    Many New World (NW) primates possess a remarkable polymorphism in an X-linked locus, which encodes for the visual pigments (opsins) used for colour vision. Females that are heterozygous for opsin alleles of different spectral sensitivity at this locus have trichromatic colour vision, whereas homozygous females and males are dichromatic, with poor colour discrimination in the red-green range. Here we describe an extensive survey of allelic variation in both exons and introns at this locus within and among species of the Callitrichines (marmosets and tamarins). All five genera of Callitrichines have the X-linked polymorphism, and only the three functional allelic classes described previously (with maximum wavelength sensitivities at about 543 nm, 556 nm and 563 nm) were found among the 16 species and 233 or more X-chromosomes sampled. In spite of the homogenizing effects of gene conversion, phylogenetic analyses provide direct evidence for trans-specific evolution of alleles over time periods of at least 5-6 million years, and up to 14 million years (estimated from independent phylogenies). These conclusions are supported by the distribution of insertions and deletions in introns. The maintenance of polymorphism over these time periods requires an adaptive explanation, which must involve a heterozygote advantage for trichromats. The lack of detection of alleles that are recombinant for spectral sensitivity suggests that such alleles are suboptimal. The two main hypotheses for the selective advantage of trichromacy in primates are frugivory for ripe fruits and folivory for young leaves. The latter can be discounted in Callitrichines, as they are not folivorous. PMID:12296957

  8. Trans-specific evolution of opsin alleles and the maintenance of trichromatic colour vision in Callitrichine primates.

    PubMed

    Surridge, Alison K; Mundy, Nicholas I

    2002-10-01

    Many New World (NW) primates possess a remarkable polymorphism in an X-linked locus, which encodes for the visual pigments (opsins) used for colour vision. Females that are heterozygous for opsin alleles of different spectral sensitivity at this locus have trichromatic colour vision, whereas homozygous females and males are dichromatic, with poor colour discrimination in the red-green range. Here we describe an extensive survey of allelic variation in both exons and introns at this locus within and among species of the Callitrichines (marmosets and tamarins). All five genera of Callitrichines have the X-linked polymorphism, and only the three functional allelic classes described previously (with maximum wavelength sensitivities at about 543 nm, 556 nm and 563 nm) were found among the 16 species and 233 or more X-chromosomes sampled. In spite of the homogenizing effects of gene conversion, phylogenetic analyses provide direct evidence for trans-specific evolution of alleles over time periods of at least 5-6 million years, and up to 14 million years (estimated from independent phylogenies). These conclusions are supported by the distribution of insertions and deletions in introns. The maintenance of polymorphism over these time periods requires an adaptive explanation, which must involve a heterozygote advantage for trichromats. The lack of detection of alleles that are recombinant for spectral sensitivity suggests that such alleles are suboptimal. The two main hypotheses for the selective advantage of trichromacy in primates are frugivory for ripe fruits and folivory for young leaves. The latter can be discounted in Callitrichines, as they are not folivorous.

  9. Allele-Specific Transcriptome and Methylome Analysis Reveals Stable Inheritance and Cis-Regulation of DNA Methylation in Nasonia

    PubMed Central

    Wang, Xu; Clark, Andrew G.

    2016-01-01

    Gene expression divergence between closely related species could be attributed to both cis- and trans- DNA sequence changes during evolution, but it is unclear how the evolutionary dynamics of epigenetic marks are regulated. In eutherian mammals, biparental DNA methylation marks are erased and reset during gametogenesis, resulting in paternal or maternal imprints, which lead to genomic imprinting. Whether DNA methylation reprogramming exists in insects is not known. Wasps of the genus Nasonia are non-social parasitoids that are emerging as a model for studies of epigenetic processes in insects. In this study, we quantified allele-specific expression and methylation genome-wide in Nasonia vitripennis and Nasonia giraulti and their reciprocal F1 hybrids. No parent-of-origin effect in allelic expression was found for >8,000 covered genes, suggesting a lack of genomic imprinting in adult Nasonia. As we expected, both significant cis- and trans- effects are responsible for the expression divergence between N. vitripennis and N. giraulti. Surprisingly, all 178 differentially methylated genes are also differentially methylated between the two alleles in F1 hybrid offspring, recapitulating the parental methylation status with nearly 100% fidelity, indicating the presence of strong cis-elements driving the target of gene body methylation. In addition, we discovered that total and allele-specific expression are positively correlated with allele-specific methylation in a subset of the differentially methylated genes. The 100% cis-regulation in F1 hybrids suggests the methylation machinery is conserved and DNA methylation is targeted by cis features in Nasonia. The lack of genomic imprinting and parent-of-origin differentially methylated regions in Nasonia, together with the stable inheritance of methylation status between generations, suggests either a cis-regulatory motif for methylation at the DNA level or highly stable inheritance of an epigenetic signal in Nasonia. PMID

  10. Allele-Specific Transcriptome and Methylome Analysis Reveals Stable Inheritance and Cis-Regulation of DNA Methylation in Nasonia.

    PubMed

    Wang, Xu; Werren, John H; Clark, Andrew G

    2016-07-01

    Gene expression divergence between closely related species could be attributed to both cis- and trans- DNA sequence changes during evolution, but it is unclear how the evolutionary dynamics of epigenetic marks are regulated. In eutherian mammals, biparental DNA methylation marks are erased and reset during gametogenesis, resulting in paternal or maternal imprints, which lead to genomic imprinting. Whether DNA methylation reprogramming exists in insects is not known. Wasps of the genus Nasonia are non-social parasitoids that are emerging as a model for studies of epigenetic processes in insects. In this study, we quantified allele-specific expression and methylation genome-wide in Nasonia vitripennis and Nasonia giraulti and their reciprocal F1 hybrids. No parent-of-origin effect in allelic expression was found for >8,000 covered genes, suggesting a lack of genomic imprinting in adult Nasonia. As we expected, both significant cis- and trans- effects are responsible for the expression divergence between N. vitripennis and N. giraulti. Surprisingly, all 178 differentially methylated genes are also differentially methylated between the two alleles in F1 hybrid offspring, recapitulating the parental methylation status with nearly 100% fidelity, indicating the presence of strong cis-elements driving the target of gene body methylation. In addition, we discovered that total and allele-specific expression are positively correlated with allele-specific methylation in a subset of the differentially methylated genes. The 100% cis-regulation in F1 hybrids suggests the methylation machinery is conserved and DNA methylation is targeted by cis features in Nasonia. The lack of genomic imprinting and parent-of-origin differentially methylated regions in Nasonia, together with the stable inheritance of methylation status between generations, suggests either a cis-regulatory motif for methylation at the DNA level or highly stable inheritance of an epigenetic signal in Nasonia. PMID

  11. Evaluation of human immunodeficiency virus type 1 reverse transcriptase primer tRNA binding by fluorescence spectroscopy: specificity and comparison to primer/template binding.

    PubMed

    Thrall, S H; Reinstein, J; Wöhrl, B M; Goody, R S

    1996-04-01

    A host cell-derived tRNA3Lys molecule is utilized by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to prime DNA synthesis from the viral RNA genome. We performed fluorescence titration experiments to characterize the interaction between RT and its natural primer, tRNA3Lys, and to address RT's putative role in the required and specific packaging of tRNA3Lys into the budding virus. Titration of RT with tRNA3Lys resulted in a 30% maximal quenching of RT tryptophan fluorescence, from which a dissociation constant (Kd) of 57.6 +/- 7.5 nM was derived. Titration of RT with Escherichia coli tRNA2Glu, E. coli tRNA2Tyr, E. coli tRNALys, yeast tRNAPhe, or in vitro-synthesized human tRNA3Lys (no base modifications) resulted in similar fluorescence changes and Kd values as obtained for the natural tRNA3Lys. The specific interaction between RT and tRNA3Lys during viral assembly suggested by previous in vivo studies is therefore not present in the fully processed, in vitro form of RT. Other factors during viral assembly must therefore cooperate in the packaging of tRNA3Lys. The nonspecific and ionic strength dependent RT-tRNA interaction detected in the present studies suggests that the overall shape and charges of tRNA constitute recognition features for RT binding. The fluorescence of the wyebutine base contained on the anticodon loop of yeast tRNAPhe was found to increase upon RT binding, supporting speculation that RT interacts with the anticodon loop of tRNA. The individual tRNAs also displaced a fluorescent DNA primer/template (p/t) substrate from RT, indicating overlapping tRNA and p/t binding sites. Cubic fit evaluation of the displacement titrations allowed further assessment of the affinities of the two competing ligands. The presence of both overlapping and separate p/t and tRNA binding regions on RT was tested by examination of the affinity of a possible RT bisubstrate type inhibitor, containing motifs proposed to be essential for both t

  12. Different aubergine alleles confirm the specificity of different RNAi pathways in Drosophila melanogaster.

    PubMed

    Specchia, Valeria; Bozzetti, Maria Pia

    2009-01-01

    The crystal-Stellate system is one of the best-known examples of heterochromatin-euchromatin interaction. The components of this system are homologous repetitive sequences clustered in three regions: 12E1 and h27 on the X and h11 on the Y. The symptom of a disrupted crystal-Stellate interaction is the presence of crystals in the spermatocytes of males lacking the crystal region. Stellate silencing is based on the RNAi process. Many modifiers of this system have been isolated and many of these are involved in RNAi. One of these modifiers is aubergine(sting); this is a "gain of function" allele in somatic tissues. Here we report the different behavior of two aubergine alleles with respect to the RNAi pathway: aub(sting) and a "loss of function" heteroallelic combination aub(HN)/aub(QC42). An increased amount of Aub interferes with the correct functioning of the somatic yellow hairpin RNAi, whereas the Aub reduction does not. We also demonstrate the different behavior of these alleles on the I transposon silencing in ovaries. Intriguingly, neither of these aubergine alleles silence the Stellate locus. We can conclude that the crystal-Stellate system reveals different RNAi pathways even though much still remains to be done to completely explain the molecular bases of the crystal-Stellate interaction. PMID:19242123

  13. Allele-specific down-regulation of RPTOR expression induced by retinoids contributes to climate adaptations.

    PubMed

    Sun, Chang; Southard, Catherine; Witonsky, David B; Kittler, Ralf; Di Rienzo, Anna

    2010-10-01

    The mechanistic target of rapamycin (MTOR) pathway regulates cell growth, energy homeostasis, apoptosis, and immune response. The regulatory associated protein of MTOR encoded by the RPTOR gene is a key component of this pathway. A previous survey of candidate genes found that RPTOR contains multiple SNPs with strong correlations between allele frequencies and climate variables, consistent with the action of selective pressures that vary across environments. Using data from a recent genome scan for selection signals, we honed in on a SNP (rs11868112) 26 kb upstream to the transcription start site of RPTOR that exhibits the strongest association with temperature variables. Transcription factor motif scanning and mining of recently mapped transcription factor binding sites identified a binding site for POU class 2 homeobox 1 (POU2F1) spanning the SNP and an adjacent retinoid acid receptor (RAR) binding site. Using expression quantification, chromatin immunoprecipitation (ChIP), and reporter gene assays, we demonstrate that POU2F1 and RARA do bind upstream of the RPTOR gene to regulate its expression in response to retinoids; this regulation is affected by the allele status at rs11868112 with the derived allele resulting in lower expression levels. We propose a model in which the derived allele influences thermogenesis or immune response by altering MTOR pathway activity and thereby increasing fitness in colder climates. Our results show that signatures of genetic adaptations can identify variants with functional effects, consistent with the idea that selection signals may be used for SNP annotation.

  14. Visualizing allele-specific expression in single cells reveals epigenetic mosaicism in an H19 loss-of-imprinting mutant

    PubMed Central

    Ginart, Paul; Kalish, Jennifer M.; Jiang, Connie L.; Yu, Alice C.; Bartolomei, Marisa S.; Raj, Arjun

    2016-01-01

    Imprinting is a classic mammalian epigenetic phenomenon that results in expression from a single parental allele. Imprinting defects can lead to inappropriate expression from the normally silenced allele, but it remains unclear whether every cell in a mutant organism follows the population average, which would have profound implications for human imprinting disorders. Here, we apply a new fluorescence in situ hybridization method that measures allele-specific expression in single cells to address this question in mutants exhibiting aberrant H19/Igf2 (insulin-like growth factor 2) imprinting. We show that mutant primary embryonic mouse fibroblasts are comprised of two subpopulations: one expressing both H19 alleles and another expressing only the maternal copy. Only in the latter cell population is Igf2 expression detected. Furthermore, the two subpopulations are stable in that cells do not interconvert between the two expression patterns. Combined small input methylation analysis and transcriptional imaging revealed that these two mutant subpopulations exhibit distinct methylation patterns at their imprinting control regions. Consistently, pharmacological inhibition of DNA methylation reduced the proportion of monoallelic cells. Importantly, we observed that the same two subpopulations are also present in vivo within murine cardiac tissue. Our results establish that imprinting disorders can display striking single-cell heterogeneity in their molecular phenotypes and suggest that such heterogeneity may underlie epigenetic mosaicism in human imprinting disorders. PMID:26944681

  15. Visualizing allele-specific expression in single cells reveals epigenetic mosaicism in an H19 loss-of-imprinting mutant.

    PubMed

    Ginart, Paul; Kalish, Jennifer M; Jiang, Connie L; Yu, Alice C; Bartolomei, Marisa S; Raj, Arjun

    2016-03-01

    Imprinting is a classic mammalian epigenetic phenomenon that results in expression from a single parental allele. Imprinting defects can lead to inappropriate expression from the normally silenced allele, but it remains unclear whether every cell in a mutant organism follows the population average, which would have profound implications for human imprinting disorders. Here, we apply a new fluorescence in situ hybridization method that measures allele-specific expression in single cells to address this question in mutants exhibiting aberrant H19/Igf2 (insulin-like growth factor 2) imprinting. We show that mutant primary embryonic mouse fibroblasts are comprised of two subpopulations: one expressing both H19 alleles and another expressing only the maternal copy. Only in the latter cell population is Igf2 expression detected. Furthermore, the two subpopulations are stable in that cells do not interconvert between the two expression patterns. Combined small input methylation analysis and transcriptional imaging revealed that these two mutant subpopulations exhibit distinct methylation patterns at their imprinting control regions. Consistently, pharmacological inhibition of DNA methylation reduced the proportion of monoallelic cells. Importantly, we observed that the same two subpopulations are also present in vivo within murine cardiac tissue. Our results establish that imprinting disorders can display striking single-cell heterogeneity in their molecular phenotypes and suggest that such heterogeneity may underlie epigenetic mosaicism in human imprinting disorders.

  16. Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients.

    PubMed

    Skotte, Niels H; Southwell, Amber L; Østergaard, Michael E; Carroll, Jeffrey B; Warby, Simon C; Doty, Crystal N; Petoukhov, Eugenia; Vaid, Kuljeet; Kordasiewicz, Holly; Watt, Andrew T; Freier, Susan M; Hung, Gene; Seth, Punit P; Bennett, C Frank; Swayze, Eric E; Hayden, Michael R

    2014-01-01

    Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.

  17. Allele-Specific Suppression of Mutant Huntingtin Using Antisense Oligonucleotides: Providing a Therapeutic Option for All Huntington Disease Patients

    PubMed Central

    Skotte, Niels H.; Southwell, Amber L.; Østergaard, Michael E.; Carroll, Jeffrey B.; Warby, Simon C.; Doty, Crystal N.; Petoukhov, Eugenia; Vaid, Kuljeet; Kordasiewicz, Holly; Watt, Andrew T.; Freier, Susan M.; Hung, Gene; Seth, Punit P.; Bennett, C. Frank; Swayze, Eric E.; Hayden, Michael R.

    2014-01-01

    Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder. PMID:25207939

  18. FACETS: allele-specific copy number and clonal heterogeneity analysis tool for high-throughput DNA sequencing.

    PubMed

    Shen, Ronglai; Seshan, Venkatraman E

    2016-09-19

    Allele-specific copy number analysis (ASCN) from next generation sequencing (NGS) data can greatly extend the utility of NGS beyond the identification of mutations to precisely annotate the genome for the detection of homozygous/heterozygous deletions, copy-neutral loss-of-heterozygosity (LOH), allele-specific gains/amplifications. In addition, as targeted gene panels are increasingly used in clinical sequencing studies for the detection of 'actionable' mutations and copy number alterations to guide treatment decisions, accurate, tumor purity-, ploidy- and clonal heterogeneity-adjusted integer copy number calls are greatly needed to more reliably interpret NGS-based cancer gene copy number data in the context of clinical sequencing. We developed FACETS, an ASCN tool and open-source software with a broad application to whole genome, whole-exome, as well as targeted panel sequencing platforms. It is a fully integrated stand-alone pipeline that includes sequencing BAM file post-processing, joint segmentation of total- and allele-specific read counts, and integer copy number calls corrected for tumor purity, ploidy and clonal heterogeneity, with comprehensive output and integrated visualization. We demonstrate the application of FACETS using The Cancer Genome Atlas (TCGA) whole-exome sequencing of lung adenocarcinoma samples. We also demonstrate its application to a clinical sequencing platform based on a targeted gene panel.

  19. FACETS: allele-specific copy number and clonal heterogeneity analysis tool for high-throughput DNA sequencing

    PubMed Central

    Shen, Ronglai; Seshan, Venkatraman E.

    2016-01-01

    Allele-specific copy number analysis (ASCN) from next generation sequencing (NGS) data can greatly extend the utility of NGS beyond the identification of mutations to precisely annotate the genome for the detection of homozygous/heterozygous deletions, copy-neutral loss-of-heterozygosity (LOH), allele-specific gains/amplifications. In addition, as targeted gene panels are increasingly used in clinical sequencing studies for the detection of ‘actionable’ mutations and copy number alterations to guide treatment decisions, accurate, tumor purity-, ploidy- and clonal heterogeneity-adjusted integer copy number calls are greatly needed to more reliably interpret NGS-based cancer gene copy number data in the context of clinical sequencing. We developed FACETS, an ASCN tool and open-source software with a broad application to whole genome, whole-exome, as well as targeted panel sequencing platforms. It is a fully integrated stand-alone pipeline that includes sequencing BAM file post-processing, joint segmentation of total- and allele-specific read counts, and integer copy number calls corrected for tumor purity, ploidy and clonal heterogeneity, with comprehensive output and integrated visualization. We demonstrate the application of FACETS using The Cancer Genome Atlas (TCGA) whole-exome sequencing of lung adenocarcinoma samples. We also demonstrate its application to a clinical sequencing platform based on a targeted gene panel. PMID:27270079

  20. FACETS: allele-specific copy number and clonal heterogeneity analysis tool for high-throughput DNA sequencing.

    PubMed

    Shen, Ronglai; Seshan, Venkatraman E

    2016-09-19

    Allele-specific copy number analysis (ASCN) from next generation sequencing (NGS) data can greatly extend the utility of NGS beyond the identification of mutations to precisely annotate the genome for the detection of homozygous/heterozygous deletions, copy-neutral loss-of-heterozygosity (LOH), allele-specific gains/amplifications. In addition, as targeted gene panels are increasingly used in clinical sequencing studies for the detection of 'actionable' mutations and copy number alterations to guide treatment decisions, accurate, tumor purity-, ploidy- and clonal heterogeneity-adjusted integer copy number calls are greatly needed to more reliably interpret NGS-based cancer gene copy number data in the context of clinical sequencing. We developed FACETS, an ASCN tool and open-source software with a broad application to whole genome, whole-exome, as well as targeted panel sequencing platforms. It is a fully integrated stand-alone pipeline that includes sequencing BAM file post-processing, joint segmentation of total- and allele-specific read counts, and integer copy number calls corrected for tumor purity, ploidy and clonal heterogeneity, with comprehensive output and integrated visualization. We demonstrate the application of FACETS using The Cancer Genome Atlas (TCGA) whole-exome sequencing of lung adenocarcinoma samples. We also demonstrate its application to a clinical sequencing platform based on a targeted gene panel. PMID:27270079

  1. Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients.

    PubMed

    Skotte, Niels H; Southwell, Amber L; Østergaard, Michael E; Carroll, Jeffrey B; Warby, Simon C; Doty, Crystal N; Petoukhov, Eugenia; Vaid, Kuljeet; Kordasiewicz, Holly; Watt, Andrew T; Freier, Susan M; Hung, Gene; Seth, Punit P; Bennett, C Frank; Swayze, Eric E; Hayden, Michael R

    2014-01-01

    Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder. PMID:25207939

  2. Linear-After-The-Exponential (LATE)-PCR: primer design criteria for high yields of specific single-stranded DNA and improved real-time detection.

    PubMed

    Pierce, Kenneth E; Sanchez, J Aquiles; Rice, John E; Wangh, Lawrence J

    2005-06-14

    Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations. The present report systematically examines the primer design parameters that affect the exponential and linear phases of LATE-PCR amplification. In particular, we investigated how altering the concentration-adjusted melting temperature (Tm) of the limiting primer (TmL) relative to that of the excess primer (TmX) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when TmL - TmX 5 degrees C. We also investigated how altering TmX relative to the higher Tm of the double-stranded amplicon (TmA) affects the rate and extent of linear amplification. Excess primers with TmX closer to TmA yielded higher rates of linear amplification and stronger signals from a hybridization probe. These design criteria maximize the yield of specific single-stranded DNA products and make LATE-PCR more robust and easier to implement. The conclusions were validated by using primer pairs that amplify sequences within the cystic fibrosis transmembrane regulator (CFTR) gene, mutations of which are responsible for cystic fibrosis.

  3. Prey choice by carabid beetles feeding on an earthworm community analysed using species- and lineage-specific PCR primers.

    PubMed

    King, R Andrew; Vaughan, Ian P; Bell, James R; Bohan, David A; Symondson, William O C

    2010-04-01

    The carabid beetle Pterostichus melanarius is a major natural enemy of pests, such as aphids and slugs in agricultural systems. Earthworms are a dominant non-pest component of the diet of P. melanarius which help sustain the beetles during periods when the pest population is low or absent. In this study we wanted to test whether this predator exercises prey choice among different earthworm species or ecological groups. High levels of genetic diversity within morphological species of earthworm necessitated the development of primers that were specific not just to species but lineages and sub-lineages within species as well. Gut samples from beetles were analysed using multiplex-PCR and fluorescent-labelled primers. Calibratory feeding trials were undertaken to calculate median detection times for prey DNA following ingestion. Extensive testing demonstrated that the primers were species-specific, that detection periods were negatively related to amplicon size and that meal size had a highly significant effect on detection periods. Monte Carlo simulations showed that, in general, worms were being predated in proportion to their densities in the field with little evidence of prey choice, other than probable avoidance of the larger, deep-living species. There was no evidence that epigeic species were being taken preferentially in comparison with endogeic species. There was also no evidence that defensive secretions by Allolobophora chlorotica reduced predation pressure on this species by P. melanarius. We concluded that any management system that increases earthworm densities generally, regardless of component species, is likely to be optimal for increasing numbers of this beneficial beetle predator. PMID:20345680

  4. Dr(a-) polymorphism of decay accelerating factor. Biochemical, functional, and molecular characterization and production of allele-specific transfectants.

    PubMed Central

    Lublin, D M; Thompson, E S; Green, A M; Levene, C; Telen, M J

    1991-01-01

    The Dra antigen belongs to the Cromer-related blood group system, a series of antigens on decay accelerating factor (DAF), a glycosyl-phosphatidylinositol-anchored membrane protein that protects host cells from complement-mediated damage. We studied the rare inherited Dr(a-) phenotype to ascertain the associated biochemical and functional changes in DAF and to characterize the basis for this polymorphism. Radioimmunoassay assay and flow cytometric analysis of Dr(a-) erythrocytes demonstrated 40% of normal surface expression of DAF but normal levels of several other glycosyl-phosphatidylinositol-anchored proteins, distinguishing this phenotype from that of paroxysmal nocturnal hemoglobinuria. Western blots confirmed this reduced DAF expression and indicated a slightly faster mobility of the molecule on SDS-PAGE. Despite the reduced DAF expression, Dr(a-) erythrocytes functioned normally in the complement lysis sensitivity assay. Utilization of the polymerase chain reaction to amplify mononuclear cell genomic DNA from three unrelated Dr(a-) individuals demonstrated that a point mutation underlies the Dr(a-) phenotype: a C to T change in nucleotide 649 resulting in a serine165 to leucine change. This defines the Drb allele of DAF, which can be distinguished from Dra by a Taq I restriction fragment length polymorphism. We created transfected Chinese hamster ovary cell lines expressing either the Dra or the Drb allelic form of DAF. These allele-specific transfectants were tested by inhibition of hemagglutination or flow cytometry and confirmed the specificity of anti-Dra alloantisera. The allele-specific transfectants could form the basis of a new serological approach to immunohematology. Images PMID:1710232

  5. Development of Primers to O-Antigen Biosynthesis Genes for Specific Detection of Escherichia coli O157 by PCR

    PubMed Central

    Maurer, John J.; Schmidt, Denise; Petrosko, Patricia; Sanchez, Susan; Bolton, Lance; Lee, Margie D.

    1999-01-01

    The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157. PMID:10388689

  6. Revealing the Diversity and Quantity of Peritrich Ciliates in Environmental Samples Using Specific Primer-based PCR and Quantitative PCR

    PubMed Central

    Liu, Xihan; Gong, Jun

    2012-01-01

    Peritrichs are a diverse, ecologically important ciliate group usually with a complex life cycle. To date, the community of the peritrichs has been investigated by using morphology-based methods such as living observation and silver staining. Here we show a molecular approach for characterizing the diversity and quantity of free-living peritrichs in environmental samples. We newly designed four peritrich-specific primers targeting 18S rRNA genes that allow clone library construction, screening and analysis. A quantitative real-time PCR (qPCR) assay was developed to quantify peritrichs in environmental samples by using rDNA copy number as an indicator. DNA extracted from four water samples of contrasting environmental gradients was analysed. The results showed that the peritrich community was differentiated among these samples, and that the diversity decreased with the increase of water salinity. The qPCR results are consistent with the library sequence analysis in terms of quantity variations from sample to sample. The development of peritrich-specific primers, for the first time, for conventional PCR and qPCR assays, provides useful molecular tools for revealing the diversity and quantity of peritrich ciliates in environmental samples. Also, our study illustrates the potential of these molecular tools to ecological studies of other ciliate groups in diverse environments. PMID:23100023

  7. TESTING THE SPECIFICITY OF PRIMERS TO ENVIRONMENTAL AMMONIA MONOOXYGENASE (AMOA) GENES IN GROUNDWATER TREATED WITH UREA TO PROMOTE CALCITE PRECIPITATION

    SciTech Connect

    Stephanie Freeman; David Reed; Yoshiko Fujita

    2006-12-01

    The diversity of bacterial ammonia monooxygenase (amoA) genes in DNA isolated from microorganisms in groundwater was characterized by amplification of amoA DNA using polymerase chain reaction (PCR), Restriction Fragment Length Polymorphism (RFLP) analysis, and sequencing. The amoA gene is characteristic of ammonia oxidizing bacteria (AOB). The DNA extracts were acquired from an experiment where dilute molasses and urea were sequentially introduced into a well in the Eastern Snake River Plain Aquifer (ESRPA) in Idaho to examine whether such amendments could stimulate enhanced ureolytic activity. The hydrolysis of urea into ammonium and carbonate serves as the basis for a potential remediation technique for trace metals and radionuclide contaminants that co-precipitate in calcite. The ammonium ion resulting from ureolysis can promote the growth of AOB. The goal of this work was to investigate the effectiveness of primers designed for quantitative PCR of environmental amoA genes and to evaluate the effect of the molasses and urea amendments upon the population diversity of groundwater AOB. PCR primers designed to target a portion of the amoA gene were used to amplify amoA gene sequences in the groundwater DNA extracts. Following PCR, amplified gene products were cloned and the clones were characterized by RFLP, a DNA restriction technique that can distinguish different DNA sequences, to gauge the initial diversity. Clones exhibiting unique RFLP patterns were subjected to DNA sequencing. Initial sequencing results suggest that the primers were successful at specific detection of amoA sequences and the RFLP analyses indicated that the diversity of detected amoA sequences in the ESRPA decreased with the additions of molasses and urea.

  8. A novel type 2 diabetes risk allele increases the promoter activity of the muscle-specific small ankyrin 1 gene

    PubMed Central

    Yan, Rengna; Lai, Shanshan; Yang, Yang; Shi, Hongfei; Cai, Zhenming; Sorrentino, Vincenzo; Du, Hong; Chen, Huimei

    2016-01-01

    Genome-wide association studies have identified Ankyrin-1 (ANK1) as a common type 2 diabetes (T2D) susceptibility locus. However, the underlying causal variants and functional mechanisms remain unknown. We screened for 8 tag single nucleotide polymorphisms (SNPs) in ANK1 between 2 case-control studies. Genotype analysis revealed significant associations of 3 SNPs, rs508419 (first identified here), rs515071, and rs516946 with T2D (P < 0.001). These SNPs were in linkage disequilibrium (r2 > 0.80); subsequent analysis indicated that the CCC haplotype associated with increased T2D susceptibility (OR 1.447, P < 0.001). Further mapping showed that rs508419 resides in the muscle-specific ANK1 gene promoter. Allele-specific mRNA and protein level measurements confirmed association of the C allele with increased small ANK1 (sAnk1) expression in human skeletal muscle (P = 0.018 and P < 0.001, respectively). Luciferase assays showed increased rs508419-C allele transcriptional activity in murine skeletal muscle C2C12 myoblasts, and electrophoretic mobility-shift assays demonstrated altered rs508419 DNA-protein complex formation. Glucose uptake was decreased with excess sAnk1 expression upon insulin stimulation. Thus, the ANK1 rs508419-C T2D-risk allele alters DNA-protein complex binding leading to increased promoter activity and sAnk1 expression; thus, increased sAnk1 expression in skeletal muscle might contribute to T2D susceptibility. PMID:27121283

  9. Validation of genome-wide association study (GWAS)-identified disease risk alleles with patient-specific stem cell lines

    PubMed Central

    Yang, Jin; Li, Yao; Chan, Lawrence; Tsai, Yi-Ting; Wu, Wen-Hsuan; Nguyen, Huy V.; Hsu, Chun-Wei; Li, Xiaorong; Brown, Lewis M.; Egli, Dieter; Sparrow, Janet R.; Tsang, Stephen H.

    2014-01-01

    While the past decade has seen great progress in mapping loci for common diseases, studying how these risk alleles lead to pathology remains a challenge. Age-related macular degeneration (AMD) affects 9 million older Americans, and is characterized by the loss of the retinal pigment epithelium (RPE). Although the closely linked genome-wide association studies ARMS2/HTRA1 genes, located at the chromosome 10q26 locus, are strongly associated with the risk of AMD, their downstream targets are unknown. Low population frequencies of risk alleles in tissue banks make it impractical to study their function in cells derived from autopsied tissue. Moreover, autopsy eyes from end-stage AMD patients, where age-related RPE atrophy and fibrosis are already present, cannot be used to determine how abnormal ARMS2/HTRA1 expression can initiate RPE pathology. Instead, induced pluripotent stem (iPS) cell-derived RPE from patients provides us with earlier stage AMD patient-specific cells and allows us to analyze the underlying mechanisms at this critical time point. An unbiased proteome screen of A2E-aged patient-specific iPS-derived RPE cell lines identified superoxide dismutase 2 (SOD2)-mediated antioxidative defense in the genetic allele's susceptibility of AMD. The AMD-associated risk haplotype (T-in/del-A) impairs the ability of the RPE to defend against aging-related oxidative stress. SOD2 defense is impaired in RPE homozygous for the risk haplotype (T-in/del-A; T-in/del-A), while the effect was less pronounced in RPE homozygous for the protective haplotype (G–Wt–G; G–Wt–G). ARMS2/HTRA1 risk alleles decrease SOD2 defense, making RPE more susceptible to oxidative damage and thereby contributing to AMD pathogenesis. PMID:24497574

  10. Use of 16S rRNA Gene-Targeted Group-Specific Primers for Real-Time PCR Analysis of Predominant Bacteria in Mouse Feces.

    PubMed

    Yang, Yun-Wen; Chen, Mang-Kun; Yang, Bing-Ya; Huang, Xian-Jie; Zhang, Xue-Rui; He, Liang-Qiang; Zhang, Jing; Hua, Zi-Chun

    2015-10-01

    Mouse models are widely used for studying gastrointestinal (GI) tract-related diseases. It is necessary and important to develop a new set of primers to monitor the mouse gut microbiota. In this study, 16S rRNA gene-targeted group-specific primers for Firmicutes, Actinobacteria, Bacteroidetes, Deferribacteres, "Candidatus Saccharibacteria," Verrucomicrobia, Tenericutes, and Proteobacteria were designed and validated for quantification of the predominant bacterial species in mouse feces by real-time PCR. After confirmation of their accuracy and specificity by high-throughput sequencing technologies, these primers were applied to quantify the changes in the fecal samples from a trinitrobenzene sulfonic acid-induced colitis mouse model. Our results showed that this approach efficiently predicted the occurrence of colitis, such as spontaneous chronic inflammatory bowel disease in transgenic mice. The set of primers developed in this study provides a simple and affordable method to monitor changes in the intestinal microbiota at the phylum level. PMID:26187967

  11. Single primer amplification reaction (SPAR) reveals inter- and intra-specific natural genetic variation in five species of Cymbidium (Orchidaceae).

    PubMed

    Sharma, Santosh Kumar; Kumaria, Suman; Tandon, Pramod; Rao, Satyawada Rama

    2011-09-01

    A total of 53 primers belonging to three SPAR methods, viz. RAPD, ISSR and DAMD, collectively produced 456 polymorphic amplicons with 96.6% polymorphism at inter-specific level in five species of Cymbidium, viz. C. aloifolium, C. mastersii, C. elegans, C. eburneum and C. tigrinum, whereas at intra-specific level, the observed polymorphism ranged from 51.2% to 77.1% among them. Three SPARs collectively revealed 25 unique species-specific amplicons; most of them were amplified with RAPD and DAMD primers besides few bands which were either missed (absent) or lost (heterozygosity). UPGMA clustering evidently distinguished the representatives of C. aloifolium and C. tigrinum, with distinct genetic distance, which may be due to their entirely different habitats as well as discrete morphological characteristics. Upon analysis of the data generated, all the three SPAR methods, either independently and/or in combination, revealed wide range of genetic variation between and within five species of Cymbidium. Comparison of matrix of individual SPAR method revealed that analysis of natural genetic variation using combination of SPAR methods, rather than an isolated approach, is highly effective. The critical analyses of the amplicon data are indicative of DAMD as the most powerful SPAR method by showing highest resolving power (Rp) followed by ISSR and RAPD. Alternatively, the total polymorphic information content was highest in case of RAPD followed by other two SPAR methods. Thus, the present investigation for the first time provides a valuable baseline data for genetic variation at inter- and intra-specific levels in horticultural Cymbidiums and also addresses conservation concerns.

  12. Compensatory embryonic response to allele-specific inactivation of the murine X-linked gene Hcfc1.

    PubMed

    Minocha, Shilpi; Sung, Tzu-Ling; Villeneuve, Dominic; Lammers, Fabienne; Herr, Winship

    2016-04-01

    Early in female mammalian embryonic development, cells randomly inactivate one of the two X chromosomes to achieve overall equal inactivation of parental X-linked alleles. Hcfc1 is a highly conserved X-linked mouse gene that encodes HCF-1 - a transcriptional co-regulator implicated in cell proliferation in tissue culture cells. By generating a Cre-recombinase inducible Hcfc1 knock-out (Hcfc1(lox)) allele in mice, we have probed the role of HCF-1 in actively proliferating embryonic cells and in cell-cycle re-entry of resting differentiated adult cells using a liver regeneration model. HCF-1 function is required for both extraembryonic and embryonic development. In heterozygous Hcfc1(lox/+) female embryos, however, embryonic epiblast-specific Cre-induced Hcfc1 deletion (creating an Hcfc1(epiKO) allele) around E5.5 is well tolerated; it leads to a mixture of HCF-1-positive and -negative epiblast cells owing to random X-chromosome inactivation of the wild-type or Hcfc1(epiKO) mutant allele. At E6.5 and E7.5, both HCF-1-positive and -negative epiblast cells proliferate, but gradually by E8.5, HCF-1-negative cells disappear owing to cell-cycle exit and apoptosis. Although generating a temporary developmental retardation, the loss of HCF-1-negative cells is tolerated, leading to viable heterozygous offspring with 100% skewed inactivation of the X-linked Hcfc1(epiKO) allele. In resting adult liver cells, the requirement for HCF-1 in cell proliferation was more evident as hepatocytes lacking HCF-1 fail to re-enter the cell cycle and thus to proliferate during liver regeneration. The survival of the heterozygous Hcfc1(epiKO/+) female embryos, even with half the cells genetically compromised, illustrates the developmental plasticity of the post-implantation mouse embryo - in this instance, permitting survival of females heterozygous for an X-linked embryonic lethal allele.

  13. Organ-specific gene expression in maize: The P-wr allele. Final report, August 15, 1993--August 14, 1996

    SciTech Connect

    Peterson, T.A.

    1997-06-01

    The ultimate aim of our work is to understand how a regulatory gene produces a specific pattern of gene expression during plant development. Our model is the P-wr gene of maize, which produces a distinctive pattern of pigmentation of maize floral organs. We are investigating this system using a combination of classical genetic and molecular approaches. Mechanisms of organ-specific gene expression are a subject of intense research interest, as it is the operation of these mechanisms during eukaryotic development which determine the characteristics of each organism Allele-specific expression has been characterized in only a few other plant genes. In maize, organ-specific pigmentation regulated by the R, B, and Pl genes is achieved by differential transcription of functionally conserved protein coding sequences. Our studies point to a strikingly different mechanism of organ-specific gene expression, involving post-transcriptional regulation of the regulatory P gene. The novel pigmentation pattern of the P-wr allele is associated with differences in the encoded protein. Furthermore, the P-wr gene itself is present as a unique tandemly amplified structure, which may affect its transcriptional regulation.

  14. Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR

    SciTech Connect

    Mesarch, M.B.; Nakatsu, C.H.; Nies, L.

    2000-02-01

    Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primes that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10{sup 2} to 10{sup 3} gene copies, which was lowered to 10{sup 0} to 10{sup 1} gene copies of hybridization. Using the dioxygenase-specific primers, an increase in catechol 2,3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR and a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.

  15. Specific PCR primers directed to identify cryI and cryIII genes within a Bacillus thuringiensis strain collection.

    PubMed Central

    Cerón, J; Ortíz, A; Quintero, R; Güereca, L; Bravo, A

    1995-01-01

    In this paper we describe a PCR strategy that can be used to rapidly identify Bacillus thuringiensis strains that harbor any of the known cryI or cryIII genes. Four general PCR primers which amplify DNA fragments from the known cryI or cryIII genes were selected from conserved regions. Once a strain was identified as an organism that contains a particular type of cry gene, it could be easily characterized by performing additional PCR with specific cryI and cryIII primers selected from variable regions. The method described in this paper can be used to identify the 10 different cryI genes and the five different cryIII genes. One feature of this screening method is that each cry gene is expected to produce a PCR product having a precise molecular weight. The genes which produce PCR products having different sizes probably represent strains that harbor a potentially novel cry gene. Finally, we present evidence that novel crystal genes can be identified by the method described in this paper. PMID:8526493

  16. Isolation of a Pseudomonas solanacearum-specific DNA probe by subtraction hybridization and construction of species-specific oligonucleotide primers for sensitive detection by the polymerase chain reaction.

    PubMed Central

    Seal, S E; Jackson, L A; Daniels, M J

    1992-01-01

    A subtraction hybridization technique was employed to make a library enriched for Pseudomonas solanacearum-specific sequences. One cloned fragment, PS2096, hybridized under stringent conditions to DNA of 82 P. solanacearum strains representing all subgroups of the species. Other plant-associated bacteria, including closely related species such as Pseudomonas capacia, Pseudomonas picketti, or Pseudomonas syzygii, did not hybridize to PS2096. A minimum number of between 4 x 10(5) and 4 x 10(6) P. solanacearum cells could routinely be detected with PS2096 labelled either with [32P]dCTP or with digoxigenin-11-dUTP. To improve the sensitivity of detection, PS2096 was sequenced to allow the construction of specific oligonucleotide primers to be used for polymerase chain reaction (PCR) amplification. After 50 cycles of amplification, 5 to 116 cells, depending on the strain, could reproducibly be detected by visualization of a 148-bp PCR product on an agarose gel. A preliminary field trial in Burundi with the probe and PCR primers has confirmed that they are sensitive tools for specifically detecting low-level infections of P. solanacearum in potato tubers. Images PMID:1482193

  17. Evaluation of sequence-specific priming and real-time polymerase chain reaction assays for detecting HLA-B*51 alleles confirmed by sequence-based typing.

    PubMed

    Park, Y; Kim, Y S; Kim, S I; Kim, H; Kim, H S

    2012-10-01

    The human leukocyte antigen (HLA)-B*51 genotype is one of the well-known genetic factors associated with the development of Behcet's disease. We evaluated three sequence-specific priming (SSP) assays and one real-time PCR assay for detecting HLA-B*51 alleles using 93 whole blood samples, which were genotyped by high-resolution sequence-based typing (SBT). All HLA-B*51 alleles determined by SBT were detected by the four evaluated assays, and the results for all HLA-B alleles other than HLA-B*51 were negative on all assays. Thus, all HLA-B51 tests showed 100% sensitivity and 100% specificity for detecting HLA-B*51 alleles. The three SSP assays and the real-time PCR test for HLA-B*51 genotyping are simple, but reliable for detecting HLA-B*51 alleles in clinical laboratories.

  18. Allele-specific RNA interference rescues the long-QT syndrome phenotype in human-induced pluripotency stem cell cardiomyocytes

    PubMed Central

    Matsa, Elena; Dixon, James E.; Medway, Christopher; Georgiou, Orestis; Patel, Minal J.; Morgan, Kevin; Kemp, Paul J.; Staniforth, Andrew; Mellor, Ian; Denning, Chris

    2014-01-01

    Aims Long-QT syndromes (LQTS) are mostly autosomal-dominant congenital disorders associated with a 1:1000 mutation frequency, cardiac arrest, and sudden death. We sought to use cardiomyocytes derived from human-induced pluripotency stem cells (hiPSCs) as an in vitro model to develop and evaluate gene-based therapeutics for the treatment of LQTS. Methods and results We produced LQTS-type 2 (LQT2) hiPSC cardiomyocytes carrying a KCNH2 c.G1681A mutation in a IKr ion-channel pore, which caused impaired glycosylation and channel transport to cell surface. Allele-specific RNA interference (RNAi) directed towards the mutated KCNH2 mRNA caused knockdown, while leaving the wild-type mRNA unaffected. Electrophysiological analysis of patient-derived LQT2 hiPSC cardiomyocytes treated with mutation-specific siRNAs showed normalized action potential durations (APDs) and K+ currents with the concurrent rescue of spontaneous and drug-induced arrhythmias (presented as early-afterdepolarizations). Conclusions These findings provide in vitro evidence that allele-specific RNAi can rescue diseased phenotype in LQTS cardiomyocytes. This is a potentially novel route for the treatment of many autosomal-dominant-negative disorders, including those of the heart. PMID:23470493

  19. Allele-specific silencing of mutant p53 attenuates dominant-negative and gain-of-function activities

    PubMed Central

    Iyer, Swathi V.; Parrales, Alejandro; Begani, Priya; Narkar, Akshay; Adhikari, Amit S.; Martinez, Luis A.; Iwakuma, Tomoo

    2016-01-01

    Many p53 hotspot mutants not only lose the transcriptional activity, but also show dominant-negative (DN) and oncogenic gain-of-function (GOF) activities. Increasing evidence indicates that knockdown of mutant p53 (mutp53) in cancer cells reduces their aggressive properties, suggesting that survival and proliferation of cancer cells are, at least partially, dependent on the presence of mutp53. However, these p53 siRNAs can downregulate both wild-type p53 (wtp53) and mutp53, which limits their therapeutic applications. In order to specifically deplete mutp53, we have developed allele-specific siRNAs against p53 hotspot mutants and validated their biological effects in the absence or presence of wtp53. First, the mutp53-specific siRNAs selectively reduced protein levels of matched p53 mutants with minimal reduction in wtp53 levels. Second, downregulation of mutp53 in cancer cells expressing a mutp53 alone (p53mut) resulted in significantly decreased cell proliferation and migration. Third, transfection of mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53 also reduced cell proliferation and migration with increased transcripts of p53 downstream target genes, which became further profound when cells were treated with an MDM2 inhibitor Nutlin-3a or a chemotherapeutic agent doxorubicin. These results indicate that depletion of mutp53 by its specific siRNA restored endogenous wtp53 activity in cells expressing both wtp53 and mutp53. This is the first study demonstrating biological effects and therapeutic potential of allele-specific silencing of mutp53 by mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53, thus providing a novel strategy towards targeted cancer therapies. PMID:26700961

  20. Powerful Identification of Cis-regulatory SNPs in Human Primary Monocytes Using Allele-Specific Gene Expression

    PubMed Central

    Almlöf, Jonas Carlsson; Lundmark, Per; Lundmark, Anders; Ge, Bing; Maouche, Seraya; Göring, Harald H. H.; Liljedahl, Ulrika; Enström, Camilla; Brocheton, Jessy; Proust, Carole; Godefroy, Tiphaine; Sambrook, Jennifer G.; Jolley, Jennifer; Crisp-Hihn, Abigail; Foad, Nicola; Lloyd-Jones, Heather; Stephens, Jonathan; Gwilliam, Rhian; Rice, Catherine M.; Hengstenberg, Christian; Samani, Nilesh J.; Erdmann, Jeanette; Schunkert, Heribert; Pastinen, Tomi; Deloukas, Panos; Goodall, Alison H.; Ouwehand, Willem H.; Cambien, François; Syvänen, Ann-Christine

    2012-01-01

    A large number of genome-wide association studies have been performed during the past five years to identify associations between SNPs and human complex diseases and traits. The assignment of a functional role for the identified disease-associated SNP is not straight-forward. Genome-wide expression quantitative trait locus (eQTL) analysis is frequently used as the initial step to define a function while allele-specific gene expression (ASE) analysis has not yet gained a wide-spread use in disease mapping studies. We compared the power to identify cis-acting regulatory SNPs (cis-rSNPs) by genome-wide allele-specific gene expression (ASE) analysis with that of traditional expression quantitative trait locus (eQTL) mapping. Our study included 395 healthy blood donors for whom global gene expression profiles in circulating monocytes were determined by Illumina BeadArrays. ASE was assessed in a subset of these monocytes from 188 donors by quantitative genotyping of mRNA using a genome-wide panel of SNP markers. The performance of the two methods for detecting cis-rSNPs was evaluated by comparing associations between SNP genotypes and gene expression levels in sample sets of varying size. We found that up to 8-fold more samples are required for eQTL mapping to reach the same statistical power as that obtained by ASE analysis for the same rSNPs. The performance of ASE is insensitive to SNPs with low minor allele frequencies and detects a larger number of significantly associated rSNPs using the same sample size as eQTL mapping. An unequivocal conclusion from our comparison is that ASE analysis is more sensitive for detecting cis-rSNPs than standard eQTL mapping. Our study shows the potential of ASE mapping in tissue samples and primary cells which are difficult to obtain in large numbers. PMID:23300628

  1. Soil clone library analyses to evaluate specificity and selectivity of PCR primers targeting fungal 18S rDNA for denaturing-gradient gel electrophoresis (DGGE).

    PubMed

    Takada Hoshino, Yuko; Morimoto, Sho

    2010-01-01

    We evaluated the fungal specificity and detection bias of four fungal 18S rRNA gene (18S rDNA) primer sets for denaturing-gradient gel electrophoresis (DGGE). We constructed and compared clone libraries amplified from upland and paddy field soils with each primer set (1, NS1/GCFung; 2, FF390/FR1-GC; 3, NS1/FR1-GC; and 4, NS1/EF3 for the first PCR and NS1/FR1-GC for the second PCR). Primer set 4 (for nested PCR) showed the highest specificity for fungi but biased specific sequences. Sets 1, 2, and 3 (for single PCR) amplified non-fungal eukaryotic sequences (from 7 to 16% for upland soil and from 20 to 31% for paddy field soil) and produced libraries with similar distributions of fungal 18S rDNA sequences at both the phylum and the class level. Set 2 tended to amplify more diverse fungal sequences, maintaining higher specificity for fungi. In addition, clone analyses revealed differences among primer sets in the frequency of chimeras. In upland field soil, the libraries amplified with primer sets 3 and 4, which targeted long fragments, contained many chimeric 18S rDNA sequences (18% and 48%, respectively), while the libraries obtained with sets 1 and 2, which targeted short fragments, contained fewer chimeras (5% and 10%, respectively).

  2. Exciton Primer-mediated SNP detection in SmartAmp2 reactions.

    PubMed

    Lezhava, Alexander; Ishidao, Takefumi; Ishizu, Yuri; Naito, Kana; Hanami, Takeshi; Katayama, Atsuko; Kogo, Yasushi; Soma, Takahiro; Ikeda, Shuji; Murakami, Kayoko; Nogawa, Chihiro; Itoh, Masayoshi; Mitani, Yasumasa; Harbers, Matthias; Okamoto, Akimitsu; Hayashizaki, Yoshihide

    2010-02-01

    Most commonly used intercalating fluorescent dyes in DNA detection are lacking any sequence specificity, whereas so-called Exciton Primers can overcome this limitation by functioning as "sequence-specific dyes." After hybridization to complementary sequences, the fluorescence of Exciton Primers provides sequence-specific signals for real-time monitoring of amplification reactions. Applied to the SmartAmp2 mutation detection process, Exciton Primers show high signal strength with low background leading to a superior specificity and sensitivity compared to SYBR Green I. Signal strength can be further enhanced using multiple dyes within one Exciton Primer or use of multiple Exciton Primers in the same amplification reaction. Here we demonstrate the use of Exciton Primers for genotyping a single nucleotide polymorphism (SNP) in the VKORC1 locus (-1639G>A) relevant for Warfarin dosing as an example for Exciton Primers mediated genotyping by SmartAmp2. The genotyping assay can use only one labeled Exciton Primer for endpoint detection, or simultaneously by real-time monitoring detect wild-type and mutant alleles in a one-tube reaction using two Exciton Primers having different dyes. Working directly from blood samples, Exciton Primer mediated genotyping by SmartAmp2 offers superior solutions for rapid point-of-care testing.

  3. ABO alleles are linked with haplotypes of an erythroid cell-specific regulatory element in intron 1 with a few exceptions attributable to genetic recombination.

    PubMed

    Nakajima, T; Sano, R; Takahashi, Y; Watanabe, K; Kubo, R; Kobayashi, M; Takahashi, K; Takeshita, H; Kominato, Y

    2016-01-01

    Recent investigation of transcriptional regulation of the ABO genes has identified a candidate erythroid cell-specific regulatory element, named the +5·8-kb site, in the first intron of ABO. Six haplotypes of the site have been reported previously. The present genetic population study demonstrated that each haplotype was mostly linked with specific ABO alleles with a few exceptions, possibly as a result of hybrid formation between common ABO alleles. Thus, investigation of these haplotypes could provide a clue to further elucidation of ABO alleles.

  4. Allele-specific silencing of EEC p63 mutant R304W restores p63 transcriptional activity

    PubMed Central

    Novelli, F; Lena, A M; Panatta, E; Nasser, W; Shalom-Feuerstein, R; Candi, E; Melino, G

    2016-01-01

    EEC (ectrodactily-ectodermal dysplasia and cleft lip/palate) syndrome is a rare genetic disease, autosomal dominant inherited. It is part of the ectodermal dysplasia disorders caused by heterozygous mutations in TP63 gene. EEC patients present limb malformations, orofacial clefting, skin and skin's appendages defects, ocular abnormalities. The transcription factor p63, encoded by TP63, is a master gene for the commitment of ectodermal-derived tissues, being expressed in the apical ectodermal ridge is critical for vertebrate limb formation and, at a later stage, for skin and skin's appendages development. The ΔNp63α isoform is predominantly expressed in epithelial cells and it is indispensable for preserving the self-renewal capacity of adult stem cells and to engage specific epithelial differentiation programs. Small interfering RNA (siRNA) offers a potential therapy approach for EEC patients by selectively silencing the mutant allele. Here, using a systemic screening based on a dual-luciferase reported gene assay, we have successfully identified specific siRNAs for repressing the EEC-causing p63 mutant, R304W. Upon siRNA treatment, we were able to restore ΔNp63-WT allele transcriptional function in induced pluripotent stem cells that were derived from EEC patient biopsy. This study demonstrates that siRNAs approach is promising and, may pave the way for curing/delaying major symptoms, such as cornea degeneration and skin erosions in young EEC patients. PMID:27195674

  5. A new mib allele with a chromosomal deletion covering foxc1a exhibits anterior somite specification defect

    PubMed Central

    Hsu, Chia-Hao; Lin, Ji-Sheng; Po Lai, Keng; Li, Jing-Woei; Chan, Ting-Fung; You, May-Su; Tse, William Ka Fai; Jiang, Yun-Jin

    2015-01-01

    mibnn2002, found from an allele screen, showed early segmentation defect and severe cell death phenotypes, which are different from previously known mib mutants. Despite distinct morphological phenotypes, the typical mib molecular phenotypes: her4 down-regulation, neurogenic phenotype and cold sensitive dlc expression pattern, still remained. The linkage analysis also indicated that mibnn2002 is a new mib allele. Failure of specification in anterior 7-10 somites is likely due to lack of foxc1a expression in mibnn2002 homozygotes. Somites and somite markers gradually appeared after 7-10 somite stage, suggesting that foxc1a is only essential for the formation of anterior 7-10 somites. Apoptosis began around 16-somite stage with p53 up-regulation. To find the possible links of mib, foxc1a and apoptosis, transcriptome analysis was employed. About 140 genes, including wnt3a, foxc1a and mib, were not detected in the homozygotes. Overexpression of foxc1a mRNA in mibnn2002 homozygotes partially rescued the anterior somite specification. In the process of characterizing mibnn2002 mutation, we integrated the scaffolds containing mib locus into chromosome 2 (or linkage group 2, LG2) based on synteny comparison and transcriptome results. Genomic PCR analysis further supported the conclusion and showed that mibnn2002 has a chromosomal deletion with the size of about 9.6 Mbp. PMID:26039894

  6. Recommendations for Accurate Resolution of Gene and Isoform Allele-Specific Expression in RNA-Seq Data

    PubMed Central

    Wood, David L. A.; Nones, Katia; Steptoe, Anita; Christ, Angelika; Harliwong, Ivon; Newell, Felicity; Bruxner, Timothy J. C.; Miller, David; Cloonan, Nicole; Grimmond, Sean M.

    2015-01-01

    Genetic variation modulates gene expression transcriptionally or post-transcriptionally, and can profoundly alter an individual’s phenotype. Measuring allelic differential expression at heterozygous loci within an individual, a phenomenon called allele-specific expression (ASE), can assist in identifying such factors. Massively parallel DNA and RNA sequencing and advances in bioinformatic methodologies provide an outstanding opportunity to measure ASE genome-wide. In this study, matched DNA and RNA sequencing, genotyping arrays and computationally phased haplotypes were integrated to comprehensively and conservatively quantify ASE in a single human brain and liver tissue sample. We describe a methodological evaluation and assessment of common bioinformatic steps for ASE quantification, and recommend a robust approach to accurately measure SNP, gene and isoform ASE through the use of personalized haplotype genome alignment, strict alignment quality control and intragenic SNP aggregation. Our results indicate that accurate ASE quantification requires careful bioinformatic analyses and is adversely affected by sample specific alignment confounders and random sampling even at moderate sequence depths. We identified multiple known and several novel ASE genes in liver, including WDR72, DSP and UBD, as well as genes that contained ASE SNPs with imbalance direction discordant with haplotype phase, explainable by annotated transcript structure, suggesting isoform derived ASE. The methods evaluated in this study will be of use to researchers performing highly conservative quantification of ASE, and the genes and isoforms identified as ASE of interest to researchers studying those loci. PMID:25965996

  7. Electrochemical detection of point mutation based on surface hybridization assay conjugated allele-specific polymerase chain reaction.

    PubMed

    Huang, Yong; Zhu, Jing; Li, Guiyin; Chen, Zhencheng; Jiang, Jian-Hui; Shen, Guo-Li; Yu, Ru-Qin

    2013-04-15

    In this work, we developed an electrochemical detection method based on allele-specific polymerase chain reaction (AS-PCR) and surface hybridization assay technique for the point mutation detection. A high-fidelity Vent(R)™(exo⁻) DNA polymerase, which eliminated the 3'→5' proofreading exonuclease activity by genetical engineering, was used to discriminate and extend the detection probe that perfectly matched with mutant target DNA and generate a redox-active DNA replica which folded into a molecular beacon structure by intramolecular hybridization. After hybridized with capture probe modified on gold electrode by self-assembly reaction, the redox tags can be closed to electrode, resulting in a substantial current with the maximized sensitivity for point mutation analysis. However, when there is an allele mismatch in the wild target DNA, and so no the redox-active replica DNA can be obtained. In this case, no remarkable current signal can be trigged. The proposed approach has been successfully implemented for the identification of single base mutation at the -28 position in human β-globin gene with a detection limit of 0.5 fM, demonstrating that this method provides a highly specific, sensitive and cost-efficient approach for point mutation detection.

  8. Characterization of the inhabitancy of mouse intestinal bacteria (MIB) in rodents and humans by real-time PCR with group-specific primers.

    PubMed

    Kibe, Ryoko; Sakamoto, Mitsuo; Yokota, Hiroshi; Benno, Yoshimi

    2007-01-01

    Mouse intestinal bacteria (MIB) is a new operational taxonomic unit (OTU) belonging to the Bacteroides subgroup in the Cytophaga-Flavobacter-Bacteroides (CFB) phylum recently found in the intestine of mice, rats and humans. However, their characters are still unknown since they have not yet been isolated by culture. To understand their habitat characteristics in intestinal tracts, the quantification assays of MIB were established using MIB group-specific primers. The MIB population in the intestine was evaluated as a percentage of the number of 16S rRNA gene copy of MIB. A real-time PCR assay using group specific primers showed the fluctuation of MIB inhabitancy and revealed that the MIB population in the small intestine of mice was significantly lower than the large intestinal contents. Moreover, MIB was found in human feces though the number was lower than in murine. This assay using group-specific primers revealed new information about host-preference of MIB. PMID:17446674

  9. Specific HLA-DRB and -DQB Alleles and Haplotypes Confer Disease Susceptibility or Resistance in Bahraini Type 1 Diabetes Patients

    PubMed Central

    Al-Harbi, Einas M.; Abbassi, Abdul-Jabbar; Tamim, Hala; al-Jenaidi, Fayza; Kooheji, Mariam; Kamal, Madeeha; al-Mahroos, Salwa; al-Nasir, Faisal; Motala, Ayesha A.; Almawi, Wassim Y.

    2004-01-01

    Insofar as genetic susceptibility to type 1 diabetes is associated with HLA class II genes, with certain allelic combinations conferring disease susceptibility or resistance, this study assessed the distributions of HLA-DR and -DQ among 107 unrelated patients with type 1 diabetes and 88 healthy controls from Bahrain, all of Arab origin. The HLA-DRB and -DQB genotypes were determined by PCR-sequence-specific priming. The following alleles showed the strongest association with type 1 diabetes among patients versus controls according to their frequencies: DRB1*030101 (0.430 versus 0.097; P < 0.001), DRB1*040101 (0.243 versus 0.034; P < 0.001), DQB1*0201 (0.467 versus 0.193; P < 0.001), and DQB1*0302 (0.229 versus 0.091; P < 0.001). When the frequencies of alleles in controls were compared to those in patients, negative associations were seen for DRB1*100101 (0.085 versus 0.014; P < 0.001), DRB1*110101 (0.210 versus 0.060; P < 0.001), DQB1*030101 (0.170 versus 0.075; P = 0.006), and DQB1*050101 (0.335 versus 0.121; P < 0.001). In addition, the DRB1*030101-DQB1*0201 (70.1 versus 22.7%; P < 0.001) and DRB1*030101-DQB1*0302 (21.5 versus 0.0%; P < 0.001) genotypes were more prevalent among patients, thereby conferring disease susceptibility, whereas the DRB1*100101-DQB1*050101 (20.5 versus 2.8%; P < 0.001), DRB1*110101-DQB1*030101 (28.4 versus 8.4%; P < 0.001), and DRB1*110101-DQB1*050101 (30.7 versus 0.9%; P < 0.001) genotypes were more prevalent among controls, thus assigning a protective role. These results confirm the association of specific HLA-DR and -DQ alleles and haplotypes with type 1 diabetes and may underline several characteristics that distinguish Bahraini patients from other Caucasians patients. PMID:15013978

  10. Specific HLA-DRB and -DQB alleles and haplotypes confer disease susceptibility or resistance in Bahraini type 1 diabetes patients.

    PubMed

    Al-Harbi, Einas M; Abbassi, Abdul-Jabbar; Tamim, Hala; al-Jenaidi, Fayza; Kooheji, Mariam; Kamal, Madeeha; al-Mahroos, Salwa; al-Nasir, Faisal; Motala, Ayesha A; Almawi, Wassim Y

    2004-03-01

    Insofar as genetic susceptibility to type 1 diabetes is associated with HLA class II genes, with certain allelic combinations conferring disease susceptibility or resistance, this study assessed the distributions of HLA-DR and -DQ among 107 unrelated patients with type 1 diabetes and 88 healthy controls from Bahrain, all of Arab origin. The HLA-DRB and -DQB genotypes were determined by PCR-sequence-specific priming. The following alleles showed the strongest association with type 1 diabetes among patients versus controls according to their frequencies: DRB1*030101 (0.430 versus 0.097; P < 0.001), DRB1*040101 (0.243 versus 0.034; P < 0.001), DQB1*0201 (0.467 versus 0.193; P < 0.001), and DQB1*0302 (0.229 versus 0.091; P < 0.001). When the frequencies of alleles in controls were compared to those in patients, negative associations were seen for DRB1*100101 (0.085 versus 0.014; P < 0.001), DRB1*110101 (0.210 versus 0.060; P < 0.001), DQB1*030101 (0.170 versus 0.075; P = 0.006), and DQB1*050101 (0.335 versus 0.121; P < 0.001). In addition, the DRB1*030101-DQB1*0201 (70.1 versus 22.7%; P < 0.001) and DRB1*030101-DQB1*0302 (21.5 versus 0.0%; P < 0.001) genotypes were more prevalent among patients, thereby conferring disease susceptibility, whereas the DRB1*100101-DQB1*050101 (20.5 versus 2.8%; P < 0.001), DRB1*110101-DQB1*030101 (28.4 versus 8.4%; P < 0.001), and DRB1*110101-DQB1*050101 (30.7 versus 0.9%; P < 0.001) genotypes were more prevalent among controls, thus assigning a protective role. These results confirm the association of specific HLA-DR and -DQ alleles and haplotypes with type 1 diabetes and may underline several characteristics that distinguish Bahraini patients from other Caucasians patients.

  11. Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in alpha-spectrin-deficient red cells.

    PubMed

    Robledo, Raymond F; Lambert, Amy J; Birkenmeier, Connie S; Cirlan, Marius V; Cirlan, Andreea Flavia M; Campagna, Dean R; Lux, Samuel E; Peters, Luanne L

    2010-03-01

    Five spontaneous, allelic mutations in the alpha-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph(1J), sph(2J), sph(2BC), sph(Dem)). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph(3J), a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sph(Ihj), a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent beta-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph(4J), a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, beta-adducin. The severity of anemia in sph(4J) indicates that the highly conserved cysteine residue at the C-terminus of alpha-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.

  12. Comparison of human platelet antigen (HPA)-1a typing by solid phase red cell adherence to HPA-1 allotypes determined by allele-specific restriction enzyme analysis.

    PubMed

    McGann, M J; Procter, J L; Honda, J; Matsuo, K; Stroncek, D F

    2000-01-01

    Phenotype results for human platelet antigen (HPA)-1 by Capture-P(R), (Immucor, Inc., Norcross, GA) solid phase red cell adherence (SPRCA) were compared to results of allele-specific restriction enzyme analysis (ASRA) for the determination of HPA-1 allotype. Because the expression of HPA-1a and HPA-1b is determined by a single nucleotide substitution of thymine --> cytosine at position 196 of the gene encoding membrane glycoprotein (GP)-IIIa, it is possible to distinguish the alternate forms of the gene using ASRA. Primers (5'- GCTCCAATGTACGGGGTAAACTC-3' and 5'-CAGACCTCCACCTTGTGCTCTATG- 3') were designed to amplify the region of DNA that contains the polymorphism and a restriction enzyme (Nci I) was used to cleave the DNA in a predictable manner. Platelet-rich plasma for immunophenotying and anticoagulated whole blood for DNA extraction were obtained from 159 platepheresis donors. Of 159 SPRCA tests, 138 were valid and 21 were invalid due to positive autologous controls. For 135 HPA-1a-positive and 2 HPA-1a-negative phenotype tests the DNA typing results correlated: 135 positive samples were either HPA-1a/a or HPA-1a/b and 2 negative samples were HPA-1b/b. One donor that typed as HPA-1b/b by ASRA had a positive result of 2+ on SPRCA. This donor had been previously typed by SPRCA as HPA-1a-negative and DNA typed as HPA-1b/b by our laboratory. Based on these findings results of = 3+ by SPRCA are interpreted as HPA-1a-positive for donor screening purposes. SPRCA test results of = 2+ are considered equivocal and the HPA-1 allotype is determined by ASRA. HPA-1a-negative donors by SPRCA must be confirmed as HPA-1b/b by ASRA prior to issue for a patient that requires HPA-1anegative platelets.

  13. Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients.

    PubMed

    Barbano, Raffaela; Pasculli, Barbara; Coco, Michelina; Fontana, Andrea; Copetti, Massimiliano; Rendina, Michelina; Valori, Vanna Maria; Graziano, Paolo; Maiello, Evaristo; Fazio, Vito Michele; Parrella, Paola

    2015-12-22

    BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients' samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAF(V600E) and BRAF(V600K) mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method.

  14. Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients

    PubMed Central

    Barbano, Raffaela; Pasculli, Barbara; Coco, Michelina; Fontana, Andrea; Copetti, Massimiliano; Rendina, Michelina; Valori, Vanna Maria; Graziano, Paolo; Maiello, Evaristo; Fazio, Vito Michele; Parrella, Paola

    2015-01-01

    BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients’ samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAFV600E and BRAFV600K mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method. PMID:26690267

  15. Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative, Ultra-High-Throughput Profiling of Fungal Communities

    PubMed Central

    Bokulich, Nicholas A.

    2013-01-01

    Ultra-high-throughput sequencing (HTS) of fungal communities has been restricted by short read lengths and primer amplification bias, slowing the adoption of newer sequencing technologies to fungal community profiling. To address these issues, we evaluated the performance of several common internal transcribed spacer (ITS) primers and designed a novel primer set and work flow for simultaneous quantification and species-level interrogation of fungal consortia. Primer comparison and validation were predicted in silico and by sequencing a “mock community” of mixed yeast species to explore the challenges of amplicon length and amplification bias for reconstructing defined yeast community structures. The amplicon size and distribution of this primer set are smaller than for all preexisting ITS primer sets, maximizing sequencing coverage of hypervariable ITS domains by very-short-amplicon, high-throughput sequencing platforms. This feature also enables the optional integration of quantitative PCR (qPCR) directly into the HTS preparatory work flow by substituting qPCR with these primers for standard PCR, yielding quantification of individual community members. The complete work flow described here, utilizing any of the qualified primer sets evaluated, can rapidly profile mixed fungal communities and capably reconstructed well-characterized beer and wine fermentation fungal communities. PMID:23377949

  16. New Design Strategy for Development of Specific Primer Sets for PCR-Based Detection of Chlorophyceae and Bacillariophyceae in Environmental Samples▿ †

    PubMed Central

    Valiente Moro, Claire; Crouzet, Olivier; Rasconi, Séréna; Thouvenot, Antoine; Coffe, Gérard; Batisson, Isabelle; Bohatier, Jacques

    2009-01-01

    Studying aquatic microalgae is essential for monitoring biodiversity and water quality. We designed new sets of 18S rRNA PCR primers for Chlorophyceae and Bacillariophyceae by using the ARB software and implementing a virtual PCR program. The results of specificity analysis showed that most of the targeted algal families were identified and nontargeted organisms, such as fungi or ciliates, were excluded. These newly developed PCR primer sets were also able to amplify microalgal rRNA genes from environmental samples with accurate specificity. These tools could be of great interest for studying freshwater microalgal ecology and for developing bioindicators of the health status of aquatic environments. PMID:19592531

  17. New design strategy for development of specific primer sets for PCR-based detection of Chlorophyceae and Bacillariophyceae in environmental samples.

    PubMed

    Moro, Claire Valiente; Crouzet, Olivier; Rasconi, Séréna; Thouvenot, Antoine; Coffe, Gérard; Batisson, Isabelle; Bohatier, Jacques

    2009-09-01

    Studying aquatic microalgae is essential for monitoring biodiversity and water quality. We designed new sets of 18S rRNA PCR primers for Chlorophyceae and Bacillariophyceae by using the ARB software and implementing a virtual PCR program. The results of specificity analysis showed that most of the targeted algal families were identified and nontargeted organisms, such as fungi or ciliates, were excluded. These newly developed PCR primer sets were also able to amplify microalgal rRNA genes from environmental samples with accurate specificity. These tools could be of great interest for studying freshwater microalgal ecology and for developing bioindicators of the health status of aquatic environments. PMID:19592531

  18. Evaluation of strain-specific primers for identification of Lactobacillus rhamnosus GG.

    PubMed

    Endo, Akihito; Aakko, Juhani; Salminen, Seppo

    2012-12-01

    Lactobacillus rhamnosus strain GG (ATCC 53103) is one of the most widely studied and commercialized probiotic strains, and thus strain-specific identification for the strain is highly valuable. In this study, two published PCR-based identification methods for strain GG, a transposase gene-targeting system and a phage-related gene-targeting system, were evaluated. The former produced amplicons from eight of the 41 strains tested and the phage-related system from five of the tested strains, including the strain GG. Fingerprinting analysis indicated that the strains LMG 18025, LMG 18030, and LMG 18038, which had an amplicon by the former system but none by the latter, were genetically distinguishable from L. rhamnosus GG at strain level. Strains LMG 23320, LMG 23325, LMG 23534, and LMG 25859 showed profiles very similar to that of the strain GG, suggesting that these strains might be identical to GG or derivative strains of it. The results here indicated that the phage-related gene-targeting system is a good tool for accurate identification of L. rhamnosus GG. This system would be able to detect both the original L. rhamnosus GG and its derivative strains.

  19. Exploring the diversity of Acinetobacter populations in river water with genus-specific primers and probes.

    PubMed

    Xin, Fangfang; Cai, Dijie; Sun, Yuhua; Guo, Dalei; Wu, Zirong; Jiang, Deming

    2014-01-01

    This study aimed to explore the diversity of river water Acinetobacter populations using culture-dependent and -independent methods. Pyrosequencing indicated that 1.5% of the total sequences from Qiandeng River water were classified as Acinetobacter. Twelve Acinetobacter strains were isolated from three different sampling sites of the Qiandeng River. Based on culture-dependent methods, A. johnsonii, A. lwoffii and A. guillouiae were the most abundantly represented Acinetobacter strains among the upper, middle and downstream populations of the river. Probing of three Acinetobacter-enriched 16S rRNA gene libraries with the Acinetobacter specific probe Act660F revealed 42 unique 16S rRNA gene sequences exhibiting a similarity of 94.9-99.9% with the known Acinetobacter strains. Among the uncultured Acinetobacter sequences, 50%, 58.3% and 68.8% of those obtained from upstream sampling site A, middle stream sampling site B and downstream sampling site C were phylogenetically located within Group I. This Group represented the most abundant strains of Acinetobacter populations in river water based on culture-independent methods. The results indicated that culture-independent methods provide more detailed information on the diversity of Acinetobacter populations than that based on culture-dependent methods. Therefore, the development of new and efficient isolation methods to identify uncultured Acinetobacter species is required.

  20. Inflammatory Bowel Disease Cause-specific Mortality: A Primer for Clinicians

    PubMed Central

    Kassam, Zain; Belga, Sara; Roifman, Idan; Hirota, Simon; Jijon, Humberto; Kaplan, Gilaad G.; Ghosh, Subrata

    2014-01-01

    Background: Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC) is perceived to harbor significant morbidity but limited excess mortality, thought to be driven by colon cancer, compared with the general population. Recent studies suggest mortality rates seem higher than previously understood, and there are emerging threats to mortality. Clinicians must be up to date and able to clearly convey the causes of mortality to arm individual patients with information to meaningfully participate in decisions regarding IBD treatment and maintenance of health. Methods: A MEDLINE search was conducted to capture all relevant articles. Keyword search included: “inflammatory bowel disease,” “Crohn's disease,” “ulcerative colitis,” and “mortality.” Results: CD and UC have slightly different causes of mortality; however, malignancy and colorectal cancer–associated mortality remains controversial in IBD. CD mortality seems to be driven by gastrointestinal disease, infection, and respiratory diseases. UC mortality was primarily attributable to gastrointestinal disease and infection. Clostridium difficile infection is an emerging cause of mortality in IBD. UC and CD patients have a marked increase in risk of thromboembolic disease. With advances in medical and surgical interventions, the exploration of treatment-associated mortality must continue to be evaluated. Conclusions: Clinicians should be aware that conventional causes of death such as malignancy do not seem to be as significant a burden as originally perceived. However, emerging threats such as infection including C. difficile are noteworthy. Although CD and UC share similar causes of death, there seems to be some differences in cause-specific mortality. PMID:25185685

  1. Transcriptome analysis revealed chimeric RNAs, single nucleotide polymorphisms and allele-specific expression in porcine prenatal skeletal muscle

    PubMed Central

    Yang, Yalan; Tang, Zhonglin; Fan, Xinhao; Xu, Kui; Mu, Yulian; Zhou, Rong; Li, Kui

    2016-01-01

    Prenatal skeletal muscle development genetically determines postnatal muscle characteristics such as growth and meat quality in pigs. However, the molecular mechanisms underlying prenatal skeletal muscle development remain unclear. Here, we performed the first genome-wide analysis of chimeric RNAs, single nuclear polymorphisms (SNPs) and allele-specific expression (ASE) in prenatal skeletal muscle in pigs. We identified 14,810 protein coding genes and 163 high-confidence chimeric RNAs expressed in prenatal skeletal muscle. More than 94.5% of the chimeric RNAs obeyed the canonical GT/AG splice rule and were trans-splicing events. Ten and two RNAs were aligned to human and mouse chimeric transcripts, respectively. We detected 106,457 high-quality SNPs (6,955 novel), which were mostly (89.09%) located within QTLs for production traits. The high proportion of non-exonic SNPs revealed the incomplete annotation status of the current swine reference genome. ASE analysis revealed that 11,300 heterozygous SNPs showed allelic imbalance, whereas 131 ASE variants were located in the chimeric RNAs. Moreover, 4 ASE variants were associated with various economically relevant traits of pigs. Taken together, our data provide a source for studies of chimeric RNAs and biomarkers for pig breeding, while illuminating the complex transcriptional events underlying prenatal skeletal muscle development in mammals. PMID:27352850

  2. Transcriptome analysis revealed chimeric RNAs, single nucleotide polymorphisms and allele-specific expression in porcine prenatal skeletal muscle.

    PubMed

    Yang, Yalan; Tang, Zhonglin; Fan, Xinhao; Xu, Kui; Mu, Yulian; Zhou, Rong; Li, Kui

    2016-01-01

    Prenatal skeletal muscle development genetically determines postnatal muscle characteristics such as growth and meat quality in pigs. However, the molecular mechanisms underlying prenatal skeletal muscle development remain unclear. Here, we performed the first genome-wide analysis of chimeric RNAs, single nuclear polymorphisms (SNPs) and allele-specific expression (ASE) in prenatal skeletal muscle in pigs. We identified 14,810 protein coding genes and 163 high-confidence chimeric RNAs expressed in prenatal skeletal muscle. More than 94.5% of the chimeric RNAs obeyed the canonical GT/AG splice rule and were trans-splicing events. Ten and two RNAs were aligned to human and mouse chimeric transcripts, respectively. We detected 106,457 high-quality SNPs (6,955 novel), which were mostly (89.09%) located within QTLs for production traits. The high proportion of non-exonic SNPs revealed the incomplete annotation status of the current swine reference genome. ASE analysis revealed that 11,300 heterozygous SNPs showed allelic imbalance, whereas 131 ASE variants were located in the chimeric RNAs. Moreover, 4 ASE variants were associated with various economically relevant traits of pigs. Taken together, our data provide a source for studies of chimeric RNAs and biomarkers for pig breeding, while illuminating the complex transcriptional events underlying prenatal skeletal muscle development in mammals. PMID:27352850

  3. Bivariate segmentation of SNP-array data for allele-specific copy number analysis in tumour samples

    PubMed Central

    2013-01-01

    Background SNP arrays output two signals that reflect the total genomic copy number (LRR) and the allelic ratio (BAF), which in combination allow the characterisation of allele-specific copy numbers (ASCNs). While methods based on hidden Markov models (HMMs) have been extended from array comparative genomic hybridisation (aCGH) to jointly handle the two signals, only one method based on change-point detection, ASCAT, performs bivariate segmentation. Results In the present work, we introduce a generic framework for bivariate segmentation of SNP array data for ASCN analysis. For the matter, we discuss the characteristics of the typically applied BAF transformation and how they affect segmentation, introduce concepts of multivariate time series analysis that are of concern in this field and discuss the appropriate formulation of the problem. The framework is implemented in a method named CnaStruct, the bivariate form of the structural change model (SCM), which has been successfully applied to transcriptome mapping and aCGH. Conclusions On a comprehensive synthetic dataset, we show that CnaStruct outperforms the segmentation of existing ASCN analysis methods. Furthermore, CnaStruct can be integrated into the workflows of several ASCN analysis tools in order to improve their performance, specially on tumour samples highly contaminated by normal cells. PMID:23497144

  4. Analysis of LMNB1 Duplications in Autosomal Dominant Leukodystrophy Provides Insights into Duplication Mechanisms and Allele-Specific Expression

    PubMed Central

    Giorgio, Elisa; Rolyan, Harshvardhan; Kropp, Laura; Chakka, Anish Baswanth; Yatsenko, Svetlana; Gregorio, Eleonora Di; Lacerenza, Daniela; Vaula, Giovanna; Talarico, Flavia; Mandich, Paola; Toro, Camilo; Pierre, Eleonore Eymard; Labauge, Pierre; Capellari, Sabina; Cortelli, Pietro; Vairo, Filippo Pinto; Miguel, Diego; Stubbolo, Danielle; Marques, Lourenco Charles; Gahl, William; Boespflug-Tanguy, Odile; Melberg, Atle; Hassin-Baer, Sharon; Cohen, Oren S; Pjontek, Rastislav; Grau, Armin; Klopstock, Thomas; Fogel, Brent; Meijer, Inge; Rouleau, Guy; Bouchard, Jean-Pierre L; Ganapathiraju, Madhavi; Vanderver, Adeline; Dahl, Niklas; Hobson, Grace; Brusco, Alfredo; Brussino, Alessandro; Padiath, Quasar Saleem

    2013-01-01

    ABSTRACT Autosomal dominant leukodystrophy (ADLD) is an adult onset demyelinating disorder that is caused by duplications of the lamin B1 (LMNB1) gene. However, as only a few cases have been analyzed in detail, the mechanisms underlying LMNB1 duplications are unclear. We report the detailed molecular analysis of the largest collection of ADLD families studied, to date. We have identified the minimal duplicated region necessary for the disease, defined all the duplication junctions at the nucleotide level and identified the first inverted LMNB1 duplication. We have demonstrated that the duplications are not recurrent; patients with identical duplications share the same haplotype, likely inherited from a common founder and that the duplications originated from intrachromosomal events. The duplication junction sequences indicated that nonhomologous end joining or replication-based mechanisms such fork stalling and template switching or microhomology-mediated break induced repair are likely to be involved. LMNB1 expression was increased in patients’ fibroblasts both at mRNA and protein levels and the three LMNB1 alleles in ADLD patients show equal expression, suggesting that regulatory regions are maintained within the rearranged segment. These results have allowed us to elucidate duplication mechanisms and provide insights into allele-specific LMNB1 expression levels. PMID:23649844

  5. Formation and characterization of precise eucaryotic transcription complexes using a semisynthetic DNA template and specific oligoribonucleotide primers.

    PubMed

    Mougey, E B; Dennis, D

    1988-06-01

    An artificial template of defined sequence which supports specific in vitro initiation and elongation by yeast RNA polymerase II has been constructed. This template is a pBR322 derivative which contains a synthetic oligonucleotide inserted into the BamHI cloning site. The sequence of this oligonucleotide is such that when the plasmid is restricted with SacI the two ends obtained are identical. The addition of an oligodeoxycytidylate chain to the 3' hydroxy termini produces a DNA template, (poly dC-p+22), with the sequence: 3'(C)nTCGA-GAGTCTCCTA. . . . The underlined position denotes the beginning of the duplex region. When initiation is primed with the diribonucleotide GpC the predicted sequence of the transcript obtained is: 5'GCUCUCAGAGGAU. . . . Kinetic and product analyses indicate that a ternary complex containing a precise length of transcript can be produced which is subsequently resistant to heparin inactivation. Initiation can also be directed to a specific position dictated by a tri or tetraribonucleotide primer.

  6. Specific amplification of bacterial DNA by optimized so-called universal bacterial primers in samples rich of plant DNA.

    PubMed

    Dorn-In, Samart; Bassitta, Rupert; Schwaiger, Karin; Bauer, Johann; Hölzel, Christina S

    2015-06-01

    Universal primers targeting the bacterial 16S-rRNA-gene allow quantification of the total bacterial load in variable sample types by qPCR. However, many universal primer pairs also amplify DNA of plants or even of archaea and other eukaryotic cells. By using these primers, the total bacterial load might be misevaluated, whenever samples contain high amounts of non-target DNA. Thus, this study aimed to provide primer pairs which are suitable for quantification and identification of bacterial DNA in samples such as feed, spices and sample material from digesters. For 42 primers, mismatches to the sequence of chloroplasts and mitochondria of plants were evaluated. Six primer pairs were further analyzed with regard to the question whether they anneal to DNA of archaea, animal tissue and fungi. Subsequently they were tested with sample matrix such as plants, feed, feces, soil and environmental samples. To this purpose, the target DNA in the samples was quantified by qPCR. The PCR products of plant and feed samples were further processed for the Single Strand Conformation Polymorphism method followed by sequence analysis. The sequencing results revealed that primer pair 335F/769R amplified only bacterial DNA in samples such as plants and animal feed, in which the DNA of plants prevailed.

  7. Designing Polymerase Chain Reaction Primers Using Primer3Plus.

    PubMed

    Hung, Jui-Hung; Weng, Zhiping

    2016-01-01

    Designing oligonucleotide primers is a crucial step for successful molecular biology experiments that require the use of the polymerase chain reaction (PCR). PCR involves cycles of three steps: denaturation, annealing, and extension. During denaturation, double-stranded DNA (dsDNA) molecules (templates) are separated into single strands. During annealing, a pair of primers is annealed to the complementary regions of the single-stranded molecules. In the extension step, DNA polymerase extends the primers to produce DNA molecules that correspond to the region bracketed by the primers (the amplicons). All of these steps are temperature sensitive, and the common choice of temperatures is 94°C, 60°C, and 70°C, respectively. Poorly designed primers may lead to no amplification product or additional undesired amplified fragments. The goals of primer design include good primer specificity, high annealing efficiency, appropriate melting temperature, proper GC content, and the prevention of primer hairpins or primer dimers. PMID:27574202

  8. Improved DNA barcoding method for Bemisia tabaci and related Aleyrodidae: development of universal and Bemisia tabaci biotype-specific mitochondrial cytochrome c oxidase I polymerase chain reaction primers.

    PubMed

    Shatters, Robert G; Powell, Charles A; Boykin, Laura M; Liansheng, He; McKenzie, C L

    2009-04-01

    Whiteflies, heteropterans in the family Aleyrodidae, are globally distributed and severe agricultural pests. The mitochondrial cytochrome c oxidase I (mtCOI) sequence has been used extensively in whitefly phylogenetic comparisons and in biotype identification of the agriculturally important Bemisia tabaci (Gennadius) whitefly. Because of the economic importance of several whitefly genera, and the invasive nature of the B and the Q biotypes of Bemisia tabaci, mtCOI sequence data are continually generated from sampled populations worldwide. Routine phylogenetic comparisons and biotype identification is done through amplification and sequencing of an approximately 800-bp mtCOI DNA fragment. Despite its routine use, published primers for amplification of this region are often inefficient for some B. tabaci biotypes and especially across whitefly species. Through new sequence generation and comparison to available whitefly mtCOI sequence data, a set of polymerase chain reaction (PCR) amplification primers (Btab-Uni primers) were identified that are more efficient at amplifying approximately 748 bp of the approximately 800-bp fragment currently used. These universal primers amplify an mtCOI fragment from numerous B. tabaci biotypes and whitefly genera by using a single amplification profile. Furthermore, mtCOI PCR primers specific for the B, Q, and New World biotypes of B. tabaci were designed that allow rapid discrimination among these biotypes. These primers produce a 478-, 405-, and 303-bp mtCOI fragment for the B, New World, and Q biotypes, respectively. By combining these primers and using rapid PCR and electrophoretic techniques, biotype determination can be made within 3 h for up to 96 samples at a time.

  9. Genotyping by Sequencing Using Specific Allelic Capture to Build a High-Density Genetic Map of Durum Wheat

    PubMed Central

    Holtz, Yan; Ardisson, Morgane; Ranwez, Vincent; Besnard, Alban; Leroy, Philippe; Poux, Gérard; Roumet, Pierre; Viader, Véronique; Santoni, Sylvain; David, Jacques

    2016-01-01

    Targeted sequence capture is a promising technology which helps reduce costs for sequencing and genotyping numerous genomic regions in large sets of individuals. Bait sequences are designed to capture specific alleles previously discovered in parents or reference populations. We studied a set of 135 RILs originating from a cross between an emmer cultivar (Dic2) and a recent durum elite cultivar (Silur). Six thousand sequence baits were designed to target Dic2 vs. Silur polymorphisms discovered in a previous RNAseq study. These baits were exposed to genomic DNA of the RIL population. Eighty percent of the targeted SNPs were recovered, 65% of which were of high quality and coverage. The final high density genetic map consisted of more than 3,000 markers, whose genetic and physical mapping were consistent with those obtained with large arrays. PMID:27171472

  10. Isolation and cloning of the raccoon (Procyon lotor) papillomavirus type 1 by using degenerate papillomavirus-specific primers.

    PubMed

    Rector, Annabel; Van Doorslaer, Koenraad; Bertelsen, Mads; Barker, Ian K; Olberg, Rolf-Arne; Lemey, Philippe; Sundberg, John P; Van Ranst, Marc

    2005-07-01

    Partial sequences of a novel papillomavirus were amplified from a cutaneous lesion biopsy of a raccoon (Procyon lotor), by using PCR with degenerate papillomavirus-specific primers. The Procyon lotor papillomavirus type 1 (PlPV-1) DNA was amplified with long template PCR in two overlapping fragments, together encompassing the entire genome, and the complete PlPV-1 genomic sequence was determined. The PlPV-1 genome consists of 8170 bp, and contains the typical papillomaviral open reading frames, encoding five early proteins and two late capsid proteins. Besides the classical non-coding region (NCR1) between the end of L1 and the start of E6, PlPV-1 contains an additional non-coding region (NCR2) of 1065 bp between the early and late protein region, which has previously also been described for the canine oral papillomavirus (COPV) and the Felis domesticus papillomavirus (FdPV-1). Phylogenetic analysis places PlPV-1 together with COPV and FdPV-1 in a monophyletic branch which encompasses the Lambda papillomavirus genus. PMID:15958682

  11. Point mutation in essential genes with loss or mutation of the second allele: relevance to the retention of tumor-specific antigens.

    PubMed

    Beck-Engeser, G B; Monach, P A; Mumberg, D; Yang, F; Wanderling, S; Schreiber, K; Espinosa, R; Le Beau, M M; Meredith, S C; Schreiber, H

    2001-08-01

    Antigens that are tumor specific yet retained by tumor cells despite tumor progression offer stable and specific targets for immunologic and possibly other therapeutic interventions. Therefore, we have studied two CD4(+) T cell-recognized tumor-specific antigens that were retained during evolution of two ultraviolet-light-induced murine cancers to more aggressive growth. The antigens are ribosomal proteins altered by somatic tumor-specific point mutations, and the progressor (PRO) variants lack the corresponding normal alleles. In the first tumor, 6132A-PRO, the antigen is encoded by a point-mutated L9 ribosomal protein gene. The tumor lacks the normal L9 allele because of an interstitial deletion from chromosome 5. In the second tumor, 6139B-PRO, both alleles of the L26 gene have point mutations, and each encodes a different tumor-specific CD4(+) T cell-recognized antigen. Thus, for both L9 and L26 genes, we observe "two hit" kinetics commonly observed in genes suppressing tumor growth. Indeed, reintroduction of the lost wild-type L9 allele into the 6132A-PRO variant suppressed the growth of the tumor cells in vivo. Since both L9 and L26 encode proteins essential for ribosomal biogenesis, complete loss of the tumor-specific target antigens in the absence of a normal allele would abrogate tumor growth.

  12. Allele Mining in Barley Genetic Resources Reveals Genes of Race-Non-Specific Powdery Mildew Resistance

    PubMed Central

    Spies, Annika; Korzun, Viktor; Bayles, Rosemary; Rajaraman, Jeyaraman; Himmelbach, Axel; Hedley, Pete E.; Schweizer, Patrick

    2012-01-01

    Race-non-specific, or quantitative, pathogen resistance is of high importance to plant breeders due to its expected durability. However, it is usually controlled by multiple quantitative trait loci (QTL) and therefore difficult to handle in practice. Knowing the genes that underlie race-non-specific resistance (NR) would allow its exploitation in a more targeted manner. Here, we performed an association-genetic study in a customized worldwide collection of spring barley accessions for candidate genes of race-NR to the powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) and combined data with results from QTL mapping as well as functional-genomics approaches. This led to the identification of 11 associated genes with converging evidence for an important role in race-NR in the presence of the Mlo gene for basal susceptibility. Outstanding in this respect was the gene encoding the transcription factor WRKY2. The results suggest that unlocking plant genetic resources and integrating functional-genomic with genetic approaches can accelerate the discovery of genes underlying race-NR in barley and other crop plants. PMID:22629270

  13. Inactive allele-specific methylation and chromatin structure of the imprinted gene U2af1-rs1 on mouse chromosome 11

    SciTech Connect

    Shibata, Hideo; Yoshino, Kiyoshi; Kamiya, Mamoru

    1996-07-01

    The imprinted U2Af1-rs1 gene that maps to mouse chromosome 11 is predominately expressed from the paternal allele. We examined the methylation of genomic sequences in and around the U2af1-rs1 locus to establish the extent of sequence modifications that accompanied the silencing of the maternal allele. The analysis of HapII or HhaI sites showed that the silent maternal allele was hypermethylated in a block of CpG sequences that covered more than 10 kb. By comparison, the expressed paternal allele was unmethylated from a CpG island upstream of the transcribed region through 2 kb. An analysis of DNaseI hypersensitivity of a putative promoter of U2af1-rs1 showed an open chromatin conformation only on the unmethylated, expressed paternal allele. These results suggest that allele-specific hypermethylation covering the gene and its upstream CpG island plays a role in maternal allele repression of U2af1-rs1, which is reflected in altered chromatin conformation of DNaseI hypersensitive sites. 9 refs., 2 figs.

  14. STR primer concordance study.

    PubMed

    Budowle, B; Masibay, A; Anderson, S J; Barna, C; Biega, L; Brenneke, S; Brown, B L; Cramer, J; DeGroot, G A; Douglas, D; Duceman, B; Eastman, A; Giles, R; Hamill, J; Haase, D J; Janssen, D W; Kupferschmid, T D; Lawton, T; Lemire, C; Llewellyn, B; Moretti, T; Neves, J; Palaski, C; Schueler, S; Sgueglia, J; Sprecher, C; Tomsey, C; Yet, D

    2001-12-15

    Over 1500 population database samples comprising African Americans, Caucasians, Hispanics, Native Americans, Chamorros and Filipinos were typed using the PowerPlex 16 and the Profiler Plus/COfiler kits. Except for the D8S1179 locus in Chamorros and Filipinos from Guam, there were eight examples in which a typing difference due to allele dropout was observed. At the D8S1179 locus in the population samples from Guam, there were 13 examples of allele dropout observed when using the Profiler Plus kit. The data support that the primers used in the PowerPlex 16, Profiler Plus, and COfiler kits are reliable for typing reference samples that are for use in CODIS. In addition, allele frequency databases have been established for the STR loci Penta D and Penta E. Both loci are highly polymorphic. PMID:11741760

  15. Target-specific PCR primers can detect and differentiate ophiostomatoid fungi from microbial communities associated with the mountain pine beetle Dendroctonus ponderosae.

    PubMed

    Khadempour, Lily; Massoumi Alamouti, Sepideh; Hamelin, Richard; Bohlmann, Jörg; Breuil, Colette

    2010-10-01

    The aim of this study was to develop DNA probes that could identify the major fungal species associated with mountain pine beetles (MPB). The beetles are closely associated with fungal species that include ophiostomatoid fungi that can be difficult to differentiate morphologically. The most frequently isolated associates are the pine pathogens Grosmannia clavigera and Leptographium longiclavatum, the less pathogenic Ophiostoma montium, and an undescribed Ceratocystiopsis species (Cop. sp.). Because growing, isolating and extracting DNA from fungi vectored by MPB can be time and labour intensive, we designed three rDNA primer sets that specifically amplify short rDNA amplicons from O. montium, Cop. sp. and the pine Leptographium clade. We also designed two primer sets on a gene of unknown function that can differentiate G. clavigera and L. longiclavatum. We tested the primers on 76 fungal isolates that included MPB associates. The primers reliably identified their targets from DNA obtained from pure fungal cultures, pulverized beetles, beetle galleries, and tree phloem inoculated with G. clavigera. The primers will facilitate large-scale work on the ecology of the MPB-fungal-lodgepole pine ecosystem, as well as phytosanitary/quarantine sample screening.

  16. Sensitive and specific detection of miRNA using an isothermal exponential amplification method using fluorescence-labeled LNA/DNA chimera primers.

    PubMed

    Huang, Jun-Fu; Zhao, Na; Xu, Han-Qing; Xia, Han; Wei, Kun; Fu, Wei-Ling; Huang, Qing

    2016-10-01

    MicroRNAs (miRNAs) are currently considered as potential biomarkers for various human diseases. In the present study, miRNA-triggered real-time fluorescent isothermal reaction with exponential amplification (ReFIRE) with or without Thermus aquaticus MutS (Taq MutS) was developed to analyze miRNAs using DNA polymerase, a nicking endonuclease, and fluorescently labeled primers. In the absence of Taq MutS, the ReFIRE system permitted the detection of 100 ymol of targeted miRNA in 80 min. However, this system enabled limited differentiation between homologous miRNA family members. Upon addition of Taq MutS to the ReFIRE system, non-specific amplification generated from the mishybridization between primers and primer dimers or primers and the template duplex was eliminated. The addition of Taq MutS enabled the ultrasensitive detection of as little as 10 ymol of targeted miRNAs in 50 min, which corresponds to less than 10 copies of miRNAs in a total volume of 20 μl. Additionally, the assay exhibited a dynamic range of up to 12 orders of magnitude. The ReFIRE system also showed high specificity, enabling differentiation between homologous miRNA family members exhibiting only single-base differences. The sensitivity, specificity, and dynamic range associated with this system were greater than most currently available miRNA isothermal amplification assays. Moreover, when target-specific primers were labeled with different fluorescent reporters, multiplex analysis was easily performed in a single tube, permitting accurate normalization of miRNA expression. This simple, fast, ultrasensitive, highly specific, and easy-to-multiplex method could significantly contribute to research investigations pertaining to the biological roles of miRNA, as well as clinical diagnosis of various diseases that involve miRNA disruptions. Graphical Abstract The principle of ReFIRE system.

  17. Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum

    PubMed Central

    2014-01-01

    Background Although sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the development of a sensitive and affordable method for identifying pathogens in clinical samples is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (m-PCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause genital infections, and the PCR method was developed. Methods The Genosensor Probe Designer (GPD) (version 1.0a) software was initially used to design highly specific and efficient primers for in-house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers for each locus in turn were added individually in subsequent amplifications until m-PCR was achieved. Amplicons of the expected size were obtained from each of the four bacterial gene fragments. Finally, the analytical specificity and limits of detection were tested. Results Because they did not amplify any product from non-STI tested species, the primers were specific. The detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum primer sets were 5.12 × 105, 3.9 × 103, 61.19 × 106 and 6.37 × 105 copies of a DNA template, respectively. Conclusions The methodology designed and standardised here could be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. This method is at least as efficient as other previously described methods; however, this method is more affordable for low-income countries. PMID:24997675

  18. Sensitive and specific detection of miRNA using an isothermal exponential amplification method using fluorescence-labeled LNA/DNA chimera primers.

    PubMed

    Huang, Jun-Fu; Zhao, Na; Xu, Han-Qing; Xia, Han; Wei, Kun; Fu, Wei-Ling; Huang, Qing

    2016-10-01

    MicroRNAs (miRNAs) are currently considered as potential biomarkers for various human diseases. In the present study, miRNA-triggered real-time fluorescent isothermal reaction with exponential amplification (ReFIRE) with or without Thermus aquaticus MutS (Taq MutS) was developed to analyze miRNAs using DNA polymerase, a nicking endonuclease, and fluorescently labeled primers. In the absence of Taq MutS, the ReFIRE system permitted the detection of 100 ymol of targeted miRNA in 80 min. However, this system enabled limited differentiation between homologous miRNA family members. Upon addition of Taq MutS to the ReFIRE system, non-specific amplification generated from the mishybridization between primers and primer dimers or primers and the template duplex was eliminated. The addition of Taq MutS enabled the ultrasensitive detection of as little as 10 ymol of targeted miRNAs in 50 min, which corresponds to less than 10 copies of miRNAs in a total volume of 20 μl. Additionally, the assay exhibited a dynamic range of up to 12 orders of magnitude. The ReFIRE system also showed high specificity, enabling differentiation between homologous miRNA family members exhibiting only single-base differences. The sensitivity, specificity, and dynamic range associated with this system were greater than most currently available miRNA isothermal amplification assays. Moreover, when target-specific primers were labeled with different fluorescent reporters, multiplex analysis was easily performed in a single tube, permitting accurate normalization of miRNA expression. This simple, fast, ultrasensitive, highly specific, and easy-to-multiplex method could significantly contribute to research investigations pertaining to the biological roles of miRNA, as well as clinical diagnosis of various diseases that involve miRNA disruptions. Graphical Abstract The principle of ReFIRE system. PMID:27485624

  19. An African ancestry-specific allele of CTLA4 confers protection against rheumatoid arthritis in African Americans.

    PubMed

    Kelley, James M; Hughes, Laura B; Faggard, Jeffrey D; Danila, Maria I; Crawford, Monica H; Edberg, Yuanqing; Padilla, Miguel A; Tiwari, Hemant K; Westfall, Andrew O; Alarcón, Graciela S; Conn, Doyt L; Jonas, Beth L; Callahan, Leigh F; Smith, Edwin A; Brasington, Richard D; Allison, David B; Kimberly, Robert P; Moreland, Larry W; Edberg, Jeffrey C; Bridges, S Louis

    2009-03-01

    Cytotoxic T-lymphocyte associated protein 4 (CTLA4) is a negative regulator of T-cell proliferation. Polymorphisms in CTLA4 have been inconsistently associated with susceptibility to rheumatoid arthritis (RA) in populations of European ancestry but have not been examined in African Americans. The prevalence of RA in most populations of European and Asian ancestry is approximately 1.0%; RA is purportedly less common in black Africans, with little known about its prevalence in African Americans. We sought to determine if CTLA4 polymorphisms are associated with RA in African Americans. We performed a 2-stage analysis of 12 haplotype tagging single nucleotide polymorphisms (SNPs) across CTLA4 in a total of 505 African American RA patients and 712 African American controls using Illumina and TaqMan platforms. The minor allele (G) of the rs231778 SNP was 0.054 in RA patients, compared to 0.209 in controls (4.462 x 10(-26), Fisher's exact). The presence of the G allele was associated with a substantially reduced odds ratio (OR) of having RA (AG+GG genotypes vs. AA genotype, OR 0.19, 95% CI: 0.13-0.26, p = 2.4 x 10(-28), Fisher's exact), suggesting a protective effect. This SNP is polymorphic in the African population (minor allele frequency [MAF] 0.09 in the Yoruba population), but is very rare in other groups (MAF = 0.002 in 530 Caucasians genotyped for this study). Markers associated with RA in populations of European ancestry (rs3087243 [+60C/T] and rs231775 [+49A/G]) were not replicated in African Americans. We found no confounding of association for rs231778 after stratifying for the HLA-DRB1 shared epitope, presence of anti-cyclic citrullinated peptide antibody, or degree of admixture from the European population. An African ancestry-specific genetic variant of CTLA4 appears to be associated with protection from RA in African Americans. This finding may explain, in part, the relatively low prevalence of RA in black African populations.

  20. Parent-of-origin-specific allelic associations among 106 genomic loci for age at menarche.

    PubMed

    Perry, John R B; Day, Felix; Elks, Cathy E; Sulem, Patrick; Thompson, Deborah J; Ferreira, Teresa; He, Chunyan; Chasman, Daniel I; Esko, Tõnu; Thorleifsson, Gudmar; Albrecht, Eva; Ang, Wei Q; Corre, Tanguy; Cousminer, Diana L; Feenstra, Bjarke; Franceschini, Nora; Ganna, Andrea; Johnson, Andrew D; Kjellqvist, Sanela; Lunetta, Kathryn L; McMahon, George; Nolte, Ilja M; Paternoster, Lavinia; Porcu, Eleonora; Smith, Albert V; Stolk, Lisette; Teumer, Alexander; Tšernikova, Natalia; Tikkanen, Emmi; Ulivi, Sheila; Wagner, Erin K; Amin, Najaf; Bierut, Laura J; Byrne, Enda M; Hottenga, Jouke-Jan; Koller, Daniel L; Mangino, Massimo; Pers, Tune H; Yerges-Armstrong, Laura M; Hua Zhao, Jing; Andrulis, Irene L; Anton-Culver, Hoda; Atsma, Femke; Bandinelli, Stefania; Beckmann, Matthias W; Benitez, Javier; Blomqvist, Carl; Bojesen, Stig E; Bolla, Manjeet K; Bonanni, Bernardo; Brauch, Hiltrud; Brenner, Hermann; Buring, Julie E; Chang-Claude, Jenny; Chanock, Stephen; Chen, Jinhui; Chenevix-Trench, Georgia; Collée, J Margriet; Couch, Fergus J; Couper, David; Coviello, Andrea D; Cox, Angela; Czene, Kamila; D'adamo, Adamo Pio; Davey Smith, George; De Vivo, Immaculata; Demerath, Ellen W; Dennis, Joe; Devilee, Peter; Dieffenbach, Aida K; Dunning, Alison M; Eiriksdottir, Gudny; Eriksson, Johan G; Fasching, Peter A; Ferrucci, Luigi; Flesch-Janys, Dieter; Flyger, Henrik; Foroud, Tatiana; Franke, Lude; Garcia, Melissa E; García-Closas, Montserrat; Geller, Frank; de Geus, Eco E J; Giles, Graham G; Gudbjartsson, Daniel F; Gudnason, Vilmundur; Guénel, Pascal; Guo, Suiqun; Hall, Per; Hamann, Ute; Haring, Robin; Hartman, Catharina A; Heath, Andrew C; Hofman, Albert; Hooning, Maartje J; Hopper, John L; Hu, Frank B; Hunter, David J; Karasik, David; Kiel, Douglas P; Knight, Julia A; Kosma, Veli-Matti; Kutalik, Zoltan; Lai, Sandra; Lambrechts, Diether; Lindblom, Annika; Mägi, Reedik; Magnusson, Patrik K; Mannermaa, Arto; Martin, Nicholas G; Masson, Gisli; McArdle, Patrick F; McArdle, Wendy L; Melbye, Mads; Michailidou, Kyriaki; Mihailov, Evelin; Milani, Lili; Milne, Roger L; Nevanlinna, Heli; Neven, Patrick; Nohr, Ellen A; Oldehinkel, Albertine J; Oostra, Ben A; Palotie, Aarno; Peacock, Munro; Pedersen, Nancy L; Peterlongo, Paolo; Peto, Julian; Pharoah, Paul D P; Postma, Dirkje S; Pouta, Anneli; Pylkäs, Katri; Radice, Paolo; Ring, Susan; Rivadeneira, Fernando; Robino, Antonietta; Rose, Lynda M; Rudolph, Anja; Salomaa, Veikko; Sanna, Serena; Schlessinger, David; Schmidt, Marjanka K; Southey, Mellissa C; Sovio, Ulla; Stampfer, Meir J; Stöckl, Doris; Storniolo, Anna M; Timpson, Nicholas J; Tyrer, Jonathan; Visser, Jenny A; Vollenweider, Peter; Völzke, Henry; Waeber, Gerard; Waldenberger, Melanie; Wallaschofski, Henri; Wang, Qin; Willemsen, Gonneke; Winqvist, Robert; Wolffenbuttel, Bruce H R; Wright, Margaret J; Boomsma, Dorret I; Econs, Michael J; Khaw, Kay-Tee; Loos, Ruth J F; McCarthy, Mark I; Montgomery, Grant W; Rice, John P; Streeten, Elizabeth A; Thorsteinsdottir, Unnur; van Duijn, Cornelia M; Alizadeh, Behrooz Z; Bergmann, Sven; Boerwinkle, Eric; Boyd, Heather A; Crisponi, Laura; Gasparini, Paolo; Gieger, Christian; Harris, Tamara B; Ingelsson, Erik; Järvelin, Marjo-Riitta; Kraft, Peter; Lawlor, Debbie; Metspalu, Andres; Pennell, Craig E; Ridker, Paul M; Snieder, Harold; Sørensen, Thorkild I A; Spector, Tim D; Strachan, David P; Uitterlinden, André G; Wareham, Nicholas J; Widen, Elisabeth; Zygmunt, Marek; Murray, Anna; Easton, Douglas F; Stefansson, Kari; Murabito, Joanne M; Ong, Ken K

    2014-10-01

    Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality. Studies of rare human disorders of puberty and animal models point to a complex hypothalamic-pituitary-hormonal regulation, but the mechanisms that determine pubertal timing and underlie its links to disease risk remain unclear. Here, using genome-wide and custom-genotyping arrays in up to 182,416 women of European descent from 57 studies, we found robust evidence (P < 5 × 10(-8)) for 123 signals at 106 genomic loci associated with age at menarche. Many loci were associated with other pubertal traits in both sexes, and there was substantial overlap with genes implicated in body mass index and various diseases, including rare disorders of puberty. Menarche signals were enriched in imprinted regions, with three loci (DLK1-WDR25, MKRN3-MAGEL2 and KCNK9) demonstrating parent-of-origin-specific associations concordant with known parental expression patterns. Pathway analyses implicated nuclear hormone receptors, particularly retinoic acid and γ-aminobutyric acid-B2 receptor signalling, among novel mechanisms that regulate pubertal timing in humans. Our findings suggest a genetic architecture involving at least hundreds of common variants in the coordinated timing of the pubertal transition. PMID:25231870

  1. Parent-of-origin specific allelic associations among 106 genomic loci for age at menarche

    PubMed Central

    Thompson, Deborah J; Ferreira, Teresa; He, Chunyan; Chasman, Daniel I; Esko, Tõnu; Thorleifsson, Gudmar; Albrecht, Eva; Ang, Wei Q; Corre, Tanguy; Cousminer, Diana L; Feenstra, Bjarke; Franceschini, Nora; Ganna, Andrea; Johnson, Andrew D; Kjellqvist, Sanela; Lunetta, Kathryn L; McMahon, George; Nolte, Ilja M; Paternoster, Lavinia; Porcu, Eleonora; Smith, Albert V; Stolk, Lisette; Teumer, Alexander; Tšernikova, Natalia; Tikkanen, Emmi; Ulivi, Sheila; Wagner, Erin K; Amin, Najaf; Bierut, Laura J; Byrne, Enda M; Hottenga, Jouke-Jan; Koller, Daniel L; Mangino, Massimo; Pers, Tune H; Yerges-Armstrong, Laura M; Zhao, Jing Hua; Andrulis, Irene L; Anton-Culver, Hoda; Atsma, Femke; Bandinelli, Stefania; Beckmann, Matthias W; Benitez, Javier; Blomqvist, Carl; Bojesen, Stig E; Bolla, Manjeet K; Bonanni, Bernardo; Brauch, Hiltrud; Brenner, Hermann; Buring, Julie E; Chang-Claude, Jenny; Chanock, Stephen; Chen, Jinhui; Chenevix-Trench, Georgia; Collée, J. Margriet; Couch, Fergus J; Couper, David; Coveillo, Andrea D; Cox, Angela; Czene, Kamila; D’adamo, Adamo Pio; Smith, George Davey; De Vivo, Immaculata; Demerath, Ellen W; Dennis, Joe; Devilee, Peter; Dieffenbach, Aida K; Dunning, Alison M; Eiriksdottir, Gudny; Eriksson, Johan G; Fasching, Peter A; Ferrucci, Luigi; Flesch-Janys, Dieter; Flyger, Henrik; Foroud, Tatiana; Franke, Lude; Garcia, Melissa E; García-Closas, Montserrat; Geller, Frank; de Geus, Eco EJ; Giles, Graham G; Gudbjartsson, Daniel F; Gudnason, Vilmundur; Guénel, Pascal; Guo, Suiqun; Hall, Per; Hamann, Ute; Haring, Robin; Hartman, Catharina A; Heath, Andrew C; Hofman, Albert; Hooning, Maartje J; Hopper, John L; Hu, Frank B; Hunter, David J; Karasik, David; Kiel, Douglas P; Knight, Julia A; Kosma, Veli-Matti; Kutalik, Zoltan; Lai, Sandra; Lambrechts, Diether; Lindblom, Annika; Mägi, Reedik; Magnusson, Patrik K; Mannermaa, Arto; Martin, Nicholas G; Masson, Gisli; McArdle, Patrick F; McArdle, Wendy L; Melbye, Mads; Michailidou, Kyriaki; Mihailov, Evelin; Milani, Lili; Milne, Roger L; Nevanlinna, Heli; Neven, Patrick; Nohr, Ellen A; Oldehinkel, Albertine J; Oostra, Ben A; Palotie, Aarno; Peacock, Munro; Pedersen, Nancy L; Peterlongo, Paolo; Peto, Julian; Pharoah, Paul DP; Postma, Dirkje S; Pouta, Anneli; Pylkäs, Katri; Radice, Paolo; Ring, Susan; Rivadeneira, Fernando; Robino, Antonietta; Rose, Lynda M; Rudolph, Anja; Salomaa, Veikko; Sanna, Serena; Schlessinger, David; Schmidt, Marjanka K; Southey, Mellissa C; Sovio, Ulla; Stampfer, Meir J; Stöckl, Doris; Storniolo, Anna M; Timpson, Nicholas J; Tyrer, Jonathan; Visser, Jenny A; Vollenweider, Peter; Völzke, Henry; Waeber, Gerard; Waldenberger, Melanie; Wallaschofski, Henri; Wang, Qin; Willemsen, Gonneke; Winqvist, Robert; Wolffenbuttel, Bruce HR; Wright, Margaret J; Boomsma, Dorret I; Econs, Michael J; Khaw, Kay-Tee; Loos, Ruth JF; McCarthy, Mark I; Montgomery, Grant W; Rice, John P; Streeten, Elizabeth A; Thorsteinsdottir, Unnur; van Duijn, Cornelia M; Alizadeh, Behrooz Z; Bergmann, Sven; Boerwinkle, Eric; Boyd, Heather A; Crisponi, Laura; Gasparini, Paolo; Gieger, Christian; Harris, Tamara B; Ingelsson, Erik; Järvelin, Marjo-Riitta; Kraft, Peter; Lawlor, Debbie; Metspalu, Andres; Pennell, Craig E; Ridker, Paul M; Snieder, Harold; Sørensen, Thorkild IA; Spector, Tim D; Strachan, David P; Uitterlinden, André G; Wareham, Nicholas J; Widen, Elisabeth; Zygmunt, Marek; Murray, Anna; Easton, Douglas F

    2014-01-01

    Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality1. Studies of rare human disorders of puberty and animal models point to a complex hypothalamic-pituitary-hormonal regulation2,3, but the mechanisms that determine pubertal timing and underlie its links to disease risk remain unclear. Here, using genome-wide and custom-genotyping arrays in up to 182,416 women of European descent from 57 studies, we found robust evidence (P<5×10−8) for 123 signals at 106 genomic loci associated with age at menarche. Many loci were associated with other pubertal traits in both sexes, and there was substantial overlap with genes implicated in body mass index and various diseases, including rare disorders of puberty. Menarche signals were enriched in imprinted regions, with three loci (DLK1/WDR25, MKRN3/MAGEL2 and KCNK9) demonstrating parent-of-origin specific associations concordant with known parental expression patterns. Pathway analyses implicated nuclear hormone receptors, particularly retinoic acid and gamma-aminobutyric acid-B2 receptor signaling, among novel mechanisms that regulate pubertal timing in humans. Our findings suggest a genetic architecture involving at least hundreds of common variants in the coordinated timing of the pubertal transition. PMID:25231870

  2. Parent-of-origin-specific allelic associations among 106 genomic loci for age at menarche.

    PubMed

    Perry, John R B; Day, Felix; Elks, Cathy E; Sulem, Patrick; Thompson, Deborah J; Ferreira, Teresa; He, Chunyan; Chasman, Daniel I; Esko, Tõnu; Thorleifsson, Gudmar; Albrecht, Eva; Ang, Wei Q; Corre, Tanguy; Cousminer, Diana L; Feenstra, Bjarke; Franceschini, Nora; Ganna, Andrea; Johnson, Andrew D; Kjellqvist, Sanela; Lunetta, Kathryn L; McMahon, George; Nolte, Ilja M; Paternoster, Lavinia; Porcu, Eleonora; Smith, Albert V; Stolk, Lisette; Teumer, Alexander; Tšernikova, Natalia; Tikkanen, Emmi; Ulivi, Sheila; Wagner, Erin K; Amin, Najaf; Bierut, Laura J; Byrne, Enda M; Hottenga, Jouke-Jan; Koller, Daniel L; Mangino, Massimo; Pers, Tune H; Yerges-Armstrong, Laura M; Hua Zhao, Jing; Andrulis, Irene L; Anton-Culver, Hoda; Atsma, Femke; Bandinelli, Stefania; Beckmann, Matthias W; Benitez, Javier; Blomqvist, Carl; Bojesen, Stig E; Bolla, Manjeet K; Bonanni, Bernardo; Brauch, Hiltrud; Brenner, Hermann; Buring, Julie E; Chang-Claude, Jenny; Chanock, Stephen; Chen, Jinhui; Chenevix-Trench, Georgia; Collée, J Margriet; Couch, Fergus J; Couper, David; Coviello, Andrea D; Cox, Angela; Czene, Kamila; D'adamo, Adamo Pio; Davey Smith, George; De Vivo, Immaculata; Demerath, Ellen W; Dennis, Joe; Devilee, Peter; Dieffenbach, Aida K; Dunning, Alison M; Eiriksdottir, Gudny; Eriksson, Johan G; Fasching, Peter A; Ferrucci, Luigi; Flesch-Janys, Dieter; Flyger, Henrik; Foroud, Tatiana; Franke, Lude; Garcia, Melissa E; García-Closas, Montserrat; Geller, Frank; de Geus, Eco E J; Giles, Graham G; Gudbjartsson, Daniel F; Gudnason, Vilmundur; Guénel, Pascal; Guo, Suiqun; Hall, Per; Hamann, Ute; Haring, Robin; Hartman, Catharina A; Heath, Andrew C; Hofman, Albert; Hooning, Maartje J; Hopper, John L; Hu, Frank B; Hunter, David J; Karasik, David; Kiel, Douglas P; Knight, Julia A; Kosma, Veli-Matti; Kutalik, Zoltan; Lai, Sandra; Lambrechts, Diether; Lindblom, Annika; Mägi, Reedik; Magnusson, Patrik K; Mannermaa, Arto; Martin, Nicholas G; Masson, Gisli; McArdle, Patrick F; McArdle, Wendy L; Melbye, Mads; Michailidou, Kyriaki; Mihailov, Evelin; Milani, Lili; Milne, Roger L; Nevanlinna, Heli; Neven, Patrick; Nohr, Ellen A; Oldehinkel, Albertine J; Oostra, Ben A; Palotie, Aarno; Peacock, Munro; Pedersen, Nancy L; Peterlongo, Paolo; Peto, Julian; Pharoah, Paul D P; Postma, Dirkje S; Pouta, Anneli; Pylkäs, Katri; Radice, Paolo; Ring, Susan; Rivadeneira, Fernando; Robino, Antonietta; Rose, Lynda M; Rudolph, Anja; Salomaa, Veikko; Sanna, Serena; Schlessinger, David; Schmidt, Marjanka K; Southey, Mellissa C; Sovio, Ulla; Stampfer, Meir J; Stöckl, Doris; Storniolo, Anna M; Timpson, Nicholas J; Tyrer, Jonathan; Visser, Jenny A; Vollenweider, Peter; Völzke, Henry; Waeber, Gerard; Waldenberger, Melanie; Wallaschofski, Henri; Wang, Qin; Willemsen, Gonneke; Winqvist, Robert; Wolffenbuttel, Bruce H R; Wright, Margaret J; Boomsma, Dorret I; Econs, Michael J; Khaw, Kay-Tee; Loos, Ruth J F; McCarthy, Mark I; Montgomery, Grant W; Rice, John P; Streeten, Elizabeth A; Thorsteinsdottir, Unnur; van Duijn, Cornelia M; Alizadeh, Behrooz Z; Bergmann, Sven; Boerwinkle, Eric; Boyd, Heather A; Crisponi, Laura; Gasparini, Paolo; Gieger, Christian; Harris, Tamara B; Ingelsson, Erik; Järvelin, Marjo-Riitta; Kraft, Peter; Lawlor, Debbie; Metspalu, Andres; Pennell, Craig E; Ridker, Paul M; Snieder, Harold; Sørensen, Thorkild I A; Spector, Tim D; Strachan, David P; Uitterlinden, André G; Wareham, Nicholas J; Widen, Elisabeth; Zygmunt, Marek; Murray, Anna; Easton, Douglas F; Stefansson, Kari; Murabito, Joanne M; Ong, Ken K

    2014-10-01

    Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality. Studies of rare human disorders of puberty and animal models point to a complex hypothalamic-pituitary-hormonal regulation, but the mechanisms that determine pubertal timing and underlie its links to disease risk remain unclear. Here, using genome-wide and custom-genotyping arrays in up to 182,416 women of European descent from 57 studies, we found robust evidence (P < 5 × 10(-8)) for 123 signals at 106 genomic loci associated with age at menarche. Many loci were associated with other pubertal traits in both sexes, and there was substantial overlap with genes implicated in body mass index and various diseases, including rare disorders of puberty. Menarche signals were enriched in imprinted regions, with three loci (DLK1-WDR25, MKRN3-MAGEL2 and KCNK9) demonstrating parent-of-origin-specific associations concordant with known parental expression patterns. Pathway analyses implicated nuclear hormone receptors, particularly retinoic acid and γ-aminobutyric acid-B2 receptor signalling, among novel mechanisms that regulate pubertal timing in humans. Our findings suggest a genetic architecture involving at least hundreds of common variants in the coordinated timing of the pubertal transition.

  3. A GWAS SNP for Schizophrenia Is Linked to the Internal MIR137 Promoter and Supports Differential Allele-Specific Expression

    PubMed Central

    Warburton, Alix; Breen, Gerome; Bubb, Vivien J.; Quinn, John P.

    2016-01-01

    Single nucleotide polymorphisms (SNPs) within the MIR137 gene locus have been shown to confer risk for schizophrenia through genome-wide association studies (GWAS). The expression levels of microRNA-137 (miR-137) and its validated gene targets have also been shown to be disrupted in several neuropsychiatric conditions, including schizophrenia. Regulation of miR-137 expression is thus imperative for normal neuronal functioning. We previously characterized an internal promoter domain within the MIR137 gene that contained a variable number tandem repeat (VNTR) polymorphism and could alter the in vitro levels of miR-137 in a stimulus-induced and allele-specific manner. We now demonstrate that haplotype tagging-SNP analysis linked the rs1625579 GWAS SNP for schizophrenia to this internal MIR137 promoter through a proxy SNP rs2660304 located at this domain. We postulated that the rs2660304 promoter SNP may act as predisposing factor for schizophrenia through altering the levels of miR-137 expression in a genotype-dependent manner. Reporter gene analysis of the internal MIR137 promoter containing the common VNTR variant demonstrated genotype-dependent differences in promoter activity with respect to rs2660304. In line with previous reports, the major allele of the rs2660304 proxy SNP, which has previously been linked with schizophrenia risk through genetic association, resulted in downregulation of reporter gene expression in a tissue culture model. The genetic influence of the rs2660304 proxy SNP on the transcriptional activity of the internal MIR137 promoter, and thus the levels of miR-137 expression, therefore offers a distinct regulatory mechanism to explain the functional significance of the rs1625579 GWAS SNP for schizophrenia risk. PMID:26429811

  4. Genome-wide identification of allele-specific expression in response to Streptococcus suis 2 infection in two differentially susceptible pig breeds.

    PubMed

    Wu, Huayu; Gaur, Uma; Mekchay, Supamit; Peng, Xianwen; Li, Lianghua; Sun, Hua; Song, Zhongxu; Dong, Binke; Li, Mingbo; Wimmers, Klaus; Ponsuksili, Siriluck; Li, Kui; Mei, Shuqi; Liu, Guisheng

    2015-11-01

    Although allele expression imbalance has been recognized in many species, and strongly linked to diseases, no whole transcriptome allele imbalance has been detected in pigs during pathogen infections. The pathogen Streptococcus suis 2 (SS2) causes serious zoonotic disease. Different pig breeds show differential susceptibility/resistance to pathogen infection, but the biological insight is little known. Here we analyzed allele-specific expression (ASE) using the spleen transcriptome of four pigs belonging to two phenotypically different breeds after SS2 infection. The comparative analysis of allele specific SNPs between control and infected animals revealed 882 and 1096 statistically significant differentially expressed allele SNPs (criteria: ratio ≧ 2 or ≦ 0.5) in Landrace and Enshi black pig, respectively. Twenty nine allelically imbalanced SNPs were further verified by Sanger sequencing, and later six SNPs were quantified by pyrosequencing assay. The pyrosequencing results are in agreement with the RNA-seq results, except two SNPs. Looking at the role of ASE in predisposition to diseases, the discovery of causative variants by ASE analysis might help the pig industry in long term to design breeding programs for improving SS2 resistance.

  5. Identification and Evolution of Functional Alleles of the Previously Described Pollen Specific Myrosinase Pseudogene AtTGG6 in Arabidopsis thaliana

    PubMed Central

    Fu, Lili; Han, Bingying; Tan, Deguan; Wang, Meng; Ding, Mei; Zhang, Jiaming

    2016-01-01

    Myrosinases are β-thioglucoside glucohydrolases and serve as defense mechanisms against insect pests and pathogens by producing toxic compounds. AtTGG6 in Arabidopsis thaliana was previously reported to be a myrosinase pseudogene but specifically expressed in pollen. However, we found that AlTGG6, an ortholog to AtTGG6 in A. lyrata (an outcrossing relative of A. thaliana) was functional, suggesting that functional AtTGG6 alleles may still exist in A. thaliana. AtTGG6 alleles in 29 A. thaliana ecotypes were cloned and sequenced. Results indicate that ten alleles were functional and encoded Myr II type myrosinase of 512 amino acids, and myrosinase activity was confirmed by overexpressing AtTGG6 in Pichia pastoris. However, the 19 other ecotypes had disabled alleles with highly polymorphic frame-shift mutations and diversified sequences. Thirteen frame-shift mutation types were identified, which occurred independently many times in the evolutionary history within a few thousand years. The functional allele was expressed specifically in pollen similar to the disabled alleles but at a higher expression level, suggesting its role in defense of pollen against insect pests such as pollen beetles. However, the defense function may have become less critical after A. thaliana evolved to self-fertilization, and thus resulted in loss of function in most ecotypes. PMID:26907263

  6. HLA-DRB1 and HLA-DQB1 allele associations in an Albanian patient population with rheumatoid arthritis: correlations with the specific autoantibody markers and inter-population DRB1 allele frequency variability.

    PubMed

    Prifti-Kurti, Margarita; Nunes, José Manuel; Shyti, Erkena; Ylli, Zamira; Sanchez-Mazas, Alicia; Sulcebe, Genc

    2014-08-01

    The prevalence of rheumatoid arthritis and its specific autoantibodies varies in different populations. This variability depends on the genetic polymorphism of the immune response genes among which the HLA system plays a major role. In this context, we studied the HLA-DRB1 and HLA-DQB1 first-level allele frequencies in 100 Albanian patients with rheumatoid arthritis (RA), and taking into account their rheumatoid factor (RF) and anticitrullinated peptide antibodies (ACPA) serologic subgroups, we compared them with the respective frequencies in a population of 191 Albanian individuals without known pathology. No differences were found between the controls and the RA patient group as a whole, but three statistically significant differences were found: an increase in DRB1*04 among ACPA+, RF+ and ACPA+/RF+ patients, a significant decrease in DRB1*11 among ACPA+/RF+ and also a decrease in DRB1*13 among RF+ patient subgroups. Comparing allele frequencies of putatively associated RA alleles in different European populations revealed a significant negative correlation between the RA predisposing DRB1*04 and protective DRB1*11 allele frequencies. A statistically significant correlation was also found between RA prevalence rates and DRB1*04 as well as DRB1*11 frequencies. The relatively low frequencies of DRB1*04 and high DRB1*11 in the Albanian population might explain the rather low positivity rate of ACPA and RF antibodies among the Albanian RA patients. These specific association patterns suggest that this first study of RA in an Albanian population should be followed up to include second level or higher definition of HLA alleles and to compare RA patterns among European populations.

  7. Linear allele-specific long-range amplification: a novel method of long-range molecular haplotyping.

    PubMed

    Wu, Wei-Ming; Tsai, Hsiang-Ju; Pang, Jong-Hwei S; Wang, Tzu-Hao; Wang, Hsin-Shih; Hong, Hong-Shang; Lee, Yun-Shien

    2005-10-01

    Haplotypes have been repeatedly shown to be more powerful than collections of single-locus markers in gene-mapping studies. Various haplotyping methods including statistical estimation are employed but molecular haplotyping, the acquisition of information directly on physical DNA sequences, has been in demand for its accuracy and independence from family pedigrees. We investigated the allelic specificity of long-range PCR, which was successful for long-range haplotyping in recent reports, and found problems of initial mispriming and crossover amplification significantly confounded its application. Based on these observations, we designed a novel method based on linear amplification of a hemizygous DNA segment with a single phosphorothioate-modified oligonucleotide. Our results revealed, with a single nucleotide polymorphism as the discriminative marker, downstream haplotypes of 14-15 kb DNA segment could be confidently scored. With two rounds of the method and five single nucleotide polymorphisms, molecular haplotypes of 29.3 kb spanning the HCR and CDSN genes, two genes associated with the susceptibility of psoriasis, of 11 members, belonging to a CEPH family, were revealed. Clear Mendelian segregation of 35 highly heterozygous SNPs confirmed the accuracy of the method. Problems of low specificity associated with long-range PCR were not observed. The simplicity, along with long-sequence accessibility and feasibility of a single nucleotide difference as the discriminative marker indicated our method holds promise for future gene-mapping studies.

  8. CYP2D6: novel genomic structures and alleles

    PubMed Central

    Kramer, Whitney E.; Walker, Denise L.; O’Kane, Dennis J.; Mrazek, David A.; Fisher, Pamela K.; Dukek, Brian A.; Bruflat, Jamie K.; Black, John L.

    2010-01-01

    Objective CYP2D6 is a polymorphic gene. It has been observed to be deleted, to be duplicated and to undergo recombination events involving the CYP2D7 pseudogene and surrounding sequences. The objective of this study was to discover the genomic structure of CYP2D6 recombinants that interfere with clinical genotyping platforms that are available today. Methods Clinical samples containing rare homozygous CYP2D6 alleles, ambiguous readouts, and those with duplication signals and two different alleles were analyzed by long-range PCR amplification of individual genes, PCR fragment analysis, allele-specific primer extension assay, and DNA sequencing to characterize alleles and genomic structure. Results Novel alleles, genomic structures, and the DNA sequence of these structures are described. Interestingly, in 49 of 50 DNA samples that had CYP2D6 gene duplications or multiplications where two alleles were detected, the chromosome containing the duplication or multiplication had identical tandem alleles. Conclusion Several new CYP2D6 alleles and genomic structures are described which will be useful for CYP2D6 genotyping. The findings suggest that the recombination events responsible for CYP2D6 duplications and multiplications are because of mechanisms other than interchromosomal crossover during meiosis. PMID:19741566

  9. Phenotypic effect of substitution of allelic variants for a histone H1 subtype specific for growing tissues in the garden pea (Pisum sativum L.).

    PubMed

    Bogdanova, Vera S; Kosterin, Oleg E; Berdnikov, Vladimir A

    2007-05-01

    In pea, subtype H1-7 of histone H1 is specific for young actively growing tissues and disappears from chromatin of mature tissues. We sequenced the alleles coding for three main variants, numbered according to the increase of the electrophoretic mobility. Allele 1 differs from the most common allele 2 by eight nucleotide substitutions, two of them associated with amino acid replacements, His->Tyr in the globular domain and Ala->Val in the C-terminal domain. Allele 3 differs from alleles 1 and 2 by a 24-bp deletion in the part coding for the C-terminal domain. In three greenhouse experiments, we compared quantitative traits in nearly isogenic lines differing by these H1-7 variants. In experiment 1, three lines bearing either of the three allelic variants were compared, the other experiments involved pairs of lines bearing variants 1 and 3. In all experiments, statistically significant differences between the lines were registered, mostly related to the plant size. The most prominent effect was associated with plant growth dynamics. Plants of line 3, carrying the 8-amino acid deletion in histone H1-7, on average grew slower. In two experiments, the differences of the mean stem length persisted throughout plant growth while in experiment 2 differences disappeared upon maturity. The H1-7 subtype is supposed to be related to maintenance of chromatin state characteristic for cell growth and division. PMID:16900316

  10. A and MdMYB1 allele-specific markers controlling apple (Malus x domestica Borkh.) skin color and suitability for marker-assisted selection.

    PubMed

    Zhang, X J; Wang, L X; Chen, X X; Liu, Y L; Meng, R; Wang, Y J; Zhao, Z Y

    2014-01-01

    Pre-selection for fruit skin color at the seedling stage would be highly advantageous, with marker-assisted selection offering a potential method for apple pre-selection. A and MdMYB1 alleles are allele-specific DNA markers that are potentially associated with apple skin color, and co-segregate with the Rf and Rni loci, respectively. Here, we assessed the potential application of these 2 alleles for marker-assisted breeding across 30 diverse cultivars and 2 apple seedling progenies. The red skin color phenotype was usually associated with the MdMYB1-1 allele and A(1) allele, respectively, while the 2 molecular markers provided approximately 91% predictability in the 'Fuji' x 'Cripps Pink' and 'Fuji' x 'Gala' progenies. The results obtained from the 30 cultivars and 2 progenies were consistent for the 2 molecular markers. Hence, the results supported that Rf and Rni could be located in a gene cluster, or even correspond to alleles of the same gene. Our results are consistent with the hypothesis that red/yellow dimorphism is controlled by a monogenic system, with the presence of the red anthocyanin pigmentation being dominant. In addition, our results supported that the practical utilization of the 2 function markers to efficiently and accurately select red-skinned apple cultivars in apple scion breeding programs.

  11. Allele-specific CAPS markers based on point mutations in resistance alleles at the pvr1 locus encoding eIF4E in Capsicum.

    PubMed

    Yeam, Inhwa; Kang, Byoung-Cheorl; Lindeman, Wouter; Frantz, James D; Faber, Nanne; Jahn, Molly M

    2005-12-01

    Marker-assisted selection has been widely implemented in crop breeding and can be especially useful in cases where the traits of interest show recessive or polygenic inheritance and/or are difficult or impossible to select directly. Most indirect selection is based on DNA polymorphism linked to the target trait, resulting in error when the polymorphism recombines away from the mutation responsible for the trait and/or when the linkage between the mutation and the polymorphism is not conserved in all relevant genetic backgrounds. In this paper, we report the generation and use of molecular markers that define loci for selection using cleaved amplified polymorphic sequences (CAPS). These CAPS markers are based on nucleotide polymorphisms in the resistance gene that are perfectly correlated with disease resistance, the trait of interest. As a consequence, the possibility that the marker will not be linked to the trait in all backgrounds or that the marker will recombine away from the trait is eliminated. We have generated CAPS markers for three recessive viral resistance alleles used widely in pepper breeding, pvr1, pvr1 (1), and pvr1 (2). These markers are based on single nucleotide polymorphisms (SNPs) within the coding region of the pvr1 locus encoding an eIF4E homolog on chromosome 3. These three markers define a system of indirect selection for potyvirus resistance in Capsicum based on genomic sequence. We demonstrate the utility of this marker system using commercially significant germplasm representing two Capsicum species. Application of these markers to Capsicum improvement is discussed.

  12. Multiplex Polymerase Chain Reaction Identification of Single Individuals of the Longidorid Nematodes Xiphinema index, X. diversicaudatum, X. vuittenezi, and X. italiae Using Specific Primers from Ribosomal Genes.

    PubMed

    Wang, Xinrong; Bosselut, Nathalie; Castagnone, Chantal; Voisin, Roger; Abad, Pierre; Esmenjaud, Daniel

    2003-02-01

    ABSTRACT The species X. index, X. diversicaudatum, X. vuittenezi, and X. italiae are established (E) or putative (P) vectors of Grapevine fanleaf virus (GFLV) (E), Arabis mosaic virus (E), Grapevine chrome mosaic virus (P), and GFLV (P) nepoviruses of grapevine, respectively. All four species are very closely related taxonomically and their low field densities make them difficult to identify from morphological and morphometrical diagnostic characters when only single or few individuals are detected. To improve diagnostic accuracy, a simple method was developed. The internal transcribed spacer 1 (ITS1) region spanning the 18S and 5.8S ribosomal genes was sequenced in one population of each species using two conserved primers from these genes. The ITS1 fragments were 1,132 bp (X. vuittenezi), 1,153 bp (X. index), 1,175 bp (X. diversicaudatum), and 1,190 bp (X. italiae), i.e., a difference of over 5% between the extremes. The sequence variability made it possible to design species-specific internal sense primers that amplified, in combination with the same antisense ITS1 primer, a single signature fragment (340 bp for X. index, 414 bp for X. italiae, 591 bp for X. vuittenezi, and 813 bp for X. diversicaudatum). Tests with DNA from a single adult or juvenile nematode confirmed the specificity of the primers from diverse isolates or populations. The primers were successfully used in a multiplex test for the reliable detection of two to four mixed species, each represented by a single individual. This multiplex-based diagnostic tool will be particularly useful for successful nematode management practices in vineyards.

  13. Pathway Analysis Using Information from Allele-Specific Gene Methylation in Genome-Wide Association Studies for Bipolar Disorder

    PubMed Central

    Chuang, Li-Chung; Kao, Chung-Feng; Shih, Wei-Liang; Kuo, Po-Hsiu

    2013-01-01

    Bipolar disorder (BPD) is a complex psychiatric trait with high heritability. Despite efforts through conducting genome-wide association (GWA) studies, the success of identifying susceptibility loci for BPD has been limited, which is partially attributed to the complex nature of its pathogenesis. Pathway-based analytic strategy is a powerful tool to explore joint effects of gene sets within specific biological pathways. Additionally, to incorporate other aspects of genomic data into pathway analysis may further enhance our understanding for the underlying mechanisms for BPD. Patterns of DNA methylation play important roles in regulating gene expression and function. A commonly observed phenomenon, allele-specific methylation (ASM) describes the associations between genetic variants and DNA methylation patterns. The present study aimed to identify biological pathways that are involve in the pathogenesis of BPD while incorporating brain specific ASM information in pathway analysis using two large-scale GWA datasets in Caucasian populations. A weighting scheme was adopted to take ASM information into consideration for each pathway. After multiple testing corrections, we identified 88 and 15 enriched pathways for their biological relevance for BPD in the Genetic Association Information Network (GAIN) and the Wellcome Trust Case Control Consortium dataset, respectively. Many of these pathways were significant only when applying the weighting scheme. Three ion channel related pathways were consistently identified in both datasets. Results in the GAIN dataset also suggest for the roles of extracellular matrix in brain for BPD. Findings from Gene Ontology (GO) analysis exhibited functional enrichment among genes of non-GO pathways in activity of gated channel, transporter, and neurotransmitter receptor. We demonstrated that integrating different data sources with pathway analysis provides an avenue to identify promising and novel biological pathways for exploring the

  14. [Screening of peafowl microsatellite primers and analysis of genetic diversity].

    PubMed

    Bao, Wen-Bin; Chen, Guo-Hong; Shu, Jing-Ting; Xu, Qi; Li, Hui-Fang

    2006-10-01

    The applicability of chicken microsatellite primers to peafowl population was analyzed in the present paper, and the results showed 14 of 29 pairs of microsatellite primers from chicken could amplify peafowl DNA and produce specific allele patterns. A mean of 1.71 alleles was found for each locus. Seven pairs were highly polymorphic, and MCW0080 and MCW0098 were ideal markers for peafowl. Genetic diversity analysis within and between the green peafowl and the blue peafowl populations demonstrated that the expected heterozygosity of two peafowl populations were 0.2482 and 0.2744, respectively. The inbreeding index (FST), Reynolds' genetic distance and gene flow between the two populations were 0.078, 0.0603 and 3.896 respectively. These results indicate that the heterozygosity and the genetic diversity of these two peafowl populations were very low, and suggest a tendency towards intermixing.

  15. [Screening of peafowl microsatellite primers and analysis of genetic diversity].

    PubMed

    Bao, Wen-Bin; Chen, Guo-Hong; Shu, Jing-Ting; Xu, Qi; Li, Hui-Fang

    2006-10-01

    The applicability of chicken microsatellite primers to peafowl population was analyzed in the present paper, and the results showed 14 of 29 pairs of microsatellite primers from chicken could amplify peafowl DNA and produce specific allele patterns. A mean of 1.71 alleles was found for each locus. Seven pairs were highly polymorphic, and MCW0080 and MCW0098 were ideal markers for peafowl. Genetic diversity analysis within and between the green peafowl and the blue peafowl populations demonstrated that the expected heterozygosity of two peafowl populations were 0.2482 and 0.2744, respectively. The inbreeding index (FST), Reynolds' genetic distance and gene flow between the two populations were 0.078, 0.0603 and 3.896 respectively. These results indicate that the heterozygosity and the genetic diversity of these two peafowl populations were very low, and suggest a tendency towards intermixing. PMID:17035182

  16. Design and application of specific 16S rDNA-targeted primers for assessing endophytic diversity in Dendrobium officinale using nested PCR-DGGE.

    PubMed

    Yu, Jie; Zhou, Xiao-Feng; Yang, Sui-Juan; Liu, Wen-Hong; Hu, Xiu-Fang

    2013-11-01

    Novel specific 16S rDNA-targeted primers were successfully designed and applied to the characterization of endophytic diversity in Dendrobium officinale. Using the popular universal bacterial primers 27f/1492r, the fragments of chloroplast and mitochondrion 16S/18S rDNA were amplified from D. officinale. They shared high nucleotide identity with the chloroplast 16S rDNAs (99-100 %) and with the mitochondrion 18S rDNAs (93-100 %) from various plants, respectively, and both shared 73-86 % identities with the bacterial 16S rDNA sequences in GenBank. The current bacterial universal primers, including 27f/1492r, match well with the chloroplast and mitochondrion 16S/18S rDNAs, which accordingly renders these primers not useful for endophytic diversity analysis. Novel 16S rDNA-targeted primers fM1 (5'-CCGCGTGNRBGAHGAAGGYYYT-3') and rC5 (5'-TAATCCTGTTTGCTCC CCAC-3') were designed, which show good specificity compared to the 16S/18S rDNAs of D. officinale, and perfect universality within bacteria except for Cyanobacteria. The primers fM1/rC5, together with 515f-GC/rC5, which overlaps the whole V4 region of 16S rDNA, were subjected to nested polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) to analyze the diversity of endophytic bacteria in D. officinale from three different sources in China. The results showed diversities in roots and stems of the plants from all three locations. Altogether, 29 bands were identified as bacteria, with the dominant group being Proteobacteria and the dominant genus being Burkholderia, some of which commonly has the function of nitrogen fixation and thus may play potentially important roles in D. officinale. Therefore, the nested PCR-DGGE method based on the novel primers provides a good alternative for investigating the communities and roles of endophytes in D. officinale.

  17. The establishment of species-specific primers for the molecular identification of ten stored-product psocids based on ITS2 rDNA

    PubMed Central

    Zhao, Zi-Hua; Cui, Bing-Yi; Li, Zhi-Hong; Jiang, Fan; Yang, Qian-Qian; Kučerová, Zuzana; Stejskal, Václav; Opit, George; Cao, Yang; Li, Fu-Jun

    2016-01-01

    Psocids are important stored product pests found worldwide that can be spread through grain trade. Most stored-product psocids, including eggs, nymphs, and adults, are very small (~1 mm) and difficult to identify morphologically. Here, we collected 10 economically important stored-product Liposcelis spp. psocids (L. bostrychophila, L. entomophila, L. decolor, L. paeta, L. brunnea, L. corrodens, L. mendax, L. rufa, L. pearmani, and L. tricolor) from 35 geographical locations in 5 countries (China, Czech Republic, Denmark, Germany, and the United States). The ITS2 rDNA gene was extracted and sequenced. The interspecific genetic distance of the stored-product psocids was significantly higher than the intraspecific genetic distance according to the barcoding gap analysis. Ten pairs of species-specific primers based on the ITS2 rDNA were developed for psocid identification. The sensitivity estimation indicated that the species-specific primers could correctly amplify the target ITS2 gene and successfully identify psocids at 1.0 ng/mL. Additionally, these species-specific primers could quantify specificity and identify 10 stored-product psocids; this approach could also be used to accurately identify other stored-product psocids. This work provides a practical approach for the precise examination of 10 stored-product psocid species and also contributes to the development of an identification method using ITS2 rDNA. PMID:26880378

  18. Allele-specific expression of mutated in colorectal cancer (MCC) gene and alternative susceptibility to colorectal cancer in schizophrenia.

    PubMed

    Wang, Yang; Cao, Yanfei; Huang, Xiaoye; Yu, Tao; Wei, Zhiyun; McGrath, John; Xu, Fei; Bi, Yan; Li, Xingwang; Yang, Fengping; Li, Weidong; Zou, Xia; Peng, Zhihai; Xiao, Yanzeng; Zhang, Yan; He, Lin; He, Guang

    2016-01-01

    Evidence has indicated that the incidence of colorectal cancer (CRC) among schizophrenia is lower than normal. To explore this potential protective effect, we employed an innovative strategy combining association study with allele-specific expression (ASE) analysis in MCC gene. We first genotyped four polymorphisms within MCC in 312 CRC patients, 270 schizophrenia patients and 270 controls. Using the MassArray technique, we performed ASE measurements in a second sample series consisting of 50 sporadic CRC patients, 50 schizophrenia patients and 52 controls. Rs2227947 showed significant differences between schizophrenia cases and controls, and haplotype analysis reported some significant discrepancies among these three subject groups. ASE values of rs2227948 and rs2227947 presented consistently differences between CRC (or schizophrenia) patients and controls. Of the three groups, highest frequencies of ASE in MCC were concordantly found in CRC group, whereas lowest frequencies of ASE were observed in schizophrenia group. Similar trends were confirmed in both haplotype frequencies and ASE frequencies (i.e. CRC > control > schizophrenia). We provide a first indication that MCC might confer alterative genetic susceptibility to CRC in individuals with schizophrenia promising to shed more light on the relationship between schizophrenia and cancer progression. PMID:27226254

  19. Endochondral ossification pathway genes and postmenopausal osteoporosis: Association and specific allele related serum bone sialoprotein levels in Han Chinese

    PubMed Central

    Zhang, Yunzhi; Liu, Haiyan; Zhang, Chen; Zhang, Tianxiao; Zhang, Bo; Li, Lu; Chen, Gang; Fu, Dongke; Wang, KunZheng

    2015-01-01

    Osteoporosis is a systemic skeletal disorder characterized by reduced bone mineral density (BMD) and disrupted bone architecture, predisposing the patient to increased fracture risk. Evidence from early genetic epidemiological studies has indicated a major role for genetics in the development of osteoporosis and the variation in BMD. In this study, we focused on two key genes in the endochondral ossification pathway, IBSP and PTHLH. Over 9,000 postmenopausal Han Chinese women were recruited, and 54 SNPs were genotyped. Two significant SNPs within IBSP, rs1054627 and rs17013181, were associated with BMD and postmenopausal osteoporosis by the two-stage strategy, and rs17013181 was also significantly associated with serum IBSP levels. Moreover, one haplotype (rs12425376-rs10843047-rs42294) covering the 5’ end of PTHLH was associated with postmenopausal osteoporosis. Our results provide evidence for the association of these two key endochondral ossification pathway genes with BMD and osteoporosis in postmenopausal Han Chinese women. Combined with previous findings, we provide evidence that a particular SNP in IBSP has an allele-specific effect on mRNA levels, which would, in turn, reflect serum IBSP levels. PMID:26568273

  20. Endochondral ossification pathway genes and postmenopausal osteoporosis: Association and specific allele related serum bone sialoprotein levels in Han Chinese.

    PubMed

    Zhang, Yunzhi; Liu, Haiyan; Zhang, Chen; Zhang, Tianxiao; Zhang, Bo; Li, Lu; Chen, Gang; Fu, Dongke; Wang, KunZheng

    2015-11-16

    Osteoporosis is a systemic skeletal disorder characterized by reduced bone mineral density (BMD) and disrupted bone architecture, predisposing the patient to increased fracture risk. Evidence from early genetic epidemiological studies has indicated a major role for genetics in the development of osteoporosis and the variation in BMD. In this study, we focused on two key genes in the endochondral ossification pathway, IBSP and PTHLH. Over 9,000 postmenopausal Han Chinese women were recruited, and 54 SNPs were genotyped. Two significant SNPs within IBSP, rs1054627 and rs17013181, were associated with BMD and postmenopausal osteoporosis by the two-stage strategy, and rs17013181 was also significantly associated with serum IBSP levels. Moreover, one haplotype (rs12425376-rs10843047-rs42294) covering the 5' end of PTHLH was associated with postmenopausal osteoporosis. Our results provide evidence for the association of these two key endochondral ossification pathway genes with BMD and osteoporosis in postmenopausal Han Chinese women. Combined with previous findings, we provide evidence that a particular SNP in IBSP has an allele-specific effect on mRNA levels, which would, in turn, reflect serum IBSP levels.

  1. Genome-wide identification and quantification of cis- and trans-regulated genes responding to Marek's disease virus infection via analysis of allele-specific expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Marek’s disease (MD) is a commercially important neoplastic disease of chickens caused by the Marek’s disease virus (MDV), a naturally-occurring oncogenic alphaherpesvirus. We attempted to identify genes conferring MD resistance, by completing a genome-wide screen for allele-specific expr...

  2. Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-Specific Assay.

    PubMed

    Zhang, Guoqing; Cai, Fangping; de Rivera, Ivette Lorenzana; Zhou, Zhiyong; Zhang, Jing; Nkengasong, John; Gao, Feng; Yang, Chunfu

    2016-01-01

    A multiplex allele-specific (MAS) assay has been developed for the detection of HIV-1 subtype C drug resistance mutations (DRMs). We have optimized the MAS assay to determine subtype B DRMs in dried blood spots (DBS) collected from patients on antiretroviral therapy. The new assay accurately detected DRMs, including low-abundance mutations that were often missed by Sanger sequencing. PMID:26560533

  3. The Length Distribution of Class I-Restricted T Cell Epitopes Is Determined by Both Peptide Supply and MHC Allele-Specific Binding Preference.

    PubMed

    Trolle, Thomas; McMurtrey, Curtis P; Sidney, John; Bardet, Wilfried; Osborn, Sean C; Kaever, Thomas; Sette, Alessandro; Hildebrand, William H; Nielsen, Morten; Peters, Bjoern

    2016-02-15

    HLA class I-binding predictions are widely used to identify candidate peptide targets of human CD8(+) T cell responses. Many such approaches focus exclusively on a limited range of peptide lengths, typically 9 aa and sometimes 9-10 aa, despite multiple examples of dominant epitopes of other lengths. In this study, we examined whether epitope predictions can be improved by incorporating the natural length distribution of HLA class I ligands. We found that, although different HLA alleles have diverse length-binding preferences, the length profiles of ligands that are naturally presented by these alleles are much more homogeneous. We hypothesized that this is due to a defined length profile of peptides available for HLA binding in the endoplasmic reticulum. Based on this, we created a model of HLA allele-specific ligand length profiles and demonstrate how this model, in combination with HLA-binding predictions, greatly improves comprehensive identification of CD8(+) T cell epitopes. PMID:26783342

  4. Evaluation of a blood-specific DNA methylated region and trial for allele-specific blood identification from mixed body fluid DNA.

    PubMed

    Watanabe, Ken; Akutsu, Tomoko; Takamura, Ayari; Sakurada, Koichi

    2016-09-01

    The identification of blood samples obtained from crime scenes has been an important step in forensic investigation. Recently, a novel approach using the blood-specific methylated CpG site cg06379435 has been reported. In this study, we developed a real-time polymerase-chain-reaction-based method that can simply and rapidly quantitate the methylation ratio of cg06379435 and its neighboring CpGs and set the threshold ratios for blood identification by analyzing various body fluid samples. Blood identification using the thresholds was successfully performed in the analysis of a small amount (1ng) of DNA from blood and various aged blood samples, including 29-year-old stains. We also demonstrated a test for allele-specific blood identification from a mixed DNA sample by bisulfite sequencing analysis of these CpG sites and their neighboring single nucleotide polymorphism, rs7359943 (A/G), which is of relevance in cases where mixed samples are obtained from crime scenes. The stability of DNA methylation in aged samples and the usefulness of neighboring genetic information shown in this study suggest that DNA-methylation-based body fluid identification will play a major role in future forensic investigations. PMID:27591539

  5. siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular Dystrophy

    PubMed Central

    Bolduc, Véronique; Zou, Yaqun; Ko, Dayoung; Bönnemann, Carsten G

    2014-01-01

    Congenital muscular dystrophy type Ullrich (UCMD) is a severe disorder of early childhood onset for which currently there is no effective treatment. UCMD commonly is caused by dominant-negative mutations in the genes coding for collagen type VI, a major microfibrillar component of the extracellular matrix surrounding the muscle fibers. To explore RNA interference (RNAi) as a potential therapy for UCMD, we designed a series of small interfering RNA (siRNA) oligos that specifically target the most common mutations resulting in skipping of exon 16 in the COL6A3 gene and tested them in UCMD-derived dermal fibroblasts. Transcript analysis by semiquantitative and quantitative reverse transcriptase PCR showed that two of these siRNAs were the most allele-specific, i.e., they efficiently knocked down the expression from the mutant allele, without affecting the normal allele. In HEK293T cells, these siRNAs selectively suppressed protein expression from a reporter construct carrying the mutation, with no or minimal suppression of the wild-type (WT) construct, suggesting that collagen VI protein levels are as also reduced in an allele-specific manner. Furthermore, we found that treating UCMD fibroblasts with these siRNAs considerably improved the quantity and quality of the collagen VI matrix, as assessed by confocal microscopy. Our current study establishes RNAi as a promising molecular approach for treating dominant COL6-related dystrophies. PMID:24518369

  6. Identification of transcriptome SNPs between Xiphophorus lines and species for assessing allele specific gene expression within F1 interspecies hybrids☆

    PubMed Central

    Shen, Yingjia; Catchen, Julian; Garcia, Tzintzuni; Amores, Angel; Beldroth, Ion; Wagner, Jonathon R; Zhang, Ziping; Postlethwait, John; Warren, Wes; Schartl, Manfred; Walter, Ronald B.

    2011-01-01

    Variations in gene expression are essential for the evolution of novel phenotypes and for speciation. Studying allelic specific gene expression (ASGE) within interspecies hybrids provides a unique opportunity to reveal underlying mechanisms of genetic variation. Using Xiphophorus interspecies hybrid fishes and high-throughput next generation sequencing technology, we were able to assess variations between two closely related vertebrate species, X. maculatus and X. couchianus, and their F1 interspecies hybrids. We constructed transcriptome-wide SNP polymorphism sets between two highly inbred X. maculatus lines (JP 163 A and B), and between X. maculatus and a second species, X. couchianus. The X. maculatus JP 163 A and B parental lines have been separated in the laboratory for ≈ 70 years and we were able to identify SNPs at a resolution of 1 SNP per 49 kb of transcriptome. In contrast, SNP polymorphisms between X. couchianus and X. maculatus species, which diverged ≈ 5–10 million years ago, were identified about every 700 bp. Using 6,524 transcripts with identified SNPs between the two parental species (X. maculatus and X. couchianus), we mapped RNA-seq reads to determine ASGE within F1 interspecies hybrids. We developed an in silico X. couchianus transcriptome by replacing 90,788 SNP bases for X. maculatus transcriptome with the consensus X. couchianus SNP bases and provide evidence that this procedure overcomes read mapping biases. Employment of the insilico reference transcriptome and tolerating 5 mismatches during read mapping allow direct assessment of ASGE in the F1 interspecies hybrids. Overall, these results show that Xiphophorus is a tractable vertebrate experimental model to investigate how genetic variations that occur during speciation may affect gene interactions and the regulation of gene expression. PMID:21466860

  7. Comprehensively evaluating cis-regulatory variation in the human prostate transcriptome by using gene-level allele-specific expression.

    PubMed

    Larson, Nicholas B; McDonnell, Shannon; French, Amy J; Fogarty, Zach; Cheville, John; Middha, Sumit; Riska, Shaun; Baheti, Saurabh; Nair, Asha A; Wang, Liang; Schaid, Daniel J; Thibodeau, Stephen N

    2015-06-01

    The identification of cis-acting regulatory variation in primary tissues has the potential to elucidate the genetic basis of complex traits and further our understanding of transcriptomic diversity across cell types. Expression quantitative trait locus (eQTL) association analysis using RNA sequencing (RNA-seq) data can improve upon the detection of cis-acting regulatory variation by leveraging allele-specific expression (ASE) patterns in association analysis. Here, we present a comprehensive evaluation of cis-acting eQTLs by analyzing RNA-seq gene-expression data and genome-wide high-density genotypes from 471 samples of normal primary prostate tissue. Using statistical models that integrate ASE information, we identified extensive cis-eQTLs across the prostate transcriptome and found that approximately 70% of expressed genes corresponded to a significant eQTL at a gene-level false-discovery rate of 0.05. Overall, cis-eQTLs were heavily concentrated near the transcription start and stop sites of affected genes, and effects were negatively correlated with distance. We identified multiple instances of cis-acting co-regulation by using phased genotype data and discovered 233 SNPs as the most strongly associated eQTLs for more than one gene. We also noted significant enrichment (25/50, p = 2E-5) of previously reported prostate cancer risk SNPs in prostate eQTLs. Our results illustrate the benefit of assessing ASE data in cis-eQTL analyses by showing better reproducibility of prior eQTL findings than of eQTL mapping based on total expression alone. Altogether, our analysis provides extensive functional context of thousands of SNPs in prostate tissue, and these results will be of critical value in guiding studies examining disease of the human prostate.

  8. Genome-wide Association Study of Subtype-Specific Epithelial Ovarian Cancer Risk Alleles Using Pooled DNA

    PubMed Central

    Earp, Madalene A.; Kelemen, Linda E.; Magliocco, Anthony M.; Swenerton, Kenneth D.; Chenevix–Trench, Georgia; Lu, Yi; Hein, Alexander; Ekici, Arif B.; Beckmann, Matthias W.; Fasching, Peter A.; Lambrechts, Diether; Despierre, Evelyn; Vergote, Ignace; Lambrechts, Sandrina; Doherty, Jennifer A.; Rossing, Mary Anne; Chang-Claude, Jenny; Rudolph, Anja; Friel, Grace; Moysich, Kirsten B.; Odunsi, Kunle; Sucheston-Campbell, Lara; Lurie, Galina; Goodman, Marc T.; Carney, Michael E.; Thompson, Pamela J.; Runnebaum, Ingo B.; Dürst, Matthias; Hillemanns, Peter; Dörk, Thilo; Antonenkova, Natalia; Bogdanova, Natalia; Leminen, Arto; Nevanlinna, Heli; Pelttari, Liisa M.; Butzow, Ralf; Bunker, Clareann H.; Modugno, Francesmary; Edwards, Robert P.; Ness, Roberta B.; du Bois, Andreas; Heitz, Florian; Schwaab, Ira; Harter, Philipp; Karlan, Beth Y.; Walsh, Christine; Lester, Jenny; Jensen, Allan; Kjær, Susanne K.; Høgdall, Claus K.; Høgdall, Estrid; Lundvall, Lene; Sellers, Thomas A.; Fridley, Brooke L.; Goode, Ellen L.; Cunningham, Julie M.; Vierkant, Robert A.; Giles, Graham G.; Baglietto, Laura; Severi, Gianluca; Southey, Melissa C.; Liang, Dong; Wu, Xifeng; Lu, Karen; Hildebrandt, Michelle A.T.; Levine, Douglas A.; Bisogna, Maria; Schildkraut, Joellen M.; Iversen, Edwin S.; Weber, Rachel Palmieri; Berchuck, Andrew; Cramer, Daniel W.; Terry, Kathryn L.; Poole, Elizabeth M.; Tworoger, Shelley S.; Bandera, Elisa V.; Chandran, Urmila; Orlow, Irene; Olson, Sara H.; Wik, Elisabeth; Salvesen, Helga B.; Bjorge, Line; Halle, Mari K.; van Altena, Anne M.; Aben, Katja K.H.; Kiemeney, Lambertus A.; Massuger, Leon F.A.G.; Pejovic, Tanja; Bean, Yukie T.; Cybulski, Cezary; Gronwald, Jacek; Lubinski, Jan; Wentzensen, Nicolas; Brinton, Louise A.; Lissowska, Jolanta; Garcia–Closas, Montserrat; Dicks, Ed; Dennis, Joe; Easton, Douglas F.; Song, Honglin; Tyrer, Jonathan P.; Pharoah, Paul D. P.; Eccles, Diana; Campbell, Ian G.; Whittemore, Alice S.; McGuire, Valerie; Sieh, Weiva; Rothstein, Joseph H.; Flanagan, James M.; Paul, James; Brown, Robert; Phelan, Catherine M.; Risch, Harvey A.; McLaughlin, John R.; Narod, Steven A.; Ziogas, Argyrios; Anton-Culver, Hoda; Gentry-Maharaj, Aleksandra; Menon, Usha; Gayther, Simon A.; Ramus, Susan J.; Wu, Anna H.; Pearce, Celeste L.; Pike, Malcolm C.; Dansonka-Mieszkowska, Agnieszka; Rzepecka, Iwona K; Szafron, Lukasz M; Kupryjanczyk, Jolanta; Cook, Linda S.; Le, Nhu D.; Brooks–Wilson, Angela

    2014-01-01

    Epithelial ovarian cancer (EOC) is a heterogeneous cancer with both genetic and environmental risk factors. Variants influencing the risk of developing the less-common EOC subtypes have not been fully investigated. We performed a genome-wide association study (GWAS) of EOC according to subtype by pooling genomic DNA from 545 cases and 398 controls of European descent, and testing for allelic associations. We evaluated for replication 188 variants from the GWAS (56 variants for mucinous, 55 for endometrioid and clear cell, 53 for low malignant potential (LMP) serous, and 24 for invasive serous EOC), selected using pre-defined criteria. Genotypes from 13,188 cases and 23,164 controls of European descent were used to perform unconditional logistic regression under the log-additive genetic model; odds ratios (OR) and 95% confidence intervals are reported. Nine variants tagging 6 loci were associated with subtype-specific EOC risk at P<0.05, and had an OR that agreed in direction of effect with the GWAS results. Several of these variants are in or near genes with a biological rationale for conferring EOC risk, including ZFP36L1 and RAD51B for mucinous EOC (rs17106154, OR=1.17, P=0.029, n=1,483 cases), GRB10 for endometrioid and clear cell EOC (rs2190503, P=0.014, n=2,903 cases), and C22orf26/BPIL2 for LMP serous EOC (rs9609538, OR=0.86, P=0.0043, n=892 cases). In analyses that included the 75 GWAS samples, the association between rs9609538 (OR=0.84, P=0.0007) and LMP serous EOC risk remained statistically significant at P<0.0012 adjusted for multiple testing. Replication in additional samples will be important to verify these results for the less-common EOC subtypes. PMID:24190013

  9. Novel genus-specific broad range primers for the detection of furoviruses, hordeiviruses and rymoviruses and their application in field surveys in South-East Australia.

    PubMed

    Zheng, Linda; Tang, Joe; Clover, Gerard R G; Spackman, Merrin E; Freeman, Angela J; Rodoni, Brendan C

    2015-03-01

    A number of viruses from the genera Furovirus, Hordeivirus and Rymovirus are known to infect and damage the four major temperate cereal crops, wheat, barley, sorghum and oats. Currently, there is no active testing in Australia for any of these viruses, which pose a significant biosecurity threat to the phytosanitary status of Australia's grains industry. To address this, broad spectrum PCR assays were developed to target virus species within the genera Furovirus, Hordeivirus and Rymovirus. Five sets of novel genus-specific primers were designed and tested in reverse-transcription polymerase chain reaction assays against a range of virus isolates in plant virus diagnostic laboratories in both Australia and New Zealand. Three of these assays were then chosen to screen samples in a three-year survey of cereal crops in western Victoria, Australia. Of the 8900 cereal plants screened in the survey, all were tested free of furoviruses, hordeiviruses and rymoviruses. To date, there were no published genus-specific primers available for the detection of furoviruses, hordeiviruses and rymoviruses. This study shows for the first time a broad-spectrum molecular test being used in a survey for exotic grain viruses in Australia. Results from this survey provide important evidence of the use of this method to demonstrate the absence of these viruses in Victoria, Australia. The primer pairs reported here are expected to detect a wide range of virus species within the three genera.

  10. Novel genus-specific broad range primers for the detection of furoviruses, hordeiviruses and rymoviruses and their application in field surveys in South-East Australia.

    PubMed

    Zheng, Linda; Tang, Joe; Clover, Gerard R G; Spackman, Merrin E; Freeman, Angela J; Rodoni, Brendan C

    2015-03-01

    A number of viruses from the genera Furovirus, Hordeivirus and Rymovirus are known to infect and damage the four major temperate cereal crops, wheat, barley, sorghum and oats. Currently, there is no active testing in Australia for any of these viruses, which pose a significant biosecurity threat to the phytosanitary status of Australia's grains industry. To address this, broad spectrum PCR assays were developed to target virus species within the genera Furovirus, Hordeivirus and Rymovirus. Five sets of novel genus-specific primers were designed and tested in reverse-transcription polymerase chain reaction assays against a range of virus isolates in plant virus diagnostic laboratories in both Australia and New Zealand. Three of these assays were then chosen to screen samples in a three-year survey of cereal crops in western Victoria, Australia. Of the 8900 cereal plants screened in the survey, all were tested free of furoviruses, hordeiviruses and rymoviruses. To date, there were no published genus-specific primers available for the detection of furoviruses, hordeiviruses and rymoviruses. This study shows for the first time a broad-spectrum molecular test being used in a survey for exotic grain viruses in Australia. Results from this survey provide important evidence of the use of this method to demonstrate the absence of these viruses in Victoria, Australia. The primer pairs reported here are expected to detect a wide range of virus species within the three genera. PMID:25497413

  11. Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae.

    PubMed

    Wang, Deguo; Liu, Yanhong

    2015-05-26

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies.

  12. Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae

    PubMed Central

    Wang, Deguo; Liu, Yanhong

    2015-01-01

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies. PMID:26016433

  13. Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae.

    PubMed

    Wang, Deguo; Liu, Yanhong

    2015-06-01

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies. PMID:26016433

  14. The genetic association of RUNX3 with ankylosing spondylitis can be explained by allele-specific effects on IRF4 recruitment that alter gene expression

    PubMed Central

    Vecellio, Matteo; Roberts, Amity R; Cohen, Carla J; Cortes, Adrian; Knight, Julian C; Bowness, Paul; Wordsworth, B Paul

    2016-01-01

    Objectives To identify the functional basis for the genetic association of single nucleotide polymorphisms (SNP), upstream of the RUNX3 promoter, with ankylosing spondylitis (AS). Methods We performed conditional analysis of genetic association data and used ENCODE data on chromatin remodelling and transcription factor (TF) binding sites to identify the primary AS-associated regulatory SNP in the RUNX3 region. The functional effects of this SNP were tested in luciferase reporter assays. Its effects on TF binding were investigated by electrophoretic mobility gel shift assays and chromatin immunoprecipitation. RUNX3 mRNA levels were compared in primary CD8+ T cells of AS risk and protective genotypes by real-time PCR. Results The association of the RUNX3 SNP rs4648889 with AS (p<7.6×10−14) was robust to conditioning on all other SNPs in this region. We identified a 2 kb putative regulatory element, upstream of RUNX3, containing rs4648889. In reporter gene constructs, the protective rs4648889 ‘G’ allele increased luciferase activity ninefold but significantly less activity (4.3-fold) was seen with the AS risk ‘A’ allele (p≤0.01). The binding of Jurkat or CD8+ T-cell nuclear extracts to the risk allele was decreased and IRF4 recruitment was reduced. The AS-risk allele also affected H3K4Me1 histone methylation and associated with an allele-specific reduction in RUNX3 mRNA (p<0.05). Conclusion We identified a regulatory region upstream of RUNX3 that is modulated by rs4648889. The risk allele decreases TF binding (including IRF4) and reduces reporter activity and RUNX3 expression. These findings may have important implications for understanding the role of T cells and other immune cells in AS. PMID:26452539

  15. Phonics Primer

    ERIC Educational Resources Information Center

    Elam, Sandra

    2007-01-01

    This primer lists the 44 sounds in the English language and then gives steps for teaching those 44 sounds and their most common spelling patterns. In addition to learning sounds and spellings, each day the student must read lists of phonetically related words and spell these words from dictation. Phonics instruction must be reinforced by having…

  16. A hybrid next generation transcript sequencing-based approach to identify allelic and homeolog-specific single nucleotide polymorphisms in allotetraploid white clover

    PubMed Central

    2013-01-01

    Background White clover (Trifolium repens L.) is an allotetraploid species possessing two highly collinear ancestral sub-genomes. The apparent existence of highly similar homeolog copies for the majority of genes in white clover is problematic for the development of genome-based resources in the species. This is especially true for the development of genetic markers based on single nucleotide polymorphisms (SNPs), since it is difficult to distinguish between homeolog-specific and allelic variants. Robust methods for categorising single nucleotide variants as allelic or homeolog-specific in large transcript datasets are required. We illustrate one potential approach in this study. Results We used 454-pyrosequencing sequencing to generate ~760,000 transcript sequences from an 8th generation white clover inbred line. These were assembled and partially annotated to yield a reference transcript set comprising 71,545 sequences. We subsequently performed Illumina sequencing on three further white clover samples, generating 14 million transcript reads from a mixed sample comprising 24 divergent white clover genotypes, and 50 million reads on two further eighth generation white clover inbred lines. Mapping these reads to the reference transcript set allowed us to develop a significant SNP resource for white clover, and to partition the SNPs from the inbred lines into categories reflecting allelic or homeolog-specific variation. The potential for using haplotype reconstruction and progenitor genome comparison to assign haplotypes to specific ancestral sub-genomes of white clover is demonstrated for sequences corresponding to genes encoding dehydration responsive element binding protein and acyl-coA oxidase. Conclusions In total, 208,854 independent SNPs in 31,715 reference sequences were discovered, approximately three quarters of which were categorised as representing allelic or homeolog-specific variation using two inbred lines. This represents a significant resource for

  17. Emaravirus-specific degenerate PCR primers allowed the identification of partial RNA-dependent RNA polymerase sequences of Maize red stripe virus and Pigeonpea sterility mosaic virus.

    PubMed

    Elbeaino, Toufic; Whitfield, Anna; Sharma, Mamta; Digiaro, Michele

    2013-03-01

    Emaravirus is a recently established viral genus that includes two approved virus species: European mountain ash ringspot-associated virus (EMARaV) and Fig mosaic virus (FMV). Other described but unclassified viruses appear to share biological characteristics similar to emaraviruses, including segmented, negative-single stranded RNA genomes with enveloped virions approximately 80-200nm in diameter. Sequence analysis of emaravirus genomes revealed the presence of conserved amino acid sequences in the RNA-dependent RNA polymerase gene (RdRp) denoted as pre-motif A, motifs A and C. Degenerate oligonucleotide primers were developed to these conserved sequences and were shown to amplify in reverse transcription-polymerase chain reaction assay (RT-PCR) DNA fragments of 276bp and 360bp in size. These primers efficiently detected emaraviruses with known sequences available in the database (FMV and EMARaV); they also detected viruses with limited sequence information such as Pigeonpea sterility mosaic virus (PPSMV) and Maize red stripe virus (MRSV). The degenerate primers designed on pre-motif A and motif A sequences successfully amplified the four species used as positive controls (276bp), whereas those of motifs A and C failed to detect only MRSV. The amino acid sequences obtained from PPSMV and MRSV shared the highest identity with those of two other tentative species of the Emaravirus genus, Rose rosette virus (RRV) (69%) and Redbud yellow ringspot virus (RYRV) (60%), respectively. The phylogenetic tree constructed with 92 amino acid-long portions of polypeptide putatively encoded by RNA1 of definitive and tentative emaravirus species clustered PPSMV and MRSV in two separate clades close to RRV and Raspberry leaf blotch virus (RLBV), respectively. The newly developed degenerate primers have proved their efficacy in amplifying new emaravirus-specific sequences; accordingly, they could be useful in identifying new emaravirus-like species in nature.

  18. Detection of minimal residual disease in patients with multiple myeloma using clonotype-specific PCR primers designed from DNA extracted from archival bone marrow slides.

    PubMed

    Takamatsu, Hiroyuki; Ogawa, Yoshiyasu; Kobayashi, Noriko; Obata, Kazue; Narisawa, Tadashi; Nakayama, Kouji; Munemoto, Saori; Aoki, Go; Ohata, Kinya; Kumano, Yoshihisa; Ozaki, Jun; Murata, Ryoichi; Kondo, Yukio; Terasaki, Yasushi; Kurokawa, Toshiro; Miyamoto, Toshihiro; Shimizu, Naomi; Fukushima, Toshihiro; Yoshida, Akira; Ueda, Takanori; Yoshida, Takashi; Nakao, Shinji

    2013-10-01

    Polymerase chain reaction (PCR)-negative molecular complete remission (mCR) can be induced by stem cell transplantation in some patients with multiple myeloma (MM) and is associated with long-term progression-free survival (PFS). The detection of molecular minimal residual disease (MRD), however, requires fresh or frozen materials for designing clone-specific primers, which are not always readily available. In this study, we used DNA extracted from archival bone marrow (BM) slides for PCR to detect MRD in 50 patients with MM who received various induction therapies and autologous peripheral blood stem cell transplantation (ASCT). Clonotype-specific immunoglobulin (Ig) H PCR primers were prepared for 32 of 50 cases (64%) using BM slides, and for 9 of 14 cases (64%) using fresh BM cells. DNA in peripheral blood stem cell autografts of the 22 patients who achieved at least a partial response after ASCT was subjected to PCR to amplify clonotype-specific rearranged IgH gene sequences. The median PFS of the eight patients with MRD-positive autografts was 18 months, whereas that of 14 patients with MRD-negative autografts was not reached at a median follow-up of 27 months (p = 0.012). Post-ASCT PFS of the four patients who achieved mCR was 100% at a median follow-up of 47 months. These results indicate that archival BM slides can serve as a source of DNA for preparing clonotype-specific primers for MRD monitoring in patients with MM whose cryopreserved myeloma cells are not available for DNA preparation. Our results also suggest that patients with MM who received MRD-negative autografts and achieved mCR have a long PFS.

  19. Primers and a specific DNA probe for detecting lactic acid bacteria producing 3-hydroxypropionaldehyde from glycerol in spoiled ciders.

    PubMed

    Claisse, O; Lonvaud-Funel, A

    2001-06-01

    Of the 40 strains isolated from several spoiled ciders where glycerol was degraded, 36 were identified as Lactobacillus collinoides, three were Lactobacillus hilgardii, and one was Lactobacillus mali. However, only 30 L. collinoides and two L. hilgardii could degrade glycerol. The glycerol dehydratase activity was shown. The main product of the transformation was 1.3 propanediol. Two DNA primers GD1 and GD2 were chosen in the region encoding one of the subunits of glycerol dehydratase of Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella Typhimurium, and Clostridium pasteurianum. A 279-bp amplicon in polymerase chain reaction amplification was obtained with the genomic L. collinoides IOEB 9527 DNA as template. The amino acid sequence deduced from the amplicon DNA sequence showed a very high similarity and identity with the gene of gram-negative and C. pasteurianum species. After labeling, the amplicon was used as DNA probe in dot-blot hybridization with the genomic DNA of all the tested strains. Only strains that could degrade glycerol hybridized. Moreover, polymerase chain reactions using GDI and GD2 revealed only glycerol dehydratase genes of positive L. collinoides and L. hilgardii strains. The primers and the amplicon proved to be suitable and reliable tools to detect the lactic acid bacteria involved in the deterioration of cider. PMID:11403134

  20. Genome-wide association study of subtype-specific epithelial ovarian cancer risk alleles using pooled DNA.

    PubMed

    Earp, Madalene A; Kelemen, Linda E; Magliocco, Anthony M; Swenerton, Kenneth D; Chenevix-Trench, Georgia; Lu, Yi; Hein, Alexander; Ekici, Arif B; Beckmann, Matthias W; Fasching, Peter A; Lambrechts, Diether; Despierre, Evelyn; Vergote, Ignace; Lambrechts, Sandrina; Doherty, Jennifer A; Rossing, Mary Anne; Chang-Claude, Jenny; Rudolph, Anja; Friel, Grace; Moysich, Kirsten B; Odunsi, Kunle; Sucheston-Campbell, Lara; Lurie, Galina; Goodman, Marc T; Carney, Michael E; Thompson, Pamela J; Runnebaum, Ingo B; Dürst, Matthias; Hillemanns, Peter; Dörk, Thilo; Antonenkova, Natalia; Bogdanova, Natalia; Leminen, Arto; Nevanlinna, Heli; Pelttari, Liisa M; Butzow, Ralf; Bunker, Clareann H; Modugno, Francesmary; Edwards, Robert P; Ness, Roberta B; du Bois, Andreas; Heitz, Florian; Schwaab, Ira; Harter, Philipp; Karlan, Beth Y; Walsh, Christine; Lester, Jenny; Jensen, Allan; Kjær, Susanne K; Høgdall, Claus K; Høgdall, Estrid; Lundvall, Lene; Sellers, Thomas A; Fridley, Brooke L; Goode, Ellen L; Cunningham, Julie M; Vierkant, Robert A; Giles, Graham G; Baglietto, Laura; Severi, Gianluca; Southey, Melissa C; Liang, Dong; Wu, Xifeng; Lu, Karen; Hildebrandt, Michelle A T; Levine, Douglas A; Bisogna, Maria; Schildkraut, Joellen M; Iversen, Edwin S; Weber, Rachel Palmieri; Berchuck, Andrew; Cramer, Daniel W; Terry, Kathryn L; Poole, Elizabeth M; Tworoger, Shelley S; Bandera, Elisa V; Chandran, Urmila; Orlow, Irene; Olson, Sara H; Wik, Elisabeth; Salvesen, Helga B; Bjorge, Line; Halle, Mari K; van Altena, Anne M; Aben, Katja K H; Kiemeney, Lambertus A; Massuger, Leon F A G; Pejovic, Tanja; Bean, Yukie T; Cybulski, Cezary; Gronwald, Jacek; Lubinski, Jan; Wentzensen, Nicolas; Brinton, Louise A; Lissowska, Jolanta; Garcia-Closas, Montserrat; Dicks, Ed; Dennis, Joe; Easton, Douglas F; Song, Honglin; Tyrer, Jonathan P; Pharoah, Paul D P; Eccles, Diana; Campbell, Ian G; Whittemore, Alice S; McGuire, Valerie; Sieh, Weiva; Rothstein, Joseph H; Flanagan, James M; Paul, James; Brown, Robert; Phelan, Catherine M; Risch, Harvey A; McLaughlin, John R; Narod, Steven A; Ziogas, Argyrios; Anton-Culver, Hoda; Gentry-Maharaj, Aleksandra; Menon, Usha; Gayther, Simon A; Ramus, Susan J; Wu, Anna H; Pearce, Celeste L; Pike, Malcolm C; Dansonka-Mieszkowska, Agnieszka; Rzepecka, Iwona K; Szafron, Lukasz M; Kupryjanczyk, Jolanta; Cook, Linda S; Le, Nhu D; Brooks-Wilson, Angela

    2014-05-01

    Epithelial ovarian cancer (EOC) is a heterogeneous cancer with both genetic and environmental risk factors. Variants influencing the risk of developing the less-common EOC subtypes have not been fully investigated. We performed a genome-wide association study (GWAS) of EOC according to subtype by pooling genomic DNA from 545 cases and 398 controls of European descent, and testing for allelic associations. We evaluated for replication 188 variants from the GWAS [56 variants for mucinous, 55 for endometrioid and clear cell, 53 for low-malignant potential (LMP) serous, and 24 for invasive serous EOC], selected using pre-defined criteria. Genotypes from 13,188 cases and 23,164 controls of European descent were used to perform unconditional logistic regression under the log-additive genetic model; odds ratios (OR) and 95 % confidence intervals are reported. Nine variants tagging six loci were associated with subtype-specific EOC risk at P < 0.05, and had an OR that agreed in direction of effect with the GWAS results. Several of these variants are in or near genes with a biological rationale for conferring EOC risk, including ZFP36L1 and RAD51B for mucinous EOC (rs17106154, OR = 1.17, P = 0.029, n = 1,483 cases), GRB10 for endometrioid and clear cell EOC (rs2190503, P = 0.014, n = 2,903 cases), and C22orf26/BPIL2 for LMP serous EOC (rs9609538, OR = 0.86, P = 0.0043, n = 892 cases). In analyses that included the 75 GWAS samples, the association between rs9609538 (OR = 0.84, P = 0.0007) and LMP serous EOC risk remained statistically significant at P < 0.0012 adjusted for multiple testing. Replication in additional samples will be important to verify these results for the less-common EOC subtypes. PMID:24190013

  1. Allele-specific PCR for detecting the deafness-associated mitochondrial 12S rRNA mutations.

    PubMed

    Ding, Yu; Xia, Bo-Hou; Liu, Qi; Li, Mei-Ya; Huang, Shui-Xian; Zhuo, Guang-Chao

    2016-10-10

    Mutations in mitochondrial 12S rRNA (MT-RNR1) are the important causes of sensorineural hearing loss. Of these mutations, the homoplasmic m.1555A>G or m.1494C>T mutation in the highly conserved A-site of MT-RNR1 gene has been found to be associated with both aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. Since the m.1555A>G and m.1494C>T mutations are sensitive to ototoxic drugs, therefore, screening for the presence of these mutations is important for early diagnosis and prevention of deafness. For this purpose, we recently developed a novel allele-specific PCR (AS-PCR) which is able to simultaneously detect these mutations. To assess its accuracy, in this study, we employed this method to screen the frequency of m.1555A>G and m.1494C>T mutations in 200 deafness patients and 120 healthy subjects. Consequently, four m.1555A>G and four m.1494C>T mutations were identified; among these, only one patient with the m.1494C>T mutation had an obvious family history of hearing loss. Strikingly, clinical evaluation showed that this family exhibited a high penetrance of hearing loss. In particular, the penetrances of hearing loss were 80% with the aminoglycoside included and 20% when excluded. PCR-Sanger sequencing of the mitochondrial genomes confirmed the presence of the m.1494C>T mutation and identified a set of polymorphisms belonging to mitochondrial haplogroup A. However, the lack of functional variants in mitochondrial and nuclear modified genes (GJB2 and TRMU) in this family indicated that mitochondrial haplogroup and nuclear genes may not play important roles in the phenotypic expression of the m.1494C>T mutation. Thus, other modification factors, such as environmental factor, aminoglycosides or epigenetic modification may have contributed to the high penetrance of hearing loss in this family. Taken together, our data showed that this assay is an effective approach that could be used for detection the deafness-associated MT-RNR1

  2. MRPrimer: a MapReduce-based method for the thorough design of valid and ranked primers for PCR

    PubMed Central

    Kim, Hyerin; Kang, NaNa; Chon, Kang-Wook; Kim, Seonho; Lee, NaHye; Koo, JaeHyung; Kim, Min-Soo

    2015-01-01

    Primer design is a fundamental technique that is widely used for polymerase chain reaction (PCR). Although many methods have been proposed for primer design, they require a great deal of manual effort to generate feasible and valid primers, including homology tests on off-target sequences using BLAST-like tools. That approach is inconvenient for many target sequences of quantitative PCR (qPCR) due to considering the same stringent and allele-invariant constraints. To address this issue, we propose an entirely new method called MRPrimer that can design all feasible and valid primer pairs existing in a DNA database at once, while simultaneously checking a multitude of filtering constraints and validating primer specificity. Furthermore, MRPrimer suggests the best primer pair for each target sequence, based on a ranking method. Through qPCR analysis using 343 primer pairs and the corresponding sequencing and comparative analyses, we showed that the primer pairs designed by MRPrimer are very stable and effective for qPCR. In addition, MRPrimer is computationally efficient and scalable and therefore useful for quickly constructing an entire collection of feasible and valid primers for frequently updated databases like RefSeq. Furthermore, we suggest that MRPrimer can be utilized conveniently for experiments requiring primer design, especially real-time qPCR. PMID:26109350

  3. A Panel of Artificial APCs Expressing Prevalent HLA Alleles Permits Generation of Cytotoxic T Cells Specific for Both Dominant and Subdominant Viral Epitopes for Adoptive Therapy1

    PubMed Central

    Hasan, Aisha N.; Kollen, Wouter J.; Trivedi, Deepa; Selvakumar, Annamalai; Dupont, Bo; Sadelain, Michel; O'Reilly, Richard J.

    2009-01-01

    Adoptive transfer of virus-specific T cells can treat infections complicating allogeneic hematopoietic cell transplants. However, autologous antigen-presenting cells (APCs) are often limited in supply. Here, we describe a panel of artificial APCs (AAPCs) consisting of murine 3T3 cells transduced to express human B7.1, ICAM-1 and LFA-3 that each stably express one of a series of 6 common HLA class I alleles. In comparative analyses, T cells sensitized with AAPCs expressing a shared HLA allele or autologous APCs loaded with a pool of 15-mers spanning the sequence of CMVpp65 produced similar yields of HLA-restricted CMVpp65 specific T cells; significantly higher yields could be achieved by sensitization with AAPCs transduced to express the CMVpp65 protein. T cells generated were CD8+, IFNγ+ and exhibited HLA-restricted CMVpp65 specific cytotoxicity. T cells sensitized with either peptide-loaded or transduced AAPCs recognized epitopes presented by each HLA allele known to be immunogenic in man. Sensitization with AAPCs also permitted expansion of IFNγ+ cytotoxic effector cells against subdominant epitopes that were either absent or in low frequencies in T cells sensitized with autologous APCs. This replenishable panel of AAPCs can be used for immediate sensitization and expansion of virus-specific T cells of desired HLA restriction for adoptive immunotherapy. It may be of particular value for recipients of transplants from HLA disparate donors. PMID:19635907

  4. CalMaTe: a method and software to improve allele-specific copy number of SNP arrays for downstream segmentation

    PubMed Central

    Ortiz-Estevez, Maria; Aramburu, Ander; Bengtsson, Henrik; Neuvial, Pierre; Rubio, Angel

    2012-01-01

    Summary: CalMaTe calibrates preprocessed allele-specific copy number estimates (ASCNs) from DNA microarrays by controlling for single-nucleotide polymorphism-specific allelic crosstalk. The resulting ASCNs are on average more accurate, which increases the power of segmentation methods for detecting changes between copy number states in tumor studies including copy neutral loss of heterozygosity. CalMaTe applies to any ASCNs regardless of preprocessing method and microarray technology, e.g. Affymetrix and Illumina. Availability: The method is available on CRAN (http://cran.r-project.org/) in the open-source R package calmate, which also includes an add-on to the Aroma Project framework (http://www.aroma-project.org/). Contact: arubio@ceit.es Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22576175

  5. Requisite analytic and diagnostic performance characteristics for the clinical detection of BRAF V600E in hairy cell leukemia: a comparison of 2 allele-specific PCR assays.

    PubMed

    Brown, Noah A; Weigelin, Helmut C; Bailey, Nathanael; Laliberte, Julie; Elenitoba-Johnson, Kojo S J; Lim, Megan S; Betz, Bryan L

    2015-09-01

    Detection of high-frequency BRAF V600E mutations in hairy cell leukemia (HCL) has important diagnostic utility. However, the requisite analytic performance for a clinical assay to routinely detect BRAF V600E mutations in HCL has not been clearly defined. In this study, we sought to determine the level of analytic sensitivity needed for formalin-fixed, paraffin-embedded (FFPE) and frozen samples and to compare the performance of 2 allele-specific polymerase chain reaction (PCR) assays. Twenty-nine cases of classic HCL, including 22 FFPE bone marrow aspirates and 7 frozen specimens from blood or bone marrow were evaluated using a laboratory-developed allele-specific PCR assay and a commercially available allele-specific quantitative PCR assay-myT BRAF Ultra. Also included were 6 HCL variant and 40 non-HCL B-cell lymphomas. Two cases of classic HCL, 1 showing CD5 expression, were truly BRAF V600E-negative based on negative results by PCR and sequencing despite high-level leukemic involvement. Among the remaining 27 specimens, V600E mutations were detected in 88.9% (17/20 FFPE; 7/7 frozen) and 81.5% (15/20 FFPE; 7/7 frozen), for the laboratory-developed and commercial assays, respectively. No mutations were detected among the 46 non-HCL lymphomas. Both assays showed an analytic sensitivity of 0.3% involvement in frozen specimens and 5% in FFPE tissue. On the basis of these results, an assay with high analytic sensitivity is required for the clinical detection of V600E mutations in HCL specimens. Two allele-specific PCR assays performed well in both frozen and FFPE bone marrow aspirates, although detection in FFPE tissue required 5% or more involvement.

  6. Artificial antigen presenting cells that express prevalent HLA alleles: A step towards the broad application of antigen-specific adoptive cell therapies.

    PubMed

    Hasan, Aisha N; Selvakumar, Annamalai; Doubrovina, Ekaterina; Riviere, Isabelle; Sadelain, Michel W; O'Reilly, Richard J

    2009-12-01

    The artificial antigen-presenting cells (AAPCs) described in this review were generated to facilitate the production of virus-specific T-cells for the treatment of infections in patients after bone marrow transplant. These AAPCs consist of murine 3T3 cells genetically modified to express critical human molecules needed for T-cell stimulation, such as the co-stimulatory molecules B7.1, ICAM-1, and LFA-3 and one of a series of 6 common HLA class I alleles. When T-cells were sensitized against cytomegalovirus (CMV) using AAPCs that express a shared HLA allele or using autologous antigen-presenting cells (APCs) loaded with the CMVpp65 antigen, they were activated and expanded to become HLA-restricted CMVpp65-specific T-cells. These T-cells demonstrated functional activity in vitro against CMV by producing IFN-gamma and inducing CMVpp65-specific cytotoxicity. T-cells sensitized with AAPCs recognized antigenic epitopes presented by each HLA allele known to be immunogenic in Man. Sensitization with AAPCs also permitted expansion of IFN-gamma+ cytotoxic T-cells against subdominant epitopes that were not effectively recognized by T-cells sensitized with autologous APCs. This panel of AAPCs provides a source of immediately accessible, standardizable, and replenishable "off the shelf" cellular reagents with the potential to make adoptive immunotherapy widely available for the treatment of lethal infections, cancer, and autoimmune diseases. PMID:20040272

  7. Development of a Melting Curve-Based Allele-Specific PCR of Apolipoprotein E (APOE) Genotyping Method for Genomic DNA, Guthrie Blood Spot, and Whole Blood.

    PubMed

    Chen, Chia-Hsiang

    2016-01-01

    Genetic polymorphisms of apolipoprotein E (APOE) are associated with various health conditions and diseases, such as Alzheimer's disease, cardiovascular diseases, type 2 diabetes, etc. Hence, genotyping of APOE has broad applications in biomedical research and clinical settings, particularly in the era of precision medicine. The study aimed to develop a convenient and accurate method with flexible throughput to genotype the APOE polymorphisms. A melting curve-based allele-specific PCR method was developed to genotype two single nucleotide polymorphisms (SNPs) of APOE, i.e. rs429358 at codon 112 and rs7412 at codon 158. These two SNPs determine the genotype of APOE2, E3, and E4. PCR-based Sanger sequencing was used as the reference method for APOE genotyping. A 100% concordance rate was obtained in 300 subjects between the melting curve-based allele-specific PCR method and the Sanger sequencing method. This method was applied to a genetic association analysis of APOE and schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects from Taiwan. However, no significant differences in the allele and genotype frequencies were detected between these two groups. Further experiments showed that DNA dissolved from blood collected on Guthrie filter paper and total blood cell lysate without DNA extraction can be used in the melting curve-based allele-specific PCR method. Thus, we suggest that this is a fast, accurate and robust APOE genotyping method with a flexible throughput and suitable for DNA template from different preparations. This convenient method shall meet the different needs of various research and clinical laboratories. PMID:27078154

  8. Explanatory chapter: PCR primer design.

    PubMed

    Álvarez-Fernández, Rubén

    2013-01-01

    This chapter is intended as a guide on polymerase chain reaction (PCR) primer design (for information on PCR, see General PCR and Explanatory Chapter: Troubleshooting PCR). In the next section, general guidelines will be provided, followed by a discussion on primer design for specific applications. A list of recommended software tools is shown at the end.

  9. Identification of lipase encoding genes from Antarctic seawater bacteria using degenerate primers: expression of a cold-active lipase with high specific activity.

    PubMed

    Parra, Loreto P; Espina, Giannina; Devia, Javier; Salazar, Oriana; Andrews, Barbara; Asenjo, Juan A

    2015-01-01

    Cold-active enzymes are valuable catalysts showing high activity at low and moderate temperatures and low thermostability. Among cold-active enzymes, lipases offer a great potential in detergent, cosmetic, biofuel and food or feed industries. In this paper we describe the identification of novel lipase coding genes and the expression of a lipase with high activity at low temperatures. The genomic DNA from Antarctic seawater bacteria showing lipolytic activity at 4°C was used to amplify five DNA fragments that partially encode novel lipases using specifically designed COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP). All the fragments were found to have a high identity with an α/β-hydrolase domain-containing protein identified by the sequencing of the complete genome of Shewanella frigidimarina NCIMB 400. The complete sequence of one of the lipase-coding gene fragments, lipE13, was obtained by genome walking. Considering that the other fragments had a high identity to the putative lipase from S. frigidimarina NCIMB 400, the complete lipase genes were amplified using oligonucleotide primers designed based on the 5' and 3' regions of the coding sequence of the related protein. This strategy allowed the amplification of 3 lipase-encoding genes of which one was expressed in the periplasm using the Escherichia coli BL21(DE3)/pET-22b(+) expression system. The recombinant protein was obtained with activity toward p-nitrophenyl caproate showing a high specific activity between 15 and 25°C.

  10. Effect of metallothionein 2A gene polymorphism on allele-specific gene expression and metal content in prostate cancer

    SciTech Connect

    Krześlak, Anna; Forma, Ewa; Jóźwiak, Paweł; Szymczyk, Agnieszka; Bryś, Magdalena

    2013-05-01

    Metallothioneins (MTs) are highly conserved, small molecular weight, cysteine rich proteins. The major physiological functions of metallothioneins include homeostasis of essential metals Zn and Cu and protection against cytotoxicity of heavy metals. The aim of this study was to determine whether there is an association between the − 5 A/G single nucleotide polymorphism (SNP; rs28366003) in core promoter region and expression of metallothionein 2A (MT2A) gene and metal concentration in prostate cancer tissues. MT2A polymorphism was determined by the polymerase chain reaction–restriction fragment length polymorphism technique (PCR–RFLP) using 412 prostate cancer tissue samples. MT2A gene expression analysis was performed by real-time RT-PCR method. A significant association between rs28366003 genotype and MT2A expression level was found. The average mRNA level was found to be lower among minor allele carriers (the risk allele) than average expression among homozygotes for the major allele. Metal levels were analyzed by flamed atomic absorption spectrometer system. Highly statistically significant associations were detected between the SNP and Cd, Zn, Cu and Pb levels. The results of Spearman's rank correlation showed that the expressions of MT2A and Cu, Pb and Ni concentrations were negatively correlated. On the basis of the results obtained in this study, we suggest that SNP polymorphism may affect the MT2A gene expression in prostate and this is associated with some metal accumulation. - Highlights: • MT2A gene expression and metal content in prostate cancer tissues • Association between SNP (rs28366003) and expression of MT2A • Significant associations between the SNP and Cd, Zn, Cu and Pb levels • Negative correlation between MT2A gene expression and Cu, Pb and Ni levels.

  11. [The Use of Specific DNA Markers for the Identification of Alleles of the FAD3 Genes in Rape (Brassica napus L.)].

    PubMed

    Lemesh, V A; Mozgova, G V; Grushetskaya, Z E; Sidorenko, E V; Pilyuk, Ya E; Bakanovskaya, A V

    2015-08-01

    A search was conducted for the alleles responsible for the quality of food-grade rapeseed oil in a collection of 21 samples of spring and winter oilseed rape of Belarusian and Russian breeding. We also developed A- and C-gene-specific DNA markers to assess the genomic polymorphisms of rape for FAD3 genes and selected plants with a low content of linolenic acid for use in the selection process. The development of a method for identifying FAD3 alleles, which control the level of linolenic acid in rapeseed oil, as well as of the design for new dCAPS markers, enabled the identification of plants homozygous for individual FAD3A and/or FAD3C genes in the F2-generation. These plants are currently involved in the selection process of new varieties with a reduced content of linolenic acid in rapeseed oil. PMID:26601489

  12. Allele-specific polymerase chain reaction for detection of a mutation in the relax circular DNA and the covalently closed circular DNA of hepatitis B virus.

    PubMed

    Pan, Wan-Long; Hu, Jie-Li; Fang, Yan; Luo, Qiang; Xu, Ge; Xu, Lei; Jing, Zhou-Hong; Shan, Xue-Feng; Zhu, Yan-Ling; Huang, Ai-Long

    2013-12-01

    The relax circle DNA (rcDNA) sequence and the covalently closed circle DNA (cccDNA) sequence in hepatitis B virus (HBV) are crucial regions for HBV infections. To analyze mutations in rcDNA and cccDNA, DNA sequencing is often used, although it is time-consuming and expensive. Herein, we report a simple, economic, albeit accurate allele-specific polymerase chain reaction (AS-PCR) to detect mutations in these regions of HBV. This method can be extensively used to screen for mutations at specific positions of HBV genome.

  13. Allele-specific loss and transcription of the miR-15a/16-1 cluster in chronic lymphocytic leukemia

    PubMed Central

    Veronese, A; Pepe, F; Chiacchia, J; Pagotto, S; Lanuti, P; Veschi, S; Di Marco, M; D'Argenio, A; Innocenti, I; Vannata, B; Autore, F; Marchisio, M; Wernicke, D; Verginelli, F; Leone, G; Rassenti, L Z; Kipps, T J; Mariani-Costantini, R; Laurenti, L; Croce, C M; Visone, R

    2015-01-01

    Deregulation of the miR-15a/16-1 cluster has a key role in the pathogenesis of chronic lymphocytic leukemia (CLL), a clinically heterogeneous disease with indolent and aggressive forms. The miR-15a/16-1 locus is located at 13q14, the most frequently deleted region in CLL. Starting from functional investigations of a rare SNP upstream the miR cluster, we identified a novel allele-specific mechanism that exploits a cryptic activator region to recruit the RNA polymerase III for miR-15a/16-1 transcription. This regulation of the miR-15a/16- locus is independent of the DLEU2 host gene, which is often transcribed monoallellically by RPII. We found that normally one allele of miR-15a/16-1 is transcribed by RNAPII, the other one by RNAPIII. In our subset of CLL patients harboring 13q14 deletions, exclusive RNA polymerase III (RPIII)-driven transcription of the miR-15a/16-1 was the consequence of loss of the RPII-regulated allele and correlated with high expression of the poor prognostic marker ZAP70 (P=0.019). Thus, our findings point to a novel biological process, characterized by double allele-specific transcriptional regulation of the miR-15a/16-1 locus by alternative mechanisms. Differential usage of these mechanisms may distinguish at onset aggressive from indolent forms of CLL. This provides a basis for the clinical heterogeneity of the CLL patients carrying 13q14 deletions. PMID:24732594

  14. RATMAC PRIMER

    SciTech Connect

    Munn, R. J.; Stewart, J. M.; Norden, A. P.; Pagoaga, M. Katherine

    1980-10-01

    The language RATMAC is a direct descendant of one of the most successful structured FORTRAN languages, rational FORTRAN, RATFOR. RATMAC has all of the characteristics of RATFOR, but is augmented by a powerful recursive macro processor which is extremely useful in generating transportable FORTRAN programs. A macro is a collection of programming steps which are associated with a keyword. This keyword uniquely identifies the macro, and whenever it appears in a RATMAC program it is replaced by the collection of steps. This primer covers the language's control and decision structures, macros, file inclusion, symbolic constants, and error messages.

  15. Testing the Specificity of Primers to Environmental Ammonia Monooxygenase (amoA) Genes in Groundwater Treated with Urea to Promote Calcite Precipitation

    SciTech Connect

    Freeman, S.; Reed, D.W.; Fujita, Y.

    2006-01-01

    Bacterial ammonia monooxygenase (amoA) genes in DNA isolated from microorganisms in groundwater were characterized by amplification of amoA DNA using polymerase chain reaction (PCR), Restriction Fragment Length Polymorphism (RFLP) analysis, and sequencing. The amoA gene is characteristic of ammonia oxidizing bacteria (AOB). The DNA extracts were acquired from an experiment where dilute molasses and urea were sequentially introduced into a well in the Eastern Snake River Plain Aquifer (ESRPA) in Idaho to examine whether such amendments could stimulate enhanced ureolytic activity. The hydrolysis of urea into ammonium and carbonate serves as the basis for a potential remediation technique for trace metals and radionuclide contaminants that can co-precipitate in calcite. The ammonium ion resulting from ureolysis can promote the growth of AOB. The goal of this work was to investigate the effectiveness of primers designed for quantitative PCR of environmental amoA genes and to evaluate the effect of the molasses and urea amendments upon the population diversity of groundwater AOB. PCR primers designed to target a portion of the amoA gene were used to amplify amoA gene sequences in the groundwater DNA extracts. Following PCR, amplified gene products were cloned and the clones were characterized by RFLP, a DNA restriction technique that can distinguish different DNA sequences, to gauge the initial diversity. Clones exhibiting unique RFLP patterns were subjected to DNA sequencing. Initial sequencing results suggest that the primers were successful at specific detection of amoA sequences and the RFLP analyses indicated that the diversity of detected amoA sequences in the ESRPA decreased with the additions of molasses and urea.

  16. Allele-specific programming of Npy and epigenetic effects of physical activity in a genetic model of depression.

    PubMed

    Melas, P A; Lennartsson, A; Vakifahmetoglu-Norberg, H; Wei, Y; Åberg, E; Werme, M; Rogdaki, M; Mannervik, M; Wegener, G; Brené, S; Mathé, A A; Lavebratt, C

    2013-05-07

    Neuropeptide Y (NPY) has been implicated in depression, emotional processing and stress response. Part of this evidence originates from human single-nucleotide polymorphism (SNP) studies. In the present study, we report that a SNP in the rat Npy promoter (C/T; rs105431668) affects in vitro transcription and DNA-protein interactions. Genotyping studies showed that the C-allele of rs105431668 is present in a genetic rat model of depression (Flinders sensitive line; FSL), while the SNP's T-allele is present in its controls (Flinders resistant line; FRL). In vivo experiments revealed binding of a transcription factor (CREB2) and a histone acetyltransferase (Ep300) only at the SNP locus of the FRL. Accordingly, the FRL had increased hippocampal levels of Npy mRNA and H3K18 acetylation; a gene-activating histone modification maintained by Ep300. Next, based on previous studies showing antidepressant-like effects of physical activity in the FSL, we hypothesized that physical activity may affect Npy's epigenetic status. In line with this assumption, physical activity was associated with increased levels of Npy mRNA and H3K18 acetylation. Physical activity was also associated with reduced mRNA levels of a histone deacetylase (Hdac5). Conclusively, the rat rs105431668 appears to be a functional Npy SNP that may underlie depression-like characteristics. In addition, the achieved epigenetic reprogramming of Npy provides molecular support for the putative effectiveness of physical activity as a non-pharmacological antidepressant.

  17. Salinas primer.

    SciTech Connect

    Walsh, Timothy Francis; Reese, Garth M.; Bhardwaj, Manoj Kumar

    2004-08-01

    Salinas provides a massively parallel implementation of structural dynamics finite element analysis. This capability is required for high fidelity, validated models used in modal, vibration, static and shock analysis of weapons systems. General capabilities for modal, statics and transient dynamics are provided. Salinas is similar to commercial codes like Nastran or Abaqus. It has some nonlinear capability, but excels in linear computation. It is different than the above commercial codes in that it is designed to operate efficiently in a massively parallel environment. Even for an experienced analyst, running a new finite element package can be a challenge. This little primer is intended to make part of this task easier by presenting the basic steps in a simple way. The analyst is referred to the theory manual for details of the mathematics behind the work. The User's Notes should be used for more complex inputs, and will have more details about the process (as well as many more examples). More information can be found on our web pages, 3 or 4. Finite element analysis can be deceptive. Any software can give the wrong answers if used improperly, and occasionally even when used properly. Certainly a solid background in structural mechanics is necessary to build an adequate finite element model and interpret the results. This primer should provide a quick start in answering some of the more common questions that come up in using Salinas.

  18. Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers.

    PubMed

    Asari, Masaru; Okuda, Katsuhiro; Hoshina, Chisato; Omura, Tomohiro; Tasaki, Yoshikazu; Shiono, Hiroshi; Matsubara, Kazuo; Shimizu, Keiko

    2016-02-01

    The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis. PMID:26505528

  19. Allele-specific recognition by LILRB3 and LILRA6 of a cytokeratin 8 - associated ligand on necrotic glandular epithelial cells

    PubMed Central

    López-Álvarez, María R.; Jahnke, Martin; Russell, Alasdair I.; Radjabova, Valeria; Trowsdale, Alice R.Z.; Trowsdale, John

    2016-01-01

    The LILRs are a family of receptors that regulate the activities of myelomonocytic cells. We found that specific allelic variants of two related members of the LILR family, LILRB3 and LILRA6, interact with a ligand exposed on necrotic glandular epithelial cells. The extracellular domains of LILRB3 and LILRA6 are very similar and their genes are highly polymorphic. A commonly occurring allele, LILRB3*12, displayed particularly strong binding of these necrotic cells and further screening of the products of LILRB3 alleles identified motifs that correlated with binding. Immunoprecipitation of the ligand from epithelial cell lysates using recombinant LILRB3*12, identified cytokeratins 8, 18 and 19. Purified proteins obtained from epithelial cell lysates, using anti-cytokeratin 8 antibodies, were able to activate LILRB3*12 reporter cells. Knock-down of cytokeratin 8 in epithelial cells abrogated expression of the LILRB3 ligand, while staining with recombinant LILRB3*12 showed co-localisation with cytokeratin 8 and 18 in permeabilised breast cancer cells. Necrosis is a common feature of tumours. The finding of a necrosis-associated ligand for these two receptors raises the possibility of a novel interaction that alters immune responses within the tumour microenvironment. Since LILRB3 and LILRA6 genes are highly polymorphic the interaction may influence an individual's immune response to tumours. PMID:26769854

  20. A Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR) for the Detection of Factor V Leiden and Prothrombin G20210A

    PubMed Central

    Bagheri, Morteza; Rad, Isa Abdi

    2011-01-01

    ABSTRACT Introduction: In order to determine the frequencies of factor V Leiden and prothrombin G20210A point mutations in the Iranian population with Azeri Turkish origin. Material and methods: 120 unrelated individuals from general population randomly selected and were examined for factor V Leiden and prothrombin G20210A mutations using a multiplex allele specific polymerase chain reaction (MAS-PCR) assay Outcomes: The frequency of prothrombin G20210A mutation was 2.08%, which means 5 chromosomes out of 240 chromosomes had prothrombin G20210A mutation. The distribution of prothrombin 20210 GG, GA, AA genotypes and prothrombin 20210A allele were 37(92.5%), 3(7.5%), 0(0%) and 3(3.75%) in males and 78(97.5%), 2(2.5%), 0(0%) and 2(1.25%) in females, respectively. Factor V Leiden was not found in our tested group (zero chromosomes out of 240 chromosomes). Analysis of the observed frequencies in the studied groups indicates that there is no statistically significant difference between females and males, regarding prothrombin G20210A mutation (p value>0.05). Conclusions: This is the first study in its own kind in this population and implies that the frequency of Factor V Leiden G1691A (R506Q, FV-Leiden) allele is extremely low but the prothrombin G20210A mutation is more frequent in the tested group. PMID:21977183

  1. From genes to phenotypes - evaluation of two methods for the SNP analysis in archaeological remains: pyrosequencing and competitive allele specific PCR (KASPar).

    PubMed

    Pruvost, Melanie; Reissmann, Monika; Benecke, Norbert; Ludwig, Arne

    2012-01-20

    The amplification length of the DNA fragments is one major limitation of most paleogenetic analyses. Routinely, only fragments below 200 bp can be amplified, significantly reducing the content of genetic information. Although overlapping PCR strategies and next generation sequencing techniques have strongly improved data mining recently, these methods are still expensive and time consuming. In contrast, SNP analyses are easy to handle, fast and cheap. In this study, we compare two methods of SNP detection as to efficiency, cost and reliability for their use in ancient DNA applications: pyrosequencing and competitive allele specific PCR (KASPar). Our sample set consisted of 16 horse bones from two Scythian graves (600-800 BC). In conclusion, both approaches produced reliable results for most allelic patterns. But an indel of 11 bp (ASIP) could not be detected in the KASPar approach and produced problems in the pyrosequencing method (70% success rate). In such cases, we recommend checking allelic distribution using a gel approach or capillary sequencing. Overall, in comparison with the traditional mode of ancient DNA investigations (PCR, cloning, capillary sequencing), both approaches are superior for SNP analyses especially of large sample sets.

  2. A simple and rapid method for HLA-DQA1 genotyping by digestion of PCR-amplified DNA with allele specific restriction endonucleases.

    PubMed

    Maeda, M; Murayama, N; Ishii, H; Uryu, N; Ota, M; Tsuji, K; Inoko, H

    1989-11-01

    The second exon of the HLA-DQA1 genes was selectively amplified from genomic DNAs of 72 HLA-homozygous B cell lines by the polymerase chain reaction (PCR). Amplified DNAs were digested with HaeIII, Ddel, ScrFI, FokI and RsaI, which recognize allelic sequence variations in the polymorphic segments of the DQA1 second exon, and then subjected to electrophoresis in polyacrylamide gels. Eight different polymorphic patterns of restriction fragments were obtained, and seven were identical to patterns predicted from the known DNA sequences, correlating with each HLA-DQw type defined by serological typing. The remaining one pattern cannot be explained from the sequence data, suggesting the presence of a novel DQA1 allele at the nucleotide level. This PCR-RFLP method provides a simple and rapid technique for accurate definition of the HLA-DQ types at the nucleotide level, eliminating the need for radioisotope as well as allele specific oligonucleotide probes and can be extended and applied to HLA-DR, -Dw DP typing. PMID:2576477

  3. An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis.

    PubMed

    Lin, Ching-Jung; Smibert, Peter; Zhao, Xiaoyu; Hu, Jennifer F; Ramroop, Johnny; Kellner, Stefanie M; Benton, Matthew A; Govind, Shubha; Dedon, Peter C; Sternglanz, Rolf; Lai, Eric C

    2015-12-01

    N(6)-threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon-codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease.

  4. Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

    PubMed

    Choudhary, Nandlal; Wei, G; Govindarajulu, A; Roy, Avijit; Li, Wenbin; Picton, Deric D; Nakhla, M K; Levy, L; Brlansky, R H

    2015-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence. PMID:26341059

  5. Small subunit ribosomal RNA gene sequence of Minchinia teredinis (Haplosporidia: Haplosporidiidae) and a specific DNA probe and PCR primers for its detection.

    PubMed

    Stokes, N A; Siddall, M E; Burreson, E M

    1995-05-01

    Minchinia teredinis is a pathogen of wood-boring molluscs (shipworms), Teredo spp., along the middle Atlantic coast of the U.S. Genomic DNA was extracted from M. teredinis spores and small subunit (SSU) rDNA was amplified by PCR, cloned, and sequenced. The sequence of M. teredinis SSU rDNA was aligned with that of Haplosporidium nelsoni and various protists in GenBank. A 22-base oligonucleotide probe unique to M. teredinis, designated MIN702, was commercially synthesized and tested for sensitivity and specificity. In dot-blot hybridizations the probe detected 500 pg of cloned M. teredinis rDNA. The probe did not hybridize with cloned SSU rDNA of Teredo spp. or H. nelsoni. The probe was further tested for specificity with in situ hybridizations on AFA-fixed, paraffin-embedded tissue sections. The probe hybridized well with M. teredinis plasmodia and immature spores, but poorly with mature spores. The probe did not hybridize with shipworm tissue or with the haplosporidians Haplosporidium louisiana from mud crabs (Panopeus spp.) or H. nelsoni and H. costale from Crassostrea virginica. The probe and a second 18-base oligonucleotide, when used as PCR primers, amplified a 536-bp fragment of the M. teredinis SSU rRNA gene. The PCR assay was able to detect 10 fg of the cloned gene and also detected the presence of M. teredinis DNA in shipworms in which infections were observed microscopically. The 536-bp amplification product was not obtained in one Teredo sp. or in one Bankia gouldi, both categorized as uninfected after microscopic inspection. The DNA probe and PCR primers appear to be specific for M. teredinis and should be useful as diagnostic tools and for life cycle investigations.

  6. Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

    PubMed

    Choudhary, Nandlal; Wei, G; Govindarajulu, A; Roy, Avijit; Li, Wenbin; Picton, Deric D; Nakhla, M K; Levy, L; Brlansky, R H

    2015-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence.

  7. Sex determination in cattle based on simultaneous amplification of a new male-specific DNA sequence and an autosomal locus using the same primers.

    PubMed

    Weikard, R; Kühn, C; Brunner, R M; Roschlau, D; Pitra, C; Laurent, P; Schwerin, M

    2001-09-01

    A PCR-based method for sex determination of bovine DNA samples and embryo biopsies is presented. Using only one primer pair both the male-specific sequence FBNY (127 bp) and a sex-independent control PCR-fragment, the microsatellite marker FBN17 (136-140 bp) are generated in the same PCR reaction. Synteny mapping assigned the male-specific sequence to bovine chromosome Y (BTA Y), whereas FBN17 was mapped to bovine chromosome 2. Localisation of FBNY on BTA Y was confirmed by fluorescence in hybridisation of two BAC clones containing the male-specific sequence. There was no amplification of the male-specific target sequence FBNY in sheep, pig, goat, mice, man, and several wild species of the tribe Bovini. The bovine male-specific fragment was detected in dilutions containing as little as 10 pg genomic DNA and in blastomeres from embryo biopsies. The PCR assay presented here does require neither restriction endonuclease digestion of the PCR product nor additional nested PCR steps. Owing to the advantage of parallel amplification of the autosomal locus FBN17 no additional control fragment is necessary to detect PCR failure. The results of sex determination in embryo biopsies using FBNY were in agreement with the outcome from a reference assay used in commercial breeding programs. PMID:11550263

  8. SAAS-CNV: A Joint Segmentation Approach on Aggregated and Allele Specific Signals for the Identification of Somatic Copy Number Alterations with Next-Generation Sequencing Data.

    PubMed

    Zhang, Zhongyang; Hao, Ke

    2015-11-01

    Cancer genomes exhibit profound somatic copy number alterations (SCNAs). Studying tumor SCNAs using massively parallel sequencing provides unprecedented resolution and meanwhile gives rise to new challenges in data analysis, complicated by tumor aneuploidy and heterogeneity as well as normal cell contamination. While the majority of read depth based methods utilize total sequencing depth alone for SCNA inference, the allele specific signals are undervalued. We proposed a joint segmentation and inference approach using both signals to meet some of the challenges. Our method consists of four major steps: 1) extracting read depth supporting reference and alternative alleles at each SNP/Indel locus and comparing the total read depth and alternative allele proportion between tumor and matched normal sample; 2) performing joint segmentation on the two signal dimensions; 3) correcting the copy number baseline from which the SCNA state is determined; 4) calling SCNA state for each segment based on both signal dimensions. The method is applicable to whole exome/genome sequencing (WES/WGS) as well as SNP array data in a tumor-control study. We applied the method to a dataset containing no SCNAs to test the specificity, created by pairing sequencing replicates of a single HapMap sample as normal/tumor pairs, as well as a large-scale WGS dataset consisting of 88 liver tumors along with adjacent normal tissues. Compared with representative methods, our method demonstrated improved accuracy, scalability to large cancer studies, capability in handling both sequencing and SNP array data, and the potential to improve the estimation of tumor ploidy and purity.

  9. SAAS-CNV: A Joint Segmentation Approach on Aggregated and Allele Specific Signals for the Identification of Somatic Copy Number Alterations with Next-Generation Sequencing Data

    PubMed Central

    Zhang, Zhongyang; Hao, Ke

    2015-01-01

    Cancer genomes exhibit profound somatic copy number alterations (SCNAs). Studying tumor SCNAs using massively parallel sequencing provides unprecedented resolution and meanwhile gives rise to new challenges in data analysis, complicated by tumor aneuploidy and heterogeneity as well as normal cell contamination. While the majority of read depth based methods utilize total sequencing depth alone for SCNA inference, the allele specific signals are undervalued. We proposed a joint segmentation and inference approach using both signals to meet some of the challenges. Our method consists of four major steps: 1) extracting read depth supporting reference and alternative alleles at each SNP/Indel locus and comparing the total read depth and alternative allele proportion between tumor and matched normal sample; 2) performing joint segmentation on the two signal dimensions; 3) correcting the copy number baseline from which the SCNA state is determined; 4) calling SCNA state for each segment based on both signal dimensions. The method is applicable to whole exome/genome sequencing (WES/WGS) as well as SNP array data in a tumor-control study. We applied the method to a dataset containing no SCNAs to test the specificity, created by pairing sequencing replicates of a single HapMap sample as normal/tumor pairs, as well as a large-scale WGS dataset consisting of 88 liver tumors along with adjacent normal tissues. Compared with representative methods, our method demonstrated improved accuracy, scalability to large cancer studies, capability in handling both sequencing and SNP array data, and the potential to improve the estimation of tumor ploidy and purity. PMID:26583378

  10. An Efficient Approach for the Development of Locus Specific Primers in Bread Wheat (Triticum aestivum L.) and Its Application to Re-Sequencing of Genes Involved in Frost Tolerance.

    PubMed

    Babben, Steve; Perovic, Dragan; Koch, Michael; Ordon, Frank

    2015-01-01

    Recent declines in costs accelerated sequencing of many species with large genomes, including hexaploid wheat (Triticum aestivum L.). Although the draft sequence of bread wheat is known, it is still one of the major challenges to developlocus specific primers suitable to be used in marker assisted selection procedures, due to the high homology of the three genomes. In this study we describe an efficient approach for the development of locus specific primers comprising four steps, i.e. (i) identification of genomic and coding sequences (CDS) of candidate genes, (ii) intron- and exon-structure reconstruction, (iii) identification of wheat A, B and D sub-genome sequences and primer development based on sequence differences between the three sub-genomes, and (iv); testing of primers for functionality, correct size and localisation. This approach was applied to single, low and high copy genes involved in frost tolerance in wheat. In summary for 27 of these genes for which sequences were derived from Triticum aestivum, Triticum monococcum and Hordeum vulgare, a set of 119 primer pairs was developed and after testing on Nulli-tetrasomic (NT) lines, a set of 65 primer pairs (54.6%), corresponding to 19 candidate genes, turned out to be specific. Out of these a set of 35 fragments was selected for validation via Sanger's amplicon re-sequencing. All fragments, with the exception of one, could be assigned to the original reference sequence. The approach presented here showed a much higher specificity in primer development in comparison to techniques used so far in bread wheat and can be applied to other polyploid species with a known draft sequence.

  11. An Efficient Approach for the Development of Locus Specific Primers in Bread Wheat (Triticum aestivum L.) and Its Application to Re-Sequencing of Genes Involved in Frost Tolerance

    PubMed Central

    Babben, Steve; Perovic, Dragan; Koch, Michael; Ordon, Frank

    2015-01-01

    Recent declines in costs accelerated sequencing of many species with large genomes, including hexaploid wheat (Triticum aestivum L.). Although the draft sequence of bread wheat is known, it is still one of the major challenges to developlocus specific primers suitable to be used in marker assisted selection procedures, due to the high homology of the three genomes. In this study we describe an efficient approach for the development of locus specific primers comprising four steps, i.e. (i) identification of genomic and coding sequences (CDS) of candidate genes, (ii) intron- and exon-structure reconstruction, (iii) identification of wheat A, B and D sub-genome sequences and primer development based on sequence differences between the three sub-genomes, and (iv); testing of primers for functionality, correct size and localisation. This approach was applied to single, low and high copy genes involved in frost tolerance in wheat. In summary for 27 of these genes for which sequences were derived from Triticum aestivum, Triticum monococcum and Hordeum vulgare, a set of 119 primer pairs was developed and after testing on Nulli-tetrasomic (NT) lines, a set of 65 primer pairs (54.6%), corresponding to 19 candidate genes, turned out to be specific. Out of these a set of 35 fragments was selected for validation via Sanger's amplicon re-sequencing. All fragments, with the exception of one, could be assigned to the original reference sequence. The approach presented here showed a much higher specificity in primer development in comparison to techniques used so far in bread wheat and can be applied to other polyploid species with a known draft sequence. PMID:26565976

  12. [Prevalence of VRN1 Locus Alleles among Spring Common Wheat Cultivars Cultivated in Western Siberia].

    PubMed

    Efremova, T T; Chumanova, E V; Trubacheeva, N V; Arbuzova, V S; Belan, I A; Pershina, L A

    2016-02-01

    With the use of allele-specific primers developed for the VRN1 loci, the allelic diversity of the VRN-A1, VRN-B1, and VRN-D1 genes was studied in 148 spring common wheat cultivars cultivated under the conditions of Western Siberia. It was demonstrated that modern Western Siberian cultivars have the VRN-A1a allele, which is widely distributed in the world (alone or in combination with the VRN-B1a and VRN-B1c alleles). It was established that the main contribution in acceleration of the.seedling-heading time is determined by a dominant VRN-A1a allele, while the VRN-A1b allele, on the contrary, determines later plant heading. Cultivars that have the VRN-A1b allele in the genotype are found with a frequency of 8%. It was shown that cultivars with different allele combinations of two dominant genes (VRN-A1a + VRN-B1c and VRN-A1a + VRN-B1a) are characterized by earlier heading and maturing.

  13. The distribution of MICA alleles in an Austrian population: evidence for increasing polymorphism.

    PubMed

    Wenda, Sabine; Faé, Ingrid; Sanchez-Mazas, Alicia; Nunes, José M; Mayr, Wolfgang R; Fischer, Gottfried F

    2013-10-01

    The Major Histocompatibility Complex Class I Chain-Related Gene A (MICA) is located 46.4 Kb centromeric to HLA-B locus on chromosome 6; 84 alleles have been described so far. To assess the distribution of MICA alleles in an Austrian population, 322 unrelated Austrian blood donors have been typed for MICA by direct sequencing of amplified exons 2-5; sequencing of exon 6 and separating alleles by haplotype specific primers or by cloning was performed to resolve ambiguities. HLA-B was typed at low level resolution and linkage disequilibrium was determined. We observed 20 already known and four novel MICA alleles. MICA*008:01/04 was the most frequent allele (42%), followed by MICA*002:01 (11%) and MICA*009:01 (9%), three alleles (MICA*029, *067 and *068) were observed only once. No deviation from the Hardy Weinberg equilibrium was observed. Linkage disequilibrium between MICA and HLA-B alleles was observed, most extensively between MICA*008:01/04 and HLA-B*07. Our population data are in agreement with other European populations. The fact that four novel alleles have been observed indicates that the polymorphism of MICA is larger than currently estimated.

  14. Transcriptome and allele specificity associated with a 3BL locus for Fusarium crown rot resistance in bread wheat.

    PubMed

    Ma, Jian; Stiller, Jiri; Zhao, Qiang; Feng, Qi; Cavanagh, Colin; Wang, Penghao; Gardiner, Donald; Choulet, Frédéric; Feuillet, Catherine; Zheng, You-Liang; Wei, Yuming; Yan, Guijun; Han, Bin; Manners, John M; Liu, Chunji

    2014-01-01

    Fusarium pathogens cause two major diseases in cereals, Fusarium crown rot (FCR) and head blight (FHB). A large-effect locus conferring resistance to FCR disease was previously located to chromosome arm 3BL (designated as Qcrs-3B) and several independent sets of near isogenic lines (NILs) have been developed for this locus. In this study, five sets of the NILs were used to examine transcriptional changes associated with the Qcrs-3B locus and to identify genes linked to the resistance locus as a step towards the isolation of the causative gene(s). Of the differentially expressed genes (DEGs) detected between the NILs, 12.7% was located on the single chromosome 3B. Of the expressed genes containing SNP (SNP-EGs) detected, 23.5% was mapped to this chromosome. Several of the DEGs and SNP-EGs are known to be involved in host-pathogen interactions, and a large number of the DEGs were among those detected for FHB in previous studies. Of the DEGs detected, 22 were mapped in the Qcrs-3B interval and they included eight which were detected in the resistant isolines only. The enrichment of DEG, and not necessarily those containing SNPs between the resistant and susceptible isolines, around the Qcrs-3B locus is suggestive of local regulation of this region by the resistance allele. Functions for 13 of these DEGs are known. Of the SNP-EGs, 28 were mapped in the Qcrs-3B interval and biological functions for 16 of them are known. These results provide insights into responses regulated by the 3BL locus and identify a tractable number of target genes for fine mapping and functional testing to identify the causative gene(s) at this QTL. PMID:25405461

  15. High-throughput RNA-seq for allelic or locus-specific expression analysis in Arabidopsis-related species, hybrids, and allotetraploids.

    PubMed

    Ng, Danny W-K; Shi, Xiaoli; Nah, Gyoungju; Chen, Z Jeffrey

    2014-01-01

    With the next generation sequencing technology, RNA-Seq (RNA sequencing) becomes one of the most powerful tools in quantification of global transcriptomes, discovery of new transcripts and alternative isoforms, as well as detection of single nucleotide polymorphisms (SNPs). RNA-Seq is advantageous over hybridization-based gene quantification methods: (1) it does not require prior information about genomic sequences, (2) it avoids high background problem caused by cross-hybridization, and (3) it is highly sensitive and avoids background and saturation of signals; and finally it is capable of detecting allelic expression differences in hybrids and allopolyploids. We used the RNA-Seq method to determine the genome-wide transcriptome changes in Arabidopsis allotetraploids and their parents, A. thaliana and A. arenosa. The use of this approach allows us to quantify transcriptome from these species and more importantly, to identify allelic or homoeologous-specific gene expression that plays a role in morphological evolution of allopolyploids. The computational pipelines developed are also applicable to the analysis of chromatin immunoprecipitation sequencing (ChIP-seq) data in Arabidopsis-related species, hybrids, and allopolyploids. Comparative analysis of RNA-Seq and ChIP-Seq data will allow us to determine the effects of chromatin modifications on nonadditive gene expression in hybrids and allopolyploids.

  16. Specific alleles at immune genes, rather than genome-wide heterozygosity, are related to immunity and survival in the critically endangered Attwater's prairie-chicken.

    PubMed

    Bateson, Zachary W; Hammerly, Susan C; Johnson, Jeff A; Morrow, Michael E; Whittingham, Linda A; Dunn, Peter O

    2016-10-01

    The negative effects of inbreeding on fitness are serious concerns for populations of endangered species. Reduced fitness has been associated with lower genome-wide heterozygosity and immune gene diversity in the wild; however, it is rare that both types of genetic measures are included in the same study. Thus, it is often unclear whether the variation in fitness is due to the general effects of inbreeding, immunity-related genes or both. Here, we tested whether genome-wide heterozygosity (20 990 SNPs) and diversity at nine immune genes were better predictors of two measures of fitness (immune response and survival) in the endangered Attwater's prairie-chicken (Tympanuchus cupido attwateri). We found that postrelease survival of captive-bred birds was related to alleles of the innate (Toll-like receptors, TLRs) and adaptive (major histocompatibility complex, MHC) immune systems, but not to genome-wide heterozygosity. Likewise, we found that the immune response at the time of release was related to TLR and MHC alleles, and not to genome-wide heterozygosity. Overall, this study demonstrates that immune genes may serve as important genetic markers when monitoring fitness in inbred populations and that in some populations specific functional genes may be better predictors of fitness than genome-wide heterozygosity.

  17. Allelic loss in colorectal carcinoma

    SciTech Connect

    Kern, S.E.; Fearon, E.R.; Tersmette, K.W.F.; Enterline, J.P.; Vogelstein, B.; Hamilton, S.R. ); Leppert, M.; Nakamura, Yusuke; White, R. )

    1989-06-02

    Clinical and pathological associations with molecular genetic alterations were studied in colorectal carcinomas from 83 patients. Fractional allelic loss, a measure of allelic deletions throughout the genome, and allelic deletions of specific chromosomal arms (the short arm of 17 and long arm of 18) each provided independent prognostic information by multivariate analysis when considered individually with Dukes' classification. Distant metastasis was significantly associated with high fractional allelic loss and with deletions of 17p and 18q. Mutations of ras proto-oncogenes and deletions of 5q had no prognostic importance. Statistically significant associations were also found between allelic losses and a family history of cancer, left-sided tumor location, and absence of extracellular tumor mucin. Allelic deletion analysis thus identified subsets of colorectal carcinoma with increased predilection for distant metastasis and cancer-related death. Further studies may define a subset of genetic alterations that can be used clinically to help assess prognosis.

  18. Branched modular primers in DNA sequencing

    SciTech Connect

    Mugasimangalam, R.C.; Shmulevitz, M. |; Ramanathan, V.

    1997-08-01

    The need to synthesize new sequencing primers, such as in primer walking, can be eliminated by assembling modular primers from oligonucleotide modules selected from presynthesized libraries. Our earlier modular primers consisted of 5-mers, 6-mers or 7-mers, annealing to the template contiguously with each other. Here we introduce a novel {open_quotes}branched{close_quotes} type of modular primer with a distinctly different specificity mechanism. The concept of a {open_quotes}branched{close_quotes} primer involves modules that are physically linked by annealing to each other as well as to the target, forming a branched structure of the 3-way junction type. While contiguous modular primers are made specific by the preference of the polymerase for longer primer, branched primers, in contrast, owe their specificity to cooperative annealing of their modules to the intended site on the template. This cooperativity of annealing to the template is provided by mutually complementary segments in the two modules that bind each other. Thus the primer-template complex is no longer limited to linear sequences, but acquires another, second dimension giving the modular primer new functionality.

  19. Determinants of specific RNA interference-mediated silencing of human beta-globin alleles differing by a single nucleotide polymorphism.

    PubMed

    Dykxhoorn, Derek M; Schlehuber, Lisa D; London, Irving M; Lieberman, Judy

    2006-04-11

    A single nucleotide polymorphism (SNP) in the sickle beta-globin gene (beta(S)) leads to sickle cell anemia. Sickling increases sharply with deoxy sickle Hb concentration and decreases with increasing fetal gamma-globin concentration. Measures that decrease sickle Hb concentration should have an antisickling effect. RNA interference (RNAi) uses small interfering (si)RNAs for sequence-specific gene silencing. A beta(S) siRNA with position 10 of the guide strand designed to align with the targeted beta(S) SNP specifically silences beta(S) gene expression without affecting the expression of the gamma-globin or normal beta-globin (beta(A)) genes. Silencing is increased by altering the 5' end of the siRNA antisense (guide) strand to enhance its binding to the RNA-induced silencing complex (RISC). Specific beta(S) silencing was demonstrated by using a luciferase reporter and full-length beta(S) cDNA transfected into HeLa cells and mouse erythroleukemia cells, where it was expressed in the context of the endogenous beta-globin gene promoter and the locus control region enhancers. When this strategy was used to target beta(E), silencing was not limited to the mutant gene but also targeted the normal beta(A) gene. siRNAs, mismatched with their target at position 10, guided mRNA cleavage in all cases except when two bulky purines were aligned. The specific silencing of the beta(S)-globin gene, as compared with beta(E), as well as studies of silencing SNP mutants in other diseases, indicates that siRNAs developed to target a disease-causing SNP will be specific if the mutant residue is a pyrimidine and the normal residue is a purine.

  20. Analysis of allele-specific expression in mouse liver by RNA-Seq: a comparison with Cis-eQTL identified using genetic linkage.

    PubMed

    Lagarrigue, Sandrine; Martin, Lisa; Hormozdiari, Farhad; Roux, Pierre-François; Pan, Calvin; van Nas, Atila; Demeure, Olivier; Cantor, Rita; Ghazalpour, Anatole; Eskin, Eleazar; Lusis, Aldons J

    2013-11-01

    We report an analysis of allele-specific expression (ASE) and parent-of-origin expression in adult mouse liver using next generation sequencing (RNA-Seq) of reciprocal crosses of heterozygous F1 mice from the parental strains C57BL/6J and DBA/2J. We found a 60% overlap between genes exhibiting ASE and putative cis-acting expression quantitative trait loci (cis-eQTL) identified in an intercross between the same strains. We discuss the various biological and technical factors that contribute to the differences. We also identify genes exhibiting parental imprinting and complex expression patterns. Our study demonstrates the importance of biological replicates to limit the number of false positives with RNA-Seq data.

  1. Mutant Allele-Specific Uncoupling of PENETRATION3 Functions Reveals Engagement of the ATP-Binding Cassette Transporter in Distinct Tryptophan Metabolic Pathways1[OPEN

    PubMed Central

    Lu, Xunli; Dittgen, Jan; Piślewska-Bednarek, Mariola; Molina, Antonio; Schneider, Bernd; Doubský, Jan; Schneeberger, Korbinian; Schulze-Lefert, Paul

    2015-01-01

    Arabidopsis (Arabidopsis thaliana) PENETRATION (PEN) genes quantitatively contribute to the execution of different forms of plant immunity upon challenge with diverse leaf pathogens. PEN3 encodes a plasma membrane-resident pleiotropic drug resistance-type ATP-binding cassette transporter and is thought to act in a pathogen-inducible and PEN2 myrosinase-dependent metabolic pathway in extracellular defense. This metabolic pathway directs the intracellular biosynthesis and activation of tryptophan-derived indole glucosinolates for subsequent PEN3-mediated efflux across the plasma membrane at pathogen contact sites. However, PEN3 also functions in abiotic stress responses to cadmium and indole-3-butyric acid (IBA)-mediated auxin homeostasis in roots, raising the possibility that PEN3 exports multiple functionally unrelated substrates. Here, we describe the isolation of a pen3 allele, designated pen3-5, that encodes a dysfunctional protein that accumulates in planta like wild-type PEN3. The specific mutation in pen3-5 uncouples PEN3 functions in IBA-stimulated root growth modulation, callose deposition induced with a conserved peptide epitope of bacterial flagellin (flg22), and pathogen-inducible salicylic acid accumulation from PEN3 activity in extracellular defense, indicating the engagement of multiple PEN3 substrates in different PEN3-dependent biological processes. We identified 4-O-β-d-glucosyl-indol-3-yl formamide (4OGlcI3F) as a pathogen-inducible, tryptophan-derived compound that overaccumulates in pen3 leaf tissue and has biosynthesis that is dependent on an intact PEN2 metabolic pathway. We propose that a precursor of 4OGlcI3F is the PEN3 substrate in extracellular pathogen defense. These precursors, the shared indole core present in IBA and 4OGlcI3F, and allele-specific uncoupling of a subset of PEN3 functions suggest that PEN3 transports distinct indole-type metabolites in distinct biological processes. PMID:26023163

  2. Mutant Allele-Specific Uncoupling of PENETRATION3 Functions Reveals Engagement of the ATP-Binding Cassette Transporter in Distinct Tryptophan Metabolic Pathways.

    PubMed

    Lu, Xunli; Dittgen, Jan; Piślewska-Bednarek, Mariola; Molina, Antonio; Schneider, Bernd; Svatoš, Aleš; Doubský, Jan; Schneeberger, Korbinian; Weigel, Detlef; Bednarek, Paweł; Schulze-Lefert, Paul

    2015-07-01

    Arabidopsis (Arabidopsis thaliana) penetration (PEN) genes quantitatively contribute to the execution of different forms of plant immunity upon challenge with diverse leaf pathogens. PEN3 encodes a plasma membrane-resident pleiotropic drug resistance-type ATP-binding cassette transporter and is thought to act in a pathogen-inducible and PEN2 myrosinase-dependent metabolic pathway in extracellular defense. This metabolic pathway directs the intracellular biosynthesis and activation of tryptophan-derived indole glucosinolates for subsequent PEN3-mediated efflux across the plasma membrane at pathogen contact sites. However, PEN3 also functions in abiotic stress responses to cadmium and indole-3-butyric acid (IBA)-mediated auxin homeostasis in roots, raising the possibility that PEN3 exports multiple functionally unrelated substrates. Here, we describe the isolation of a pen3 allele, designated pen3-5, that encodes a dysfunctional protein that accumulates in planta like wild-type PEN3. The specific mutation in pen3-5 uncouples PEN3 functions in IBA-stimulated root growth modulation, callose deposition induced with a conserved peptide epitope of bacterial flagellin (flg22), and pathogen-inducible salicylic acid accumulation from PEN3 activity in extracellular defense, indicating the engagement of multiple PEN3 substrates in different PEN3-dependent biological processes. We identified 4-O-β-D-glucosyl-indol-3-yl formamide (4OGlcI3F) as a pathogen-inducible, tryptophan-derived compound that overaccumulates in pen3 leaf tissue and has biosynthesis that is dependent on an intact PEN2 metabolic pathway. We propose that a precursor of 4OGlcI3F is the PEN3 substrate in extracellular pathogen defense. These precursors, the shared indole core present in IBA and 4OGlcI3F, and allele-specific uncoupling of a subset of PEN3 functions suggest that PEN3 transports distinct indole-type metabolites in distinct biological processes.

  3. Collagen Specific T-Cell Repertoire and HLA-DR Alleles: Biomarkers of Active Refractory Rheumatoid Arthritis

    PubMed Central

    Di Sante, Gabriele; Tolusso, Barbara; Fedele, Anna Laura; Gremese, Elisa; Alivernini, Stefano; Nicolò, Chiara; Ria, Francesco; Ferraccioli, Gianfranco

    2015-01-01

    Rheumatoid arthritis (RA) is characterized by chronic joint inflammation and associates with HLA-DRB1*04. The Collagen IIp261-273-specific T cell repertoire in the peripheral blood of DR4 + patients at the onset of the disease shows a restricted TCR-beta chain usage among which the most frequent is TRBV25. To define whether this group of DR4-restricted collagen-specific shared T cell could represent markers of active-severe disease and response to therapy, 90 subjects affected by early-RA were enrolled in the study; peripheral blood mononuclear cells were cultured with or without the human collagen II peptide p261-273 and were examined by immunoscope analysis for the usage of the previously identified shared TCR-beta chains. We report that the presence of T cells carrying rearrangement TRBV25 associated with HLA-DR haplotype and disease activity. HLA-DRB1* haplotypes 04–04, 04–01 and 04–11 were significantly associated with usage of TRBV25, higher disease activity at the onset of disease and poor response to DMARDs. Finally, the HLA-DRB1* haplotype appeared complementary with current serologic tools to predict good and poor responders in a treat to target strategy. The data reported here offer clues to predict the course of the disease and to foresee personalized treatments in RA patients. PMID:26844284

  4. Collagen Specific T-Cell Repertoire and HLA-DR Alleles: Biomarkers of Active Refractory Rheumatoid Arthritis.

    PubMed

    Di Sante, Gabriele; Tolusso, Barbara; Fedele, Anna Laura; Gremese, Elisa; Alivernini, Stefano; Nicolò, Chiara; Ria, Francesco; Ferraccioli, Gianfranco

    2015-12-01

    Rheumatoid arthritis (RA) is characterized by chronic joint inflammation and associates with HLA-DRB1*04. The Collagen IIp261-273-specific T cell repertoire in the peripheral blood of DR4 + patients at the onset of the disease shows a restricted TCR-beta chain usage among which the most frequent is TRBV25. To define whether this group of DR4-restricted collagen-specific shared T cell could represent markers of active-severe disease and response to therapy, 90 subjects affected by early-RA were enrolled in the study; peripheral blood mononuclear cells were cultured with or without the human collagen II peptide p261-273 and were examined by immunoscope analysis for the usage of the previously identified shared TCR-beta chains. We report that the presence of T cells carrying rearrangement TRBV25 associated with HLA-DR haplotype and disease activity. HLA-DRB1* haplotypes 04-04, 04-01 and 04-11 were significantly associated with usage of TRBV25, higher disease activity at the onset of disease and poor response to DMARDs. Finally, the HLA-DRB1* haplotype appeared complementary with current serologic tools to predict good and poor responders in a treat to target strategy. The data reported here offer clues to predict the course of the disease and to foresee personalized treatments in RA patients.

  5. Deletion endpoint allele-specificity in the developmentally regulated elimination of an internal sequence (IES) in Paramecium.

    PubMed

    Dubrana, K; Le Mouël, A; Amar, L

    1997-06-15

    Ciliated protozoa undergo thousands of site-specific DNA deletion events during the programmed development of micronuclear genomes to macronuclear genomes. Two deletion elements, W1 and W2, were identified in the Paramecium primaurelia wild-type 156 strain. Here, we report the characterization of both elements in wild-type strain 168 and show that they display variant deletion patterns when compared with those of strain 156. The W1 ( 168 ) element is defective for deletion. The W2 ( 168 ) element is excised utilizing two alternative boundaries on one side, both are different from the boundary utilized to excise the W2156 element. By crossing the 156 and 168 strains, we demonstrate that the definition of all deletion endpoints are each controlled by cis -acting determinant(s) rather than by strain-specific trans-acting factor(s). Sequence comparison of all deleted DNA segments indicates that the 5'-TA-3'terminal sequence is strictly required at their ends. Furthermore the identity of the first eight base pairs of these ends to a previously established consensus sequence correlates with the frequency of the corresponding deletion events. Our data implies the existence of an adaptive convergent evolution of these Paramecium deleted DNA segment end sequences. PMID:9171098

  6. Negotiations Primer.

    ERIC Educational Resources Information Center

    New Jersey Principals and Supervisors Association, Trenton.

    This booklet provides step-by-step advice for New Jersey school administrators on the organization of bargaining units and on negotiation. The section covering organizing reviews briefly the rights of school employee associations to negotiate, the values of collective negotiation, and the specific requirements and procedures involved in…

  7. Diskette Primer.

    ERIC Educational Resources Information Center

    Chien, Philip

    1986-01-01

    This detailed look into proper diskette maintenance presents general precautions on temperature, humidity, lifespan, labeling, storage, and disk drive maintenance. The Apple UniDisk 3.5 disk drive is described, tips on purchasing diskettes are presented, and information on specific data retrieval and backup software and diskettes is included. (MBR)

  8. Detecting Allelic Expression Imbalance at Candidate Genes Using 5' Exonuclease Genotyping Technology.

    PubMed

    Gahan, Jillian M; Byrne, Mikaela M; Hill, Matthew; Quinn, Emma M; Murphy, Ross T; Anney, Richard J L; Ryan, Anthony W

    2015-01-01

    Genetic variation along the length of a chromosome can influence the transcription of a gene. In a heterozygous individual, this may lead to one chromosome producing different levels of RNA, compared to its paired chromosome, for a given gene. Allelic differences in gene expression can offer insight into the role of variation in transcription, and subsequently infer a route to conferring disease risk. This phenomenon is known as allele expression imbalance or AEI, which may be assayed using a PCR-based method that includes the quantification of the relative dosage of each allele (e.g., 5' exonuclease assays, TaqMan™). Importantly, in heterozygous individuals the resolution of expression imbalance is performed within a controlled system; the comparison of the alternate allele is reported relative to the wild-type, as the experiment can be performed within a single sample, controlled for background genetic information. Alternative methods for the detection of AEI include Primer-extension MALDI-TOF (Sequenom MassARRAY(®)), Next-Generation Sequencing, and SNP genotyping arrays. Here we present the methods used for the TaqMan™ approach and include a description of the SNP identification, allele-specific PCR, and analytic methods to convert allele amplification metrics to relative allele dosage.

  9. Specific primer sets used to amplify by PCR the hepatitis B virus overlapping S/Pol region select different viral variants.

    PubMed

    Cuestas, M L; Mathet, V L; Oubiña, J R

    2012-10-01

    PCR detection of viral genomes has provided new insights into viral diagnosis. Nowadays, it is the most frequently used nucleic acid testing (qualitative and quantitative) technique. The aim of this study was to analyse the major circulating hepatitis B virus (HBV) variants PCR-amplified by three sets of primers in a patient infected with genotype E. The HBV S/Pol overlapping genomic region was amplified from the serum of an infected child using three primer sets previously described. Sequence analysis corresponding to the HBV S/Pol region revealed the presence of different viral populations depending on the set of primers used. D144A S-escape mutant was detected with two of the primer sets, while the rtL217R mutant within the Pol - conferring resistance to Adefovir - could be picked up with a different pair of primer sets. This study undoubtedly implies that the description of viral polymorphisms should be stated together with the sequence of the primers used for PCR amplification when studies of escape and/or antiviral-resistant HBV mutants are carried out.

  10. Specific primer sets used to amplify by PCR the hepatitis B virus overlapping S/Pol region select different viral variants.

    PubMed

    Cuestas, M L; Mathet, V L; Oubiña, J R

    2012-10-01

    PCR detection of viral genomes has provided new insights into viral diagnosis. Nowadays, it is the most frequently used nucleic acid testing (qualitative and quantitative) technique. The aim of this study was to analyse the major circulating hepatitis B virus (HBV) variants PCR-amplified by three sets of primers in a patient infected with genotype E. The HBV S/Pol overlapping genomic region was amplified from the serum of an infected child using three primer sets previously described. Sequence analysis corresponding to the HBV S/Pol region revealed the presence of different viral populations depending on the set of primers used. D144A S-escape mutant was detected with two of the primer sets, while the rtL217R mutant within the Pol - conferring resistance to Adefovir - could be picked up with a different pair of primer sets. This study undoubtedly implies that the description of viral polymorphisms should be stated together with the sequence of the primers used for PCR amplification when studies of escape and/or antiviral-resistant HBV mutants are carried out. PMID:22967107

  11. Identification of FAD2 and FAD3 genes in Brassica napus genome and development of allele-specific markers for high oleic and low linolenic acid contents.

    PubMed

    Yang, Qingyong; Fan, Chuchuan; Guo, Zhenhua; Qin, Jie; Wu, Jianzhong; Li, Qingyuan; Fu, Tingdong; Zhou, Yongming

    2012-08-01

    Modification of oleic acid (C18:1) and linolenic acid (C18:3) contents in seeds is one of the major goals for quality breeding after removal of erucic acid in oilseed rape (Brassica napus). The fatty acid desaturase genes FAD2 and FAD3 have been shown as the major genes for the control of C18:1 and C18:3 contents. However, the genome structure and locus distributions of the two gene families in amphidiploid B. napus are still not completely understood to date. In the present study, all copies of FAD2 and FAD3 genes in the A- and C-genome of B. napus and its two diploid progenitor species, Brassica rapa and Brassica oleracea, were identified through bioinformatic analysis and extensive molecular cloning. Two FAD2 genes exist in B. rapa and B. oleracea, and four copies of FAD2 genes exist in B. napus. Three and six copies of FAD3 genes were identified in diploid species and amphidiploid species, respectively. The genetic control of high C18:1 and low C18:3 contents in a double haploid population was investigated through mapping of the quantitative trait loci (QTL) for the traits and the molecular cloning of the underlying genes. One major QTL of BnaA.FAD2.a located on A5 chromosome was responsible for the high C18:1 content. A deleted mutation in the BnaA.FAD2.a locus was uncovered, which represented a previously unidentified allele for the high oleic variation in B. napus species. Two major QTLs on A4 and C4 chromosomes were found to be responsible for the low C18:3 content in the DH population as well as in SW Hickory. Furthermore, several single base pair changes in BnaA.FAD3.b and BnaC.FAD3.b were identified to cause the phenotype of low C18:3 content. Based on the results of genetic mapping and identified sequences, allele-specific markers were developed for FAD2 and FAD3 genes. Particularly, single-nucleotide amplified polymorphisms markers for FAD3 alleles were demonstrated to be a reliable type of SNP markers for unambiguous identification of genotypes with

  12. Identification of FAD2 and FAD3 genes in Brassica napus genome and development of allele-specific markers for high oleic and low linolenic acid contents.

    PubMed

    Yang, Qingyong; Fan, Chuchuan; Guo, Zhenhua; Qin, Jie; Wu, Jianzhong; Li, Qingyuan; Fu, Tingdong; Zhou, Yongming

    2012-08-01

    Modification of oleic acid (C18:1) and linolenic acid (C18:3) contents in seeds is one of the major goals for quality breeding after removal of erucic acid in oilseed rape (Brassica napus). The fatty acid desaturase genes FAD2 and FAD3 have been shown as the major genes for the control of C18:1 and C18:3 contents. However, the genome structure and locus distributions of the two gene families in amphidiploid B. napus are still not completely understood to date. In the present study, all copies of FAD2 and FAD3 genes in the A- and C-genome of B. napus and its two diploid progenitor species, Brassica rapa and Brassica oleracea, were identified through bioinformatic analysis and extensive molecular cloning. Two FAD2 genes exist in B. rapa and B. oleracea, and four copies of FAD2 genes exist in B. napus. Three and six copies of FAD3 genes were identified in diploid species and amphidiploid species, respectively. The genetic control of high C18:1 and low C18:3 contents in a double haploid population was investigated through mapping of the quantitative trait loci (QTL) for the traits and the molecular cloning of the underlying genes. One major QTL of BnaA.FAD2.a located on A5 chromosome was responsible for the high C18:1 content. A deleted mutation in the BnaA.FAD2.a locus was uncovered, which represented a previously unidentified allele for the high oleic variation in B. napus species. Two major QTLs on A4 and C4 chromosomes were found to be responsible for the low C18:3 content in the DH population as well as in SW Hickory. Furthermore, several single base pair changes in BnaA.FAD3.b and BnaC.FAD3.b were identified to cause the phenotype of low C18:3 content. Based on the results of genetic mapping and identified sequences, allele-specific markers were developed for FAD2 and FAD3 genes. Particularly, single-nucleotide amplified polymorphisms markers for FAD3 alleles were demonstrated to be a reliable type of SNP markers for unambiguous identification of genotypes with

  13. Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction

    SciTech Connect

    Day, D.J.; Barany, F.; Speiser, P.W.

    1995-09-01

    Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia, an inherited inability to synthesize cortisol that occurs in 1 in 10,000-15,000 births. Affected females are born with ambiguous genitalia, a condition that can be ameliorated by administering dexamethasone to the mother for most of gestation. Prenatal diagnosis is required for accurate treatment of affected females as well as for genetic counseling purposes. Approximately 95% of mutations causing this disorder result from recombinations between the gene encoding the 21-hydroxylase enzyme (CYP21) and a linked, highly homologous pseudogene (CYP21P). Approximately 20% of these mutations are gene deletions, and the remainder are gene conversions that transfer any of nine deleterious mutations from the CYP21P pseudogene to CYP21. We describe a methodology for genetic diagnosis of 21-hydroxylase deficiency that utilizes gene-specific PCR amplification in conjunction with thermostable DNA ligase to discriminate single nucleotide variations in a multiplexed ligation detection assay. The assay has been designed to be used with either fluorescent or radioactive detection of ligation products by electrophoresis on denaturing acrylamide gels and is readily adaptable for use in other disease systems. 30 refs., 5 figs.

  14. CEP290 alleles in mice disrupt tissue-specific cilia biogenesis and recapitulate features of syndromic ciliopathies.

    PubMed

    Rachel, Rivka A; Yamamoto, Erin A; Dewanjee, Mrinal K; May-Simera, Helen L; Sergeev, Yuri V; Hackett, Alice N; Pohida, Katherine; Munasinghe, Jeeva; Gotoh, Norimoto; Wickstead, Bill; Fariss, Robert N; Dong, Lijin; Li, Tiansen; Swaroop, Anand

    2015-07-01

    Distinct mutations in the centrosomal-cilia protein CEP290 lead to diverse clinical findings in syndromic ciliopathies. We show that CEP290 localizes to the transition zone in ciliated cells, precisely to the region of Y-linkers between central microtubules and plasma membrane. To create models of CEP290-associated ciliopathy syndromes, we generated Cep290(ko/ko) and Cep290(gt/gt) mice that produce no or a truncated CEP290 protein, respectively. Cep290(ko/ko) mice exhibit early vision loss and die from hydrocephalus. Retinal photoreceptors in Cep290(ko/ko) mice lack connecting cilia, and ciliated ventricular ependyma fails to mature. The minority of Cep290(ko/ko) mice that escape hydrocephalus demonstrate progressive kidney pathology. Cep290(gt/gt) mice die at mid-gestation, and the occasional Cep290(gt/gt) mouse that survives shows hydrocephalus and severely cystic kidneys. Partial loss of CEP290-interacting ciliopathy protein MKKS mitigates lethality and renal pathology in Cep290(gt/gt) mice. Our studies demonstrate domain-specific functions of CEP290 and provide novel therapeutic paradigms for ciliopathies. PMID:25859007

  15. Genetic linkage analysis using pooled DNA and infrared detection of tailed STRP primer patterns

    NASA Astrophysics Data System (ADS)

    Oetting, William S.; Wildenberg, Scott C.; King, Richard A.

    1996-04-01

    The mapping of a disease locus to a specific chromosomal region is an important step in the eventual isolation and analysis of a disease causing gene. Conventional mapping methods analyze large multiplex families and/or smaller nuclear families to find linkage between the disease and a chromosome marker that maps to a known chromosomal region. This analysis is time consuming and tedious, typically requiring the determination of 30,000 genotypes or more. For appropriate populations, we have instead utilized pooled DNA samples for gene mapping which greatly reduces the amount of time necessary for an initial chromosomal screen. This technique assumes a common founder for the disease locus of interest and searches for a region of a chromosome shared between affected individuals. Our analysis involves the PCR amplification of short tandem repeat polymorphisms (STRP) to detect these shared regions. In order to reduce the cost of genotyping, we have designed unlabeled tailed PCR primers which, when combined with a labeled universal primer, provides for an alternative to synthesizing custom labeled primers. The STRP pattern is visualized with an infrared fluorescence based automated DNA sequencer and the patterns quantitated by densitometric analysis of the allele pattern. Differences in the distribution of alleles between pools of affected and unaffected individuals, including a reduction in the number of alleles in the affected pool, indicate the sharing of a region of a chromosome. We have found this method effective for markers 10 - 15 cM away from the disease locus for a recessive genetic disease.

  16. Parental Allele-Specific Chromatin Configuration in a Boundary–Imprinting-Control Element Upstream of the Mouse H19 Gene

    PubMed Central

    Khosla, Sanjeev; Aitchison, Alan; Gregory, Richard; Allen, Nicholas D.; Feil, Robert

    1999-01-01

    The mouse H19 gene is expressed from the maternal chromosome exclusively. A 2-kb region at 2 to 4 kb upstream of H19 is paternally methylated throughout development, and these sequences are necessary for the imprinted expression of both H19 and the 5′-neighboring Igf2 gene. In particular, on the maternal chromosome this element appears to insulate the Igf2 gene from enhancers located downstream of H19. We analyzed the chromatin organization of this element by assaying its sensitivity to nucleases in nuclei. Six DNase I hypersensitive sites (HS sites) were detected on the unmethylated maternal chromosome exclusively, the two most prominent of which mapped 2.25 and 2.75 kb 5′ to the H19 transcription initiation site. Five of the maternal HS sites were present in expressing and nonexpressing tissues and in embryonic stem (ES) cells. They seem, therefore, to reflect the maternal origin of the chromosome rather than the expression of H19. A sixth maternal HS site, at 3.45 kb upstream of H19, was detected in ES cells only. The nucleosomal organization of this element was analyzed in tissues and ES cells by micrococcal nuclease digestion. Specifically on the maternal chromosome, an unusual and strong banding pattern was obtained, suggestive of a nonnucleosomal organization. From our studies, it appears that the unusual chromatin organization with the presence of HS sites (maternal chromosome) and DNA methylation (paternal chromosome) in this element are mutually exclusive and reflect alternate epigenetic states. In addition, our data suggest that nonhistone proteins are associated with the maternal chromosome and that these might be involved in its boundary function. PMID:10082521

  17. Design of an F1 hybrid breeding strategy for ryegrasses based on selection of self-incompatibility locus-specific alleles.

    PubMed

    Pembleton, Luke W; Shinozuka, Hiroshi; Wang, Junping; Spangenberg, German C; Forster, John W; Cogan, Noel O I

    2015-01-01

    Relatively modest levels of genetic gain have been achieved in conventional ryegrass breeding when compared to cereal crops such as maize, current estimates indicating an annual improvement of 0.25-0.6% in dry matter production. This property is partially due to an inability to effectively exploit heterosis through the formation of F1 hybrids. Controlled crossing of ryegrass lines from geographically distant origins has demonstrated the occurrence of heterosis, which can result in increases of dry matter production in the order of 25%. Although capture of hybrid vigor offers obvious advantages for ryegrass cultivar production, to date there have been no effective and commercially suitable methods for obtaining high proportions of F1 hybrid seed. Continued advances in fine-scale genetic and physical mapping of the gametophytic self-incompatibility (SI) loci (S and Z) of ryegrasses are likely in the near future to permit the identification of closely linked genetic markers that define locus-specific haplotypes, allowing prediction of allelic variants and hence compatibility between different plant genotypes. Given the availability of such information, a strategy for efficient generation of ryegrass cultivars with a high proportion of F1 hybrid individuals has been simulated, which is suitable for commercial implementation. Through development of two parental pools with restricted diversity at the SI loci, relative crossing compatibility between pools is increased. Based on simulation of various levels of SI allele diversity restriction, the most effective scheme will generate 83.33% F1 hybrids. Results from the study, including the impact of varying flowering time, are discussed along with a proposed breeding design for commercial application. PMID:26442077

  18. Electromobility Shift Assay Reveals Evidence in Favor of Allele-Specific Binding of RUNX1 to the 5' Hypersensitive Site 4-Locus Control Region.

    PubMed

    Dehghani, Hossein; Ghobakhloo, Sepideh; Neishabury, Maryam

    2016-08-01

    In our previous studies on the Iranian β-thalassemia (β-thal) patients, we identified an association between the severity of the β-thal phenotype and the polymorphic palindromic site at the 5' hypersensitive site 4-locus control region (5'HS4-LCR) of the β-globin gene cluster. Furthermore, a linkage disequilibrium was observed between this region and XmnI-HBG2 in the patient population. Based on this data, it was suggested that the well-recognized phenotype-ameliorating role assigned to positive XmnI could be associated with its linked elements in the LCR. To investigate the functional significance of polymorphisms at the 5'HS4-LCR, we studied its influence on binding of transcription factors. Web-based predictions of transcription factor binding revealed a binding site for runt-related transcription factor 1 (RUNX1), when the allele at the center of the palindrome (TGGGG(A/G)CCCCA) was A but not when it was G. Furthermore, electromobility shift assay (EMSA) presented evidence in support of allele-specific binding of RUNX1 to 5'HS4. Considering that RUNX1 is a well-known regulator of hematopoiesis, these preliminary data suggest the importance of further studies to confirm this interaction and consequently investigate its functional and phenotypical relevance. These studies could help us to understand the molecular mechanism behind the phenotype modifying role of the 5'HS4-LCR polymorphic palindromic region (rs16912979), which has been observed in previous studies. PMID:27492765

  19. RNA-Seq Analysis of Allele-Specific Expression, Hybrid Effects, and Regulatory Divergence in Hybrids Compared with Their Parents from Natural Populations

    PubMed Central

    Bell, Graeme D.M.; Kane, Nolan C.; Rieseberg, Loren H.; Adams, Keith L.

    2013-01-01

    Hybridization is a prominent process among natural plant populations that can result in phenotypic novelty, heterosis, and changes in gene expression. The effects of intraspecific hybridization on F1 hybrid gene expression were investigated using parents from divergent, natural populations of Cirsium arvense, an invasive Compositae weed. Using an RNA-seq approach, the expression of 68,746 unigenes was quantified in parents and hybrids. The expression levels of 51% of transcripts differed between parents, a majority of which had less than 1.25× fold-changes. More unigenes had higher expression in the invasive parent (P1) than the noninvasive parent (P2). Of those that were divergently expressed between parents, 10% showed additive and 81% showed nonadditive (transgressive or dominant) modes of gene action in the hybrids. A majority of the dominant cases had P2-like expression patterns in the hybrids. Comparisons of allele-specific expression also enabled a survey of cis- and trans-regulatory effects. Cis- and trans-regulatory divergence was found at 70% and 68% of 62,281 informative single-nucleotide polymorphism sites, respectively. Of the 17% of sites exhibiting both cis- and trans-effects, a majority (70%) had antagonistic regulatory interactions (cis x trans); trans-divergence tended to drive higher expression of the P1 allele, whereas cis-divergence tended to increase P2 transcript abundance. Trans-effects correlated more highly than cis with parental expression divergence and accounted for a greater proportion of the regulatory divergence at sites with additive compared with nonadditive inheritance patterns. This study explores the nature of, and types of mechanisms underlying, expression changes that occur in upon intraspecific hybridization in natural populations. PMID:23677938

  20. Major histocompatibility complex class I chain related (MIC) A gene, TNFa microsatellite alleles and TNFB alleles in juvenile idiopathic arthritis patients from Latvia.

    PubMed

    Nikitina Zake, Liene; Cimdina, Ija; Rumba, Ingrida; Dabadghao, Preethi; Sanjeevi, Carani B

    2002-05-01

    In order to analyze involvement of major histocompatibility complex class I chain-related gene A (MICA) and tumor necrosis factor a (TNFa) microsatellite polymorphisms as well as TNFB gene in juvenile idiopathic arthritis (JIA), we studied 128 patients divided into groups according to clinical features [monoarthritis (n = 14), oligoarthritis (n = 58), polyarthritis (n = 50), and systemic (n = 6)], and 114 age- and sex-matched healthy controls from Latvia. DNA samples were amplified with specific primers and used for genotyping of MICA and TNFa microsatellite. Typing for a biallelic NcoI polymerase chain reaction RFLP polymorphism located at the first intron of TNFB gene was done as follows: restriction digests generated fragments of 555bp and 185bp for TNFB*1 allele, and 740bp for TNFB*2 allele. The results were compared between cases and controls. We found significant increase of MICA allele A4 (p = 0.009; odds ratio [OR] = 2.3) and allele TNFa2 (p = 0.0001; OR = 4.4) in patients compared with controls. The frequency of allele TNFa9 was significantly decreased (p = 0.0001; OR = 0.1) in patients with JIA. No significant differences of TNFB allele frequency were found. Our data suggest that MICA and TNFa microsatellite polymorphisms may be used as markers for determination of susceptibility and protection from JIA.

  1. Laser Doppler velocimetry primer

    NASA Technical Reports Server (NTRS)

    Bachalo, William D.

    1985-01-01

    Advanced research in experimental fluid dynamics required a familiarity with sophisticated measurement techniques. In some cases, the development and application of new techniques is required for difficult measurements. Optical methods and in particular, the laser Doppler velocimeter (LDV) are now recognized as the most reliable means for performing measurements in complex turbulent flows. And such, the experimental fluid dynamicist should be familiar with the principles of operation of the method and the details associated with its application. Thus, the goals of this primer are to efficiently transmit the basic concepts of the LDV method to potential users and to provide references that describe the specific areas in greater detail.

  2. Frequency of HLA-A alleles in the Syrian population genotyped by sequence-based typing.

    PubMed

    Madania, A; Ghoury, I; Al-Ashkar, W; Nweder, S; Zarzour, H

    2014-10-01

    HLA-A molecules are highly polymorphic. Their accurate typing at a high-resolution level is crucial for successful organ, bone marrow and cord blood transplantation. Furthermore, several HLA alleles have been involved in susceptibility to autoimmune diseases, allergies, cancers and inflammations. In order to determine common HLA-A alleles in Syria and their frequencies, sequence-based typing (SBT) was used to genotype HLA-A alleles at high resolution (four digit level) among one hundred and thirty randomly selected Syrian individuals. Exons 2, 3 and 4 of the HLA-A gene were amplified by PCR and sequenced. The sbt-engine software was used for allele assignment. Ambiguities were solved using group-specific sequencing primers (GSSPs). We could identify 32 different HLA-A alleles which were divided into 3 groups: high frequency (approximately 10%, A*01:01; A*24:02; A*03:01; A*02:01), moderate frequency (approximately 3%, such as A*02:05, A*31:01 and A*33:01), and low frequency (approximately 1%, such as A*02:11, A*29:01, A*02:02 and A*36:01). Homozygosity rate was higher than expected (11.5% vs. 7.15%). For high frequency alleles, our results show similarity to neighbouring countries. However, 15 alleles (such as A*02:04, A*02:06, A*02:11 and A*02:17) found in our cohort in low frequencies were never reported in some or all neighbouring countries. This is the first report on HLA-A allele frequencies in Syria. In spite of the relatively low number of tested subjects, our results revealed a high degree of diversity, with 32 different alleles, reflecting the high ethnic heterogeneity of the Syrian population. The identification of alleles rarely or never reported in neighbouring countries indicates a higher genetic diversity in Syria.

  3. SBE primer : multiplexing minisequencing-based genotyping

    SciTech Connect

    Kaderali, L.; Deshpande, A.; Uribe-Romeo, F. J.; Schliep, A.; Torney, D. C.

    2002-01-01

    Single-nucleotide polymorphism (SNP) analysis is a powerful tool for mapping and diagnosing disease-related alleles. Most of the known genetic diseases are caused by point mutations, and a growing number of SNPs will be routinely analyzed to diagnose genetic disorders. Mutation analysis by polymerase mediated single-base primer extension (minisequencing) can be massively parallelized using for example DNA microchips or flow cytometry with microspheres as solid support. By adding a unique oligonucleotide tag to the 5-inch end of the minisequencing primer and attaching the complementary anti-tag to the array or bead surface, the assay can be 'demultiplexed'. However, such high-throughput scoring of SNPs requires a high level of primer multiplexing in order to analyze multiple loci in one assay, thus enabling inexpensive and fast polymorphism scoring. Primers can be chosen from either the plus or the minus strand, and primers used in the same experiment must not bind to one another. To genotype a given number of polymorphic sites, the question is which primer to use for each SNP, and which primers to group into the same experiment. Furthermore, a crosshybridization-free tag/anti-tag code is required in order to sort the extended primers to the corresponding microspheres or chip spots. These problems pose challenging algorithmic questions. We present a computer program lo automate the design process for the assay. Oligonucleotide primers for the reaction are automatically selected by the software, a unique DNA tag/anti-tag system is generated, and the pairing of primers and DNA-Tags is automatically done in a way to avoid any crossreactivity. We report first results on a 45-plex genotyping assay, indicating that minisequencing can be adapted to be a powerful tool for high-throughput, massively parallel genotyping.

  4. HLA allele distribution distinguishes sporadic inclusion body myositis from hereditary inclusion body myopathies.

    PubMed

    Koffman, B M; Sivakumar, K; Simonis, T; Stroncek, D; Dalakas, M C

    1998-04-15

    We studied the HLA class II associations in patients with sporadic inclusion body myositis (s-IBM) and hereditary inclusion body myopathies (h-IBM) and attempted to distinguish these myopathies on the basis of HLA allele assignments. Forty-five patients, 30 with s-IBM and 15 with h-IBM, underwent HLA class II allele-specific typing using polymerase chain reaction sequence-specific primers for 71 alleles contained in the DRbeta1, DRbeta3-5, and DQbeta1 loci. In s-IBM, we found a high (up to 77%) frequency of DRbeta1*0301, DRbeta3*0101 (or DRbeta3*0202) and DQbeta1*0201 alleles. No significant association with alleles in the DR and DQ haplotypes was found among the 15 h-IBM patients. The strong association of prominent alleles with s-IBM, but not h-IBM, suggests that s-IBM is a distinct disorder with an immunogenetic background that differs from h-IBM.

  5. Allele-specific qRT-PCR demonstrates superior detection of single nucleotide polymorphisms as genetic markers for West Nile virus compared to Luminex® and quantitative sequencing.

    PubMed

    Worwa, Gabriella; Andrade, Christy C; Thiemann, Tara C; Park, Bborie; Maharaj, Payal D; Anishchenko, Michael; Brault, Aaron C; Reisen, William K

    2014-01-01

    To enable in vivo and in vitro competitive fitness comparisons among West Nile viruses (WNV), three reference viruses were marked genetically by site-directed mutagenesis with five synonymous nucleotide substitutions in the envelope gene region of the genome. Phenotypic neutrality of the mutants was assessed experimentally by competitive replication in cell culture and genetic stability of the substituted nucleotides was confirmed by direct sequencing. Luminex(®) technology, quantitative sequencing and quantitative RT-PCR (qRT-PCR) were compared in regard to specificity, sensitivity and accuracy for quantitation of wildtype and genetically marked viruses in mixed samples based on RNA obtained from samples of known viral titers. Although Luminex(®) technology and quantitative sequencing provided semi-quantitative or qualitative measurements, a sequence-specific primer extension approach using a specific reverse primer set in singleplex qRT-PCR demonstrated the best quantitation and specificity in the detection of RNA from wildtype and mutant viruses.

  6. [DNA amplification using PCR with abutting primers].

    PubMed

    Garafutdinov, R R; Galimova, A A; Sakhabutdinova, A R; Vakhitov, V A; Chemeris, A V

    2015-01-01

    DNA analysis of ñîmplex biological objects (wastewater, soil, archaeological and forensic samples, etc.) is currently of great interest. DNA of these objects is characterized by low suitability for research due to the violation of its integrity and chemical structure; thus, the detection of specific nucleic acid fragments can be achieved by PCR with contiguous primers. In this paper, we present the results that clarify the specific characteristics of PCR with abutting primers. The 3'-ends of these primers are annealed at adjacent nucleotides of complementary chains of DNA target. It has been shown that the proximity of primers enables the formation of specific reaction products with a higher sensitivity and less reaction time. Using artificially damaged DNA and DNA from the soil we demonstrated that the abutting primers provide assured detection of specific DNA fragments. The results of this work may be taken into account in PCR with degraded (fragmented) DNA.

  7. Concordance between allele-specific PCR and ultra-deep pyrosequencing for the detection of HIV-1 non-nucleoside reverse transcriptase inhibitor resistance mutations

    PubMed Central

    Hunt, Gillian M; Morris, Lynn; Moorthy, Anitha; Coovadia, Ashraf; Abrams, Elaine J; Strehlau, Renate; Kuhn, Louise; Persaud, Deborah

    2014-01-01

    Recent advances in genotyping technologies have allowed for detection of HIV-1 drug resistance mutations present at low levels. The presence and percentage of Y181C and K103N drug-resistant variants in the blood of 105 subtype C HIV-infected infants who failed single-dose nevirapine prophylaxis for HIV transmission were compared using two highly sensitive genotyping methods, allele-specific PCR (AS-PCR) and ultra-deep pyrosequencing. Significant correlations in detection between both methods were found for both Y181C (correlation coefficients of 0.94 [95% CI 0.91-0.96]) and K103N (0.89 [95% CI 0.84 – 0.92]) mutations. The majority of discordant specimens (3/5 Y181C and 8/11 K103N) had wild-type variants when population sequencing was used, but mutant variants were detectable at very low levels (≤5%) with either assay. This difference is most likely due to stochastic variations in the appearance of mutant variants. Overall, both AS-PCR and ultra-deep pyrosequencing methods have proven to be sensitive and accurate, and may confidently be used where feasible. PMID:25034127

  8. Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody

    SciTech Connect

    Ying, Tianlei; Prabakaran, Ponraj; Du, Lanying; Shi, Wei; Feng, Yang; Wang, Yanping; Wang, Lingshu; Li, Wei; Jiang, Shibo; Dimitrov, Dimiter S.; Zhou, Tongqing

    2015-09-15

    The MERS-CoV is an emerging virus, which already infected more than 1,300 humans with high (~36%) mortality. Here, we show that m336, an exceptionally potent human anti-MERS-CoV antibody, is almost germline with only one somatic mutation in the heavy chain. The structure of Fab m336 in complex with the MERS-CoV receptor-binding domain reveals that its IGHV1-69-derived heavy chain provides more than 85% binding surface and that its epitope almost completely overlaps with the receptor-binding site. Analysis of antibodies from 69 healthy humans suggests an important role of the V(D)J recombination-generated junctional and allele-specific residues for achieving high affinity of binding at such low levels of somatic hypermutation. Our results also have important implications for development of vaccine immunogens based on the newly identified m336 epitope as well as for elucidation of mechanisms of neutralization by m336-like antibodies and their elicitation in vivo.

  9. Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody

    DOE PAGES

    Ying, Tianlei; Prabakaran, Ponraj; Du, Lanying; Shi, Wei; Feng, Yang; Wang, Yanping; Wang, Lingshu; Li, Wei; Jiang, Shibo; Dimitrov, Dimiter S.; et al

    2015-09-15

    The MERS-CoV is an emerging virus, which already infected more than 1,300 humans with high (~36%) mortality. Here, we show that m336, an exceptionally potent human anti-MERS-CoV antibody, is almost germline with only one somatic mutation in the heavy chain. The structure of Fab m336 in complex with the MERS-CoV receptor-binding domain reveals that its IGHV1-69-derived heavy chain provides more than 85% binding surface and that its epitope almost completely overlaps with the receptor-binding site. Analysis of antibodies from 69 healthy humans suggests an important role of the V(D)J recombination-generated junctional and allele-specific residues for achieving high affinity of bindingmore » at such low levels of somatic hypermutation. Our results also have important implications for development of vaccine immunogens based on the newly identified m336 epitope as well as for elucidation of mechanisms of neutralization by m336-like antibodies and their elicitation in vivo.« less

  10. Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody

    PubMed Central

    Ying, Tianlei; Prabakaran, Ponraj; Du, Lanying; Shi, Wei; Feng, Yang; Wang, Yanping; Wang, Lingshu; Li, Wei; Jiang, Shibo; Dimitrov, Dimiter S.; Zhou, Tongqing

    2015-01-01

    The MERS-CoV is an emerging virus, which already infected more than 1,300 humans with high (∼36%) mortality. Here, we show that m336, an exceptionally potent human anti-MERS-CoV antibody, is almost germline with only one somatic mutation in the heavy chain. The structure of Fab m336 in complex with the MERS-CoV receptor-binding domain reveals that its IGHV1-69-derived heavy chain provides more than 85% binding surface and that its epitope almost completely overlaps with the receptor-binding site. Analysis of antibodies from 69 healthy humans suggests an important role of the V(D)J recombination-generated junctional and allele-specific residues for achieving high affinity of binding at such low levels of somatic hypermutation. Our results also have important implications for development of vaccine immunogens based on the newly identified m336 epitope as well as for elucidation of mechanisms of neutralization by m336-like antibodies and their elicitation in vivo. PMID:26370782

  11. Rapid KRAS, EGFR, BRAF and PIK3CA Mutation Analysis of Fine Needle Aspirates from Non-Small-Cell Lung Cancer Using Allele-Specific qPCR

    PubMed Central

    Schrumpf, Melanie; Talebian Yazdi, Mehrdad; Ruano, Dina; Forte, Giusi I.; Nederlof, Petra M.; Veselic, Maud; Rabe, Klaus F.; Annema, Jouke T.; Smit, Vincent; Morreau, Hans; van Wezel, Tom

    2011-01-01

    Endobronchial Ultrasound Guided Transbronchial Needle Aspiration (EBUS-TBNA) and Trans-esophageal Ultrasound Scanning with Fine Needle Aspiration (EUS-FNA) are important, novel techniques for the diagnosis and staging of non-small cell lung cancer (NSCLC) that have been incorporated into lung cancer staging guidelines. To guide and optimize treatment decisions, especially for NSCLC patients in stage III and IV, EGFR and KRAS mutation status is often required. The concordance rate of the mutation analysis between these cytological aspirates and histological samples obtained by surgical staging is unknown. Therefore, we studied the extent to which allele-specific quantitative real-time PCR with hydrolysis probes could be reliably performed on EBUS and EUS fine needle aspirates by comparing the results with histological material from the same patient. We analyzed a series of 43 NSCLC patients for whom cytological and histological material was available. We demonstrated that these standard molecular techniques can be accurately applied on fine needle cytological aspirates from NSCLC patients. Importantly, we show that all mutations detected in the histological material of primary tumor were also identified in the cytological samples. We conclude that molecular profiling can be reliably performed on fine needle cytology aspirates from NSCLC patients. PMID:21408138

  12. Allele-Specific Virulence Attenuation of the Pseudomonas syringae HopZ1a Type III Effector via the Arabidopsis ZAR1 Resistance Protein

    PubMed Central

    Lewis, Jennifer D.; Wu, Ronald

    2010-01-01

    Plant resistance (R) proteins provide a robust surveillance system to defend against potential pathogens. Despite their importance in plant innate immunity, relatively few of the ∼170 R proteins in Arabidopsis have well-characterized resistance specificity. In order to identify the R protein responsible for recognition of the Pseudomonas syringae type III secreted effector (T3SE) HopZ1a, we assembled an Arabidopsis R gene T–DNA Insertion Collection (ARTIC) from publicly available Arabidopsis thaliana insertion lines and screened it for plants lacking HopZ1a-induced immunity. This reverse genetic screen revealed that the Arabidopsis R protein HOPZ-ACTIVATED RESISTANCE 1 (ZAR1; At3g50950) is required for recognition of HopZ1a in Arabidopsis. ZAR1 belongs to the coiled-coil (CC) class of nucleotide binding site and leucine-rich repeat (NBS–LRR) containing R proteins; however, the ZAR1 CC domain phylogenetically clusters in a clade distinct from other related Arabidopsis R proteins. ZAR1–mediated immunity is independent of several genes required by other R protein signaling pathways, including NDR1 and RAR1, suggesting that ZAR1 possesses distinct signaling requirements. The closely-related T3SE protein, HopZ1b, is still recognized by zar1 Arabidopsis plants indicating that Arabidopsis has evolved at least two independent R proteins to recognize the HopZ T3SE family. Also, in Arabidopsis zar1 plants HopZ1a promotes P. syringae growth indicative of an ancestral virulence function for this T3SE prior to the evolution of recognition by the host resistance protein ZAR1. Our results demonstrate that the Arabidopsis resistance protein ZAR1 confers allele-specific recognition and virulence attenuation of the Pseudomonas syringae T3SE protein HopZ1a. PMID:20368970

  13. Trans-ethnic fine-mapping of lipid loci identifies population-specific signals and allelic heterogeneity that increases the trait variance explained.

    PubMed

    Wu, Ying; Waite, Lindsay L; Jackson, Anne U; Sheu, Wayne H-H; Buyske, Steven; Absher, Devin; Arnett, Donna K; Boerwinkle, Eric; Bonnycastle, Lori L; Carty, Cara L; Cheng, Iona; Cochran, Barbara; Croteau-Chonka, Damien C; Dumitrescu, Logan; Eaton, Charles B; Franceschini, Nora; Guo, Xiuqing; Henderson, Brian E; Hindorff, Lucia A; Kim, Eric; Kinnunen, Leena; Komulainen, Pirjo; Lee, Wen-Jane; Le Marchand, Loic; Lin, Yi; Lindström, Jaana; Lingaas-Holmen, Oddgeir; Mitchell, Sabrina L; Narisu, Narisu; Robinson, Jennifer G; Schumacher, Fred; Stančáková, Alena; Sundvall, Jouko; Sung, Yun-Ju; Swift, Amy J; Wang, Wen-Chang; Wilkens, Lynne; Wilsgaard, Tom; Young, Alicia M; Adair, Linda S; Ballantyne, Christie M; Bůžková, Petra; Chakravarti, Aravinda; Collins, Francis S; Duggan, David; Feranil, Alan B; Ho, Low-Tone; Hung, Yi-Jen; Hunt, Steven C; Hveem, Kristian; Juang, Jyh-Ming J; Kesäniemi, Antero Y; Kuusisto, Johanna; Laakso, Markku; Lakka, Timo A; Lee, I-Te; Leppert, Mark F; Matise, Tara C; Moilanen, Leena; Njølstad, Inger; Peters, Ulrike; Quertermous, Thomas; Rauramaa, Rainer; Rotter, Jerome I; Saramies, Jouko; Tuomilehto, Jaakko; Uusitupa, Matti; Wang, Tzung-Dau; Boehnke, Michael; Haiman, Christopher A; Chen, Yii-Der I; Kooperberg, Charles; Assimes, Themistocles L; Crawford, Dana C; Hsiung, Chao A; North, Kari E; Mohlke, Karen L

    2013-03-01

    Genome-wide association studies (GWAS) have identified ~100 loci associated with blood lipid levels, but much of the trait heritability remains unexplained, and at most loci the identities of the trait-influencing variants remain unknown. We conducted a trans-ethnic fine-mapping study at 18, 22, and 18 GWAS loci on the Metabochip for their association with triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), respectively, in individuals of African American (n = 6,832), East Asian (n = 9,449), and European (n = 10,829) ancestry. We aimed to identify the variants with strongest association at each locus, identify additional and population-specific signals, refine association signals, and assess the relative significance of previously described functional variants. Among the 58 loci, 33 exhibited evidence of association at P<1 × 10(-4) in at least one ancestry group. Sequential conditional analyses revealed that ten, nine, and four loci in African Americans, Europeans, and East Asians, respectively, exhibited two or more signals. At these loci, accounting for all signals led to a 1.3- to 1.8-fold increase in the explained phenotypic variance compared to the strongest signals. Distinct signals across ancestry groups were identified at PCSK9 and APOA5. Trans-ethnic analyses narrowed the signals to smaller sets of variants at GCKR, PPP1R3B, ABO, LCAT, and ABCA1. Of 27 variants reported previously to have functional effects, 74% exhibited the strongest association at the respective signal. In conclusion, trans-ethnic high-density genotyping and analysis confirm the presence of allelic heterogeneity, allow the identification of population-specific variants, and limit the number of candidate SNPs for functional studies. PMID:23555291

  14. Fine mapping of QTL and genomic prediction using allele-specific expression SNPs demonstrates that the complex trait of genetic resistance to Marek’s disease is predominantly determined by transcriptional regulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The hypothesis that polymorphisms associated with transcriptional regulation are critical for viral disease resistance was tested by selecting birds using SNPs exhibiting allele-specific expression (ASE) in response to viral challenge. Analysis indicates ASE markers account for 83% of the disease re...

  15. Allele-specific marker development and selection efficiencies for both flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase genes in soybean subgenus soja.

    PubMed

    Guo, Yong; Qiu, Li-Juan

    2013-06-01

    Color is one of the phenotypic markers mostly used to study soybean (Glycine max L. Merr.) genetic, molecular and biochemical processes. Two P450-dependent mono-oxygenases, flavonoid 3'-hydroxylase (F3'H; EC1.14.3.21) and flavonoid 3',5'-hydroxylase (F3'5'H, EC1.14.13.88), both catalyzing the hydroxylation of the B-ring in flavonoids, play an important role in coloration. Previous studies showed that the T locus was a gene encoding F3'H and the W1 locus co-segregated with a gene encoding F3'5'H in soybean. These two genetic loci have identified to control seed coat, flower and pubescence colors. However, the allelic distributions of both F3'H and F3'5'H genes in soybean were unknown. In this study, three novel alleles were identified (two of four alleles for GmF3'H and one of three alleles for GmF3'5'H). A set of gene-tagged markers was developed and verified based on the sequence diversity of all seven alleles. Furthermore, the markers were used to analyze soybean accessions including 170 cultivated soybeans (G. max) from a mini core collection and 102 wild soybeans (G. soja). For both F3'H and F3'5'H, the marker selection efficiencies for pubescence color and flower color were determined. The results showed that one GmF3'H allele explained 92.2 % of the variation in tawny and two gmf3'h alleles explained 63.8 % of the variation in gray pubescence colors. In addition, two GmF3'5'H alleles and one gmF3'5'h allele explained 94.0 % of the variation in purple and 75.3 % in white flowers, respectively. By the combination of the two loci, seed coat color was determined. In total, 90.9 % of accessions possessing both the gmf3'h-b and gmf3'5'h alleles had yellow seed coats. Therefore, seed coat colors are controlled by more than two loci.

  16. Variants in the 3′ untranslated region of the KCNQ1-encoded Kv7.1 potassium channel modify disease severity in patients with type 1 long QT syndrome in an allele-specific manner

    PubMed Central

    Amin, Ahmad S.; Giudicessi, John R.; Tijsen, Anke J.; Spanjaart, Anne M.; Reckman, Yolan J.; Klemens, Christine A.; Tanck, Michael W.; Kapplinger, Jamie D.; Hofman, Nynke; Sinner, Moritz F.; Müller, Martina; Wijnen, Wino J.; Tan, Hanno L.; Bezzina, Connie R.; Creemers, Esther E.; Wilde, Arthur A. M.; Ackerman, Michael J.; Pinto, Yigal M.

    2012-01-01

    Aims Heterozygous mutations in KCNQ1 cause type 1 long QT syndrome (LQT1), a disease characterized by prolonged heart rate-corrected QT interval (QTc) and life-threatening arrhythmias. It is unknown why disease penetrance and expressivity is so variable between individuals hosting identical mutations. We aimed to study whether this can be explained by single nucleotide polymorphisms (SNPs) in KCNQ1's 3′ untranslated region (3′UTR). Methods and results This study was performed in 84 LQT1 patients from the Academic Medical Center in Amsterdam and validated in 84 LQT1 patients from the Mayo Clinic in Rochester. All patients were genotyped for SNPs in KCNQ1's 3′UTR, and six SNPs were found. Single nucleotide polymorphisms rs2519184, rs8234, and rs10798 were associated in an allele-specific manner with QTc and symptom occurrence. Patients with the derived SNP variants on their mutated KCNQ1 allele had shorter QTc and fewer symptoms, while the opposite was also true: patients with the derived SNP variants on their normal KCNQ1 allele had significantly longer QTc and more symptoms. Luciferase reporter assays showed that the expression of KCNQ1's 3′UTR with the derived SNP variants was lower than the expression of the 3′UTR with the ancestral SNP variants. Conclusion Our data indicate that 3′UTR SNPs potently modify disease severity in LQT1. The allele-specific effects of the SNPs on disease severity and gene expression strongly suggest that they are functional variants that directly alter the expression of the allele on which they reside, and thereby influence the balance between proteins stemming from either the normal or the mutant KCNQ1 allele. PMID:22199116

  17. Proper Use of Allele-Specific Expression Improves Statistical Power for cis-eQTL Mapping with RNA-Seq Data

    PubMed Central

    HU, Yi-Juan; SUN, Wei; TZENG, Jung-Ying; PEROU, Charles M.

    2015-01-01

    Studies of expression quantitative trait loci (eQTLs) offer insight into the molecular mechanisms of loci that were found to be associated with complex diseases and the mechanisms can be classified into cis- and trans-acting regulation. At present, high-throughput RNA sequencing (RNA-seq) is rapidly replacing expression microarrays to assess gene expression abundance. Unlike microarrays that only measure the total expression of each gene, RNA-seq also provides information on allele-specific expression (ASE), which can be used to distinguish cis-eQTLs from trans-eQTLs and, more importantly, enhance cis-eQTL mapping. However, assessing the cis-effect of a candidate eQTL on a gene requires knowledge of the haplotypes connecting the candidate eQTL and the gene, which cannot be inferred with certainty. The existing two-stage approach that first phases the candidate eQTL against the gene and then treats the inferred phase as observed in the association analysis tends to attenuate the estimated cis-effect and reduce the power for detecting a cis-eQTL. In this article, we provide a maximum-likelihood framework for cis-eQTL mapping with RNA-seq data. Our approach integrates the inference of haplotypes and the association analysis into a single stage, and is thus unbiased and statistically powerful. We also develop a pipeline for performing a comprehensive scan of all local eQTLs for all genes in the genome by controlling for false discovery rate, and implement the methods in a computationally efficient software program. The advantages of the proposed methods over the existing ones are demonstrated through realistic simulation studies and an application to empirical breast cancer data from The Cancer Genome Atlas project. PMID:26568645

  18. The feather-degrading bacterial community in two soils as revealed by a specific primer targeting serine-type keratinolytic proteases.

    PubMed

    Gu, Zhenhong; Zhu, Honghui; Xie, Xiaolin; Wang, Yonghong; Liu, Xiaodi; Yao, Qing

    2016-10-01

    Feather waste represents a huge resource of protein, but is underutilized due to its recalcitrant nature. Feather-degrading bacteria can biologically degrade feathers and have great potential for industries. In this study, we first designed a primer set (BC) suitable for exploring the diversity of the keratinolytic bacterial community with denatured gradient gel electrophoresis (DGGE). With the BC primer set, the difference in the keratinolytic bacterial community between a feather-dumping (FD) soil and a non feather-dumping (NFD) soil and the influence of feather addition (enrichment culture) on the keratinolytic bacterial community were investigated. DGGE and sequencing showed that keratinolytic bacteria in these soils belong to 2 phyla (Actinobacteria and Proteobacteria) and 9 genera (Micromonospora, Verrucosispora, Actinopolymorpha, Knoellia, Hyalangium, Stigmatella, Archangium, Cystobacter, and Luteimonas). Feather addition decreased the species richness of the keratinolytic bacteria in FD soil, but greatly increased the diversity, species richness and abundance in NFD soil. Moreover, feather addition to NFD soil induced some keratinolytic bacteria that were absent in all of the other soils. Collectively, these data indicate that keratinolytic bacteria are diverse in both FD and NFD soil, and some novel keratinolytic bacteria taxa might be revealed by using the BC primer set.

  19. The feather-degrading bacterial community in two soils as revealed by a specific primer targeting serine-type keratinolytic proteases.

    PubMed

    Gu, Zhenhong; Zhu, Honghui; Xie, Xiaolin; Wang, Yonghong; Liu, Xiaodi; Yao, Qing

    2016-10-01

    Feather waste represents a huge resource of protein, but is underutilized due to its recalcitrant nature. Feather-degrading bacteria can biologically degrade feathers and have great potential for industries. In this study, we first designed a primer set (BC) suitable for exploring the diversity of the keratinolytic bacterial community with denatured gradient gel electrophoresis (DGGE). With the BC primer set, the difference in the keratinolytic bacterial community between a feather-dumping (FD) soil and a non feather-dumping (NFD) soil and the influence of feather addition (enrichment culture) on the keratinolytic bacterial community were investigated. DGGE and sequencing showed that keratinolytic bacteria in these soils belong to 2 phyla (Actinobacteria and Proteobacteria) and 9 genera (Micromonospora, Verrucosispora, Actinopolymorpha, Knoellia, Hyalangium, Stigmatella, Archangium, Cystobacter, and Luteimonas). Feather addition decreased the species richness of the keratinolytic bacteria in FD soil, but greatly increased the diversity, species richness and abundance in NFD soil. Moreover, feather addition to NFD soil induced some keratinolytic bacteria that were absent in all of the other soils. Collectively, these data indicate that keratinolytic bacteria are diverse in both FD and NFD soil, and some novel keratinolytic bacteria taxa might be revealed by using the BC primer set. PMID:27562599

  20. Marker-assisted selection for recognizing wheat mutant genotypes carrying HMW glutenin alleles related to baking quality.

    PubMed

    Zamani, Mohammad Javad; Bihamta, Mohammad Reza; Naserian Khiabani, Behnam; Tahernezhad, Zahra; Hallajian, Mohammad Taher; Shamsi, Marzieh Varasteh

    2014-01-01

    Allelic diversity of HMW glutenin loci in several studies revealed that allelic combinations affect dough quality. Dx5 + Dy10 subunits are related to good baking quality and Dx2 + Dy12 are related to undesirable baking quality. One of the most regular methods to evaluate the baking quality is SDS-PAGE which is used to improve baking quality labs. Marker-assisted selection is the method which can recognize the alleles related to baking quality and this method is based on polymerase chain reaction. 10 pairs of specific primers related to Dx2, Dx2.1, Dx5, Dy10, and Dy12 subunits were used for recognizing baking quality of some wheat varieties and some mutant genotypes. Only 5 pairs of them could show the specific bands. All subunits were recognized by the primers except Dx2.1. Some of the primers were extracted from previous studies and the others were designed based on D genome subunits of wheat. SDS-PAGE method accomplished having confidence in these marker's results. To realize the effect of mutation, seed storage proteins were measured. It showed that mutation had effect on the amount of seed storage protein on the mutant seeds (which showed polymorphism).

  1. Marker-Assisted Selection for Recognizing Wheat Mutant Genotypes Carrying HMW Glutenin Alleles Related to Baking Quality

    PubMed Central

    Zamani, Mohammad Javad; Bihamta, Mohammad Reza; Naserian Khiabani, Behnam; Tahernezhad, Zahra; Hallajian, Mohammad Taher; Shamsi, Marzieh Varasteh

    2014-01-01

    Allelic diversity of HMW glutenin loci in several studies revealed that allelic combinations affect dough quality. Dx5 + Dy10 subunits are related to good baking quality and Dx2 + Dy12 are related to undesirable baking quality. One of the most regular methods to evaluate the baking quality is SDS-PAGE which is used to improve baking quality labs. Marker-assisted selection is the method which can recognize the alleles related to baking quality and this method is based on polymerase chain reaction. 10 pairs of specific primers related to Dx2, Dx2.1, Dx5, Dy10, and Dy12 subunits were used for recognizing baking quality of some wheat varieties and some mutant genotypes. Only 5 pairs of them could show the specific bands. All subunits were recognized by the primers except Dx2.1. Some of the primers were extracted from previous studies and the others were designed based on D genome subunits of wheat. SDS-PAGE method accomplished having confidence in these marker's results. To realize the effect of mutation, seed storage proteins were measured. It showed that mutation had effect on the amount of seed storage protein on the mutant seeds (which showed polymorphism). PMID:24883389

  2. Application of Self-Quenched JH Consensus Primers for Real-Time Quantitative PCR of IGH Gene to Minimal Residual Disease Evaluation in Multiple Myeloma

    PubMed Central

    Martinez-Lopez, Joaquin; Martinez-Sanchez, Pilar; Garcia-Sanz, Ramon; Sarasquete, Maria Eugenia; Ayala, Rosa; Gonzalez, Marcos; Bautista, Jose Manuel; Gonzalez, David; Miguel, Jesus San; Garcia-Effron, Guillermo; Lahuerta, Juan Jose

    2006-01-01

    Monitoring multiple myeloma patients for relapse requires sensitive methods to measure minimal residual disease and to establish a more precise prognosis. The present study aimed to standardize a real-time quantitative polymerase chain reaction (PCR) test for the IgH gene with a JH consensus self-quenched fluorescence reverse primer and a VDJH or DJH allele-specific sense primer (self-quenched PCR). This method was compared with allele-specific real-time quantitative PCR test for the IgH gene using a TaqMan probe and a JH consensus primer (TaqMan PCR). We studied nine multiple myeloma patients from the Spanish group treated with the MM2000 therapeutic protocol. Self-quenched PCR demonstrated sensitivity of ≥10−4 or 16 genomes in most cases, efficiency was 1.71 to 2.14, and intra-assay and interassay reproducibilities were 1.18 and 0.75%, respectively. Sensitivity, efficiency, and residual disease detection were similar with both PCR methods. TaqMan PCR failed in one case because of a mutation in the JH primer binding site, and self-quenched PCR worked well in this case. In conclusion, self-quenched PCR is a sensitive and reproducible method for quantifying residual disease in multiple myeloma patients; it yields similar results to TaqMan PCR and may be more effective than the latter when somatic mutations are present in the JH intronic primer binding site. PMID:16825510

  3. Biochemical comparison of major histocompatibility complex molecules from different subspecies of Mus musculus: evidence for trans-specific evolution of alleles.

    PubMed

    Arden, B; Klein, J

    1982-04-01

    H-2 haplotypes were extracted from wild mice of three subspecies, Mus musculus domesticus, M. m. molossinus, and M. m. castaneus, that are known to have been separated from one another for some 1 to 2 million years. Serologically indistinguishable molecules controlled by some of the polymorphic H-2 loci were compared by tryptic peptide mapping, and the maps were found to be identical. In addition, a number of instances of biochemically indistinguishable H-2 molecules were found among wild mice and inbred strains of the M. m. domesticus subspecies. These findings suggest that some of the H-2 alleles have not altered for greater than 1 million years. To reconcile this apparent stability of H-2 genes with their extraordinary polymorphism (some 100 alleles at each of the polymorphic H-2 loci), it is proposed that the H-2 alleles evolve as if they were separate loci.

  4. Brief communication: Evolution of a specific O allele (O1vG542A) supports unique ancestry of Native Americans.

    PubMed

    Villanea, Fernando A; Bolnick, Deborah A; Monroe, Cara; Worl, Rosita; Cambra, Rosemary; Leventhal, Alan; Kemp, Brian M

    2013-08-01

    In this study, we explore the geographic and temporal distribution of a unique variant of the O blood group allele called O1v(G542A) , which has been shown to be shared among Native Americans but is rare in other populations. O1v(G542A) was previously reported in Native American populations in Mesoamerica and South America, and has been proposed as an ancestry informative marker. We investigated whether this allele is also found in the Tlingit and Haida, two contemporary indigenous populations from Alaska, and a pre-Columbian population from California. If O1v(G542A) is present in Na-Dene speakers (i.e., Tlingits), it would indicate that Na-Dene speaking groups share close ancestry with other Native American groups and support a Beringian origin of the allele, consistent with the Beringian Incubation Model. If O1v(G542A) is found in pre-Columbian populations, it would further support a Beringian origin of the allele, rather than a more recent introduction of the allele into the Americas via gene flow from one or more populations which have admixed with Native Americans over the past five centuries. We identified this allele in one Na-Dene population at a frequency of 0.11, and one ancient California population at a frequency of 0.20. Our results support a Beringian origin of O1v(G542A) , which is distributed today among all Native American groups that have been genotyped in appreciable numbers at this locus. This result is consistent with the hypothesis that Na-Dene and other Native American populations primarily derive their ancestry from a single source population. PMID:23868176

  5. Polyacid macromolecule primers

    DOEpatents

    Sugama, Toshifumi.

    1989-12-26

    Hydrophilic polyacids are described, such as macromolecules of polyitaconic acid and polyacrylic acid, where such macromolecules have molecular weights >50,000 as primers between a polymeric top coating, such as polyurethane, and an oxidized aluminum or aluminum alloy. A near monolayer of primer is used in polymeric adhesive/oxidized aluminum adhered joint systems in 0.05% primer concentration to give superior results in standard peel tests. 2 figs.

  6. Polyacid macromolecule primers

    DOEpatents

    Sugama, Toshifumi

    1989-01-01

    Hydrophylic polyacids, such as macromolecules of polyitaconic acid and polyacrylic acid, where such macromolecules have molecular weights >50,000 as primers between a polymeric top coating, such as polyurethane, and an oxidized aluminum or aluminum alloy. A near monolayer of primer is used in polymeric adhesive/oxidized aluminum adhered joint systems in 0.05% primer concentration to give superior results in standard peel tests.

  7. An improved assay for the determination of Huntington`s disease allele size

    SciTech Connect

    Reeves, C.; Klinger, K.; Miller, G.

    1994-09-01

    The hallmark of Huntington`s disease (HD) is the expansion of a polymorphic (CAG)n repeat. Several methods have been published describing PCR amplification of this region. Most of these assays require a complex PCR reaction mixture to amplify this GC-rich region. A consistent problem with trinucleotide repeat PCR amplification is the presence of a number of {open_quotes}stutter bands{close_quotes} which may be caused by primer or amplicon slippage during amplification or insufficient polymerase processivity. Most assays for HD arbitrarily select a particular band for diagnostic purposes. Without a clear choice for band selection such an arbitrary selection may result in inconsistent intra- or inter-laboratory findings. We present an improved protocol for the amplification of the HD trinucleotide repeat region. This method simplifies the PCR reaction buffer and results in a set of easily identifiable bands from which to determine allele size. HD alleles were identified by selecting bands of clearly greater signal intensity. Stutter banding was much reduced thus permitting easy identification of the most relevant PCR product. A second set of primers internal to the CCG polymorphism was used in selected samples to confirm allele size. The mechanism of action of N,N,N trimethylglycine in the PCR reaction is not clear. It may be possible that the minimal isostabilizing effect of N,N,N trimethylglycine at 2.5 M is significant enough to affect primer specificity. The use of N,N,N trimethylglycine in the PCR reaction facilitated identification of HD alleles and may be appropriate for use in other assays of this type.

  8. DNA typing for HLA class I alleles: I. Subsets of HLA-A2 and of -A28.

    PubMed

    Fernandez-Viña, M A; Falco, M; Sun, Y; Stastny, P

    1992-03-01

    A group of HLA-A locus alleles known to be comprised of approximately 14 closely related variants are collectively called HLA-A2 and -A28. Variations among these alleles are given by differences in only a few codons, and in the case of A*6901, elements of A*6801 (exons 1 and 2) and of A*0201 are combined. The purpose of these experiments was to determine the possibility of designing oligonucleotide probes to identify and develop a typing method for all or most of the A2 and A28 variants. Because the regions of interest are also shared by alleles of other groups, allele-specific or group-specific primers were needed to amplify only the alleles under study. HLA-A2-specific amplification of exon 2 and selective amplification of portions of exon 3 of the A2-A28 group were accomplished with sequence-specific primers and after appropriate adjustments of the PCR conditions. Hybridization patterns using products of four PCR reactions with our set of probes distinguished 11 alleles. Two other alleles might be recognized with the reagents used, but were not found in the panels in this study. A*0201 and A*0209, which are different in exon 4, were not resolved because exon 4 was not tested. A new variant of Aw68, defined by a hybridization pattern obtained with our probes, was different from A*6801 only in that it was negative with probe A6. It was called A*68.3. Population studies were performed in North American whites, blacks, and Indians and in a sample of subjects from North China. HLA-A*0201 was the most frequent allele. A*0202 was found only in blacks, and A*0203 and A*0207 were found only in Chinese. Among the A28-positive subjects, Caucasoids were predominantly A*6801 or A*68.3; A*6802 was the most frequent subtype in American blacks; among American Indians the predominant type was A*68.3. The two A28-positive Chinese subjects studied had A*6901. The results obtained demonstrate that DNA typing is an efficient method for determining these alleles. The methodology

  9. Serologic and nucleotide sequencing analyses of a novel DR52-associated DRB1 allele with the DR 'NJ25' specificity, designated DRB1*1307.

    PubMed

    Kaneshige, T; Hashimoto, M; Matsumoto, Y; Kinoshita, T; Hirasawa, T; Uchida, K; Inoko, H

    1994-10-01

    A novel DR52-associated DRB1* allele, designated DRB1*1307, was encountered in the course of our HLA-DRB1 genotyping study in a Japanese population by PCR-RFLP. Comparison of the nucleotide sequence of its second exon with those of the other known DRB1 alleles revealed that DRB1*1307 was most similar to DRB1*1101, differing by two amino acid substitutions. From a family study, DRB1*1307 was found to segregate with a haplotype of DRB3*0202-DQA1*0501-DQB1*0301, which was also observed with DRB1*1101 in a Japanese population. DRB1*1307 was recognized in three of 652 healthy Japanese controls (gene frequency: 0.24%) with the same DR-DQ haplotype, indicating that DRB1*1307 arose from DRB1*1101 by a gene conversionlike event(s) and/or point mutations. Further, it was also observed that this allele had a strong linkage disequilibrium with HLA-B70 (p < 0.001). This new DRB1*1307 allele was serologically defined as DR 'NJ25,' and it gave an almost identical serologic pattern to DRB1*1406. On sequence comparison, however, no unique amino acid residues conserved in DRB1*1406 and DRB1*1307 but absent in all the other DRB1 alleles could be found, indicating that two amino acid changes at positions 47 and 58 abolished the reactivity against the DR11 antisera.

  10. T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire

    PubMed Central

    1991-01-01

    We report here the first extensive study of a T cell repertoire for a class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) response. We have found that the T cell receptors (TCRs) carried by 28 H-2Kd-restricted CTL clones specific for a single Plasmodium berghei circumsporozoite nonapeptide are highly diverse in terms of V alpha, J alpha, and J beta segments and aminoacid composition of the junctional regions. However, despite this extensive diversity, a high proportion of the TCRs contain the same V beta segment. These results are in contrast to most previously reported T cell responses towards class II MHC-peptide complexes, where the TCR repertoires appeared to be much more limited. In our study, the finding of a dominant V beta in the midst of otherwise highly diverse TCRs suggests the importance of the V beta segment in shaping the T cell repertoire specific for a given MHC-peptide complex. As an additional finding, we observed that nearly all clones have rearranged both TCR alpha loci. Moreover, as many as one-third of the CTL clones that we analyzed apparently display two productive alpha rearrangements. This argues against a regulated model of sequential recombination at the alpha locus and consequently raises the question of whether allelic exclusion of the TCR alpha chain is achieved at all. PMID:1836010

  11. Education Vouchers. A Primer.

    ERIC Educational Resources Information Center

    Richter, Philip C.; Hollender, Mary Jo

    This document is intended to serve both as a primer and as an annotated bibliography about educational vouchers. As a primer, it introduces the reader to the concept of vouchers and to the variety of issues--including political, economic, legal, and educational issues--associated with vouchers. As an annotated bibliography, it provides a summary…

  12. PHUSER (Primer Help for USER): a novel tool for USER fusion primer design.

    PubMed

    Olsen, Lars Rønn; Hansen, Niels Bjørn; Bonde, Mads Tvillinggaard; Genee, Hans Jasper; Holm, Dorte Koefoed; Carlsen, Simon; Hansen, Bjarne Gram; Patil, Kiran Raosaheb; Mortensen, Uffe Hasbro; Wernersson, Rasmus

    2011-07-01

    Uracil-Specific Exision Reagent (USER) fusion is a recently developed technique that allows for assembly of multiple DNA fragments in a few simple steps. However, designing primers for USER fusion is both tedious and time consuming. Here, we present the Primer Help for USER (PHUSER) software, a novel tool for designing primers specifically for USER fusion and USER cloning applications. We also present proof-of-concept experimental validation of its functionality. PHUSER offers quick and easy design of PCR optimized primers ensuring directionally correct fusion of fragments into a plasmid containing a customizable USER cassette. Designing primers using PHUSER ensures that the primers have similar annealing temperature (T(m)), which is essential for efficient PCR. PHUSER also avoids identical overhangs, thereby ensuring correct order of assembly of DNA fragments. All possible primers are individually analysed in terms of GC content, presence of GC clamp at 3'-end, the risk of primer dimer formation, the risk of intra-primer complementarity (secondary structures) and the presence of polyN stretches. Furthermore, PHUSER offers the option to insert linkers between DNA fragments, as well as highly flexible cassette options. PHUSER is publicly available at http://www.cbs.dtu.dk/services/phuser/. PMID:21622660

  13. Allele-specific germ cell epimutation in the spacer promoter of the 45S ribosomal RNA gene after Cr(III) exposure

    SciTech Connect

    Shiao, Y.-H. . E-mail: shiao@mail.ncifrcf.gov; Crawford, Erik B.; Anderson, Lucy M.; Patel, Pritesh; Ko, Kinarm

    2005-06-15

    Paternal exposure of mice to Cr(III) causes increased tumor risk in offspring; an epigenetic mechanism has been hypothesized. Representational difference analysis of gene methylation in sperm revealed hypomethylation in the 45S ribosomal RNA (rRNA) gene after Cr(III) exposure, compared with controls. The most striking effects were seen in the rRNA spacer promoter, a region in the intergenic region of rRNA gene clusters that can influence transcription. Methylation of the rRNA spacer promoter has not been studied heretofore. Sperm DNAs from Cr(III)-treated and control mice were modified by the bisulfite method followed by PCR amplification of the spacer promoter, including 27 CpG sites. Cloning and dideoxy sequencing identified sequence variants (T or G at base -2214) in the spacer promoter. The T allele had less DNA methylation than the G allele in control mice (17 of 17 clones vs. 42 of 72 clones, P = 0.0004). In spite of diversity of sperm DNA methylation patterns, the DNA clones from Cr(III)-exposed mice had fewer methylated CpG sites, by an average of 19% (P < 0.0001). This difference was limited to the G allele. The pyrosequencing technique was applied to quantify the percentage of methylation directly from amplified PCR products. Strikingly, for nine CpG sites including the spacer promoter core region, hypomethylation was highly significant in the Cr(III)-treated group (paired T test, P < 0.0001). Thus, one allele of the 45S rRNA spacer promoter is hypomethylated in sperm germ cells after Cr(III) exposure. This epimutation may lead to increase of tumor risk in the offspring.

  14. A Mutant S3 RNase of Petunia inflata Lacking RNase Activity Has an Allele-Specific Dominant Negative Effect on Self-Incompatibility Interactions.

    PubMed Central

    McCubbin, A. G.; Chung, Y. Y.; Kao, Th.

    1997-01-01

    Gametophytic self-incompatibility in the Solanaceae is controlled by a multiallelic locus called the S locus. Growth of pollen tubes in the pistil is inhibited when the pollen has one of the two S alleles carried by the pistil. The products of a number of pistil S alleles[mdash]S proteins or S RNases[mdash]have been identified, and their role in controlling the pistil's ability to reject self-pollen has been positively established. In contrast, the existence of pollen S allele products has so far been inferred entirely from genetic evidence. Here, we introduced a modified S3 gene of Petunia inflata encoding an S3 RNase lacking RNase activity into P. inflata plants of the S2S3 genotype to determine whether the production of the mutant protein, designated S3(H93R), would have any effect on the ability of the transgenic plants to reject S2 and S3 pollen. Analysis of the self-incompatibility behavior of 49 primary transgenic plants and the progeny of three plants (H30, H37, and H40) that produced S3(H93R) in addition to producing wild-type levels of endogenous S2 and S3 RNases revealed that S3(H93R) had a dominant negative effect on the function of the S3 RNase in rejecting self-pollen; however, it had no effect on the function of the S2 RNase. One likely explanation of the results is that S3(H93R) competes with the S3 RNase for binding to a common molecule, which is presumably the product of the pollen S3 allele. PMID:12237345

  15. AHR promoter variant modulates its transcription and downstream effectors by allele-specific AHR-SP1 interaction functioning as a genetic marker for vitiligo.

    PubMed

    Wang, Xiaowen; Li, Kai; Liu, Ling; Shi, Qiong; Song, Pu; Jian, Zhe; Guo, Sen; Wang, Gang; Li, Chunying; Gao, Tianwen

    2015-09-15

    Vitiligo is an acquired depigmentation disorder largely caused by defective melanocyte- or autoimmunity-induced melanocyte destruction. The aryl hydrocarbon receptor (AHR) is essential for melanocyte homeostasis and immune process, and abnormal AHR was observed in vitiligo. We previously identified the T allele of AHR -129C > T variant as a protective factor against vitiligo. However, biological characterization underlying such effects is not fully certain, further validation by mechanistic research is warranted and was conducted in the present study. We showed that -129T allele promoted AHR transcriptional activity through facilitating its interaction with SP1 transcription factor (SP1) compared with -129C allele. We subsequently found reduced peripheral AHR and SP1 transcript expressions in vitiligo and a negative correlation of AHR level with disease duration. We also investigated AHR-related cytokines and observed increased serum TNF-α concentration and diminished serum levels of IL-10 and TGF-β1 in vitiligo. Further genetic analysis showed that -129T carriers possessed higher levels of AHR and IL-10 than -129C carriers. Therefore, our study indicates that the modulation of AHR transcription by a promoter variant has a profound influence on vitiligo, not only advancing our understanding on AHR function but also providing novel insight into the pathogenesis of degenerative or autoimmune diseases including vitiligo.

  16. AHR promoter variant modulates its transcription and downstream effectors by allele-specific AHR-SP1 interaction functioning as a genetic marker for vitiligo

    PubMed Central

    Wang, Xiaowen; Li, Kai; Liu, Ling; Shi, Qiong; Song, Pu; Jian, Zhe; Guo, Sen; Wang, Gang; Li, Chunying; Gao, Tianwen

    2015-01-01

    Vitiligo is an acquired depigmentation disorder largely caused by defective melanocyte- or autoimmunity-induced melanocyte destruction. The aryl hydrocarbon receptor (AHR) is essential for melanocyte homeostasis and immune process, and abnormal AHR was observed in vitiligo. We previously identified the T allele of AHR −129C > T variant as a protective factor against vitiligo. However, biological characterization underlying such effects is not fully certain, further validation by mechanistic research is warranted and was conducted in the present study. We showed that −129T allele promoted AHR transcriptional activity through facilitating its interaction with SP1 transcription factor (SP1) compared with −129C allele. We subsequently found reduced peripheral AHR and SP1 transcript expressions in vitiligo and a negative correlation of AHR level with disease duration. We also investigated AHR-related cytokines and observed increased serum TNF-α concentration and diminished serum levels of IL-10 and TGF-β1 in vitiligo. Further genetic analysis showed that -129T carriers possessed higher levels of AHR and IL-10 than −129C carriers. Therefore, our study indicates that the modulation of AHR transcription by a promoter variant has a profound influence on vitiligo, not only advancing our understanding on AHR function but also providing novel insight into the pathogenesis of degenerative or autoimmune diseases including vitiligo. PMID:26370050

  17. Influence of HLA-DRB alleles on haemorrhagic fever with renal syndrome in a Chinese Han population in Hubei Province, China.

    PubMed

    Zhu, N; Luo, F; Chen, Q; Li, N; Xiong, H; Feng, Y; Yang, Z; Hou, W

    2015-01-01

    Specific human leucocyte antigen (HLA) alleles are considered a genetic risk factor for the progression of haemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses. The aim of this study was to establish whether HLA-DRB alleles are associated with the severity of HFRS caused by different types of hantaviruses in a Chinese Han population from Hubei Province of central China. Twenty-two specific HLA-DRB alleles were analysed by sequence-specific primer-polymerase chain reaction (SSP-PCR) in 100 HFRS patients and 213 healthy volunteers. Associations of HLA-DRB alleles with the severity and clinical parameters of HFRS caused by Hantaan virus (HTNV) or Seoul virus (SEOV) infection were evaluated. Six alleles (HLA-DRB1*0401-0411, HLA-DRB1*1001, HLA-DRB1*1101-1105, HLA-DRB1*1201-1202, HLA-DRB1*1305 and DRB5*0101-0201) demonstrated strong associations with HFRS caused by HTNV and SEOV infections. Further comparison of these HLA-DRB1 allele frequencies between HFRS patients with differing severities and healthy controls demonstrated that the HLA-DRB1*0401-0411, HLA-DRB1*1001 and DRB1*1305 alleles were more frequent in the moderate course of HTNV-infected HFRS. Meanwhile, the DRB1*1101-1105 allele was more frequently observed in the severe course of HTNV-infected HFRS. We also found that the HLA-DRB1*1201-1202 allele frequency was higher in the moderate course of SEOV-infected HFRS, whereas the DRB5*0101-0201 allele may play a protective role in moderate HFRS caused by both HTNV and SEOV infections. These results provide evidence of the influence of HLA-DRB on the severity of HFRS and confirm the effect of HLA-DRB on HFRS during different types of hantavirus infection in a Chinese Han population in Hubei Province, China.

  18. Increased TNFA*2, but not TNFB*1, allele frequency in Spanish atopic patients.

    PubMed

    Castro, J; Tellería, J J; Linares, P; Blanco-Quirós, A

    2000-01-01

    Tumor necrosis factor (TNF) is a potent proinflammatory cytokine involved in asthma and atopy. Increased TNF-alpha levels have been found in airway biopsies and bronchoalveolar lavage fluids from asthmatic patients. Constitutional variations in the TNF-alpha secretion levels in vitro are associated with molecular polymorphisms located within and around the TNF loci. Our study objective was to investigate the association between atopy and two described di-allelic polymorphisms in the TNF locus: a G to A transition at position -308 in the 5'-promoter region of the TNFA gene (TNFA*1 and TNFA*2 alleles) and an Ncol restriction fragment length polymorphism (RFLP) in the first intron of the TNFB gene (TNFB*1 and TNFB*2 alleles). The genetic study was performed in 65 unrelated atopic patients and 60 healthy controls. The regions of interest were amplified from genomic DNA using specific primers and polymerase chain reaction. SSP-PCR analysis for TNFA -308 polymorphism genotyping and endonuclease digestion analysis for the TNFB Ncol RFLP were used. The frequency of the TNFA*2 allele was significantly higher in atopic subjects compared to the control group (38.5% vs. 10.5%; chi2 = 32.06; p <0.0001). The TNFA*2 allele is associated with a higher risk for the development of atopy (risk ratio = 9.44; EF = 0.65; chi2 = 30.06 p <0.0005). On the other hand, no significant association between the TNFB alleles and atopy was found. In conclusion, the TNFA*2 allele could be also a genetic risk marker for the predisposition to atopy in our population, as has been reported in other studies. Either the TNFA gene itself or a linked gene on chromosome region 6p21, which has yet to be identified, is a candidate gene for susceptibility to atopy. PMID:10923589

  19. Quick spacecraft charging primer

    SciTech Connect

    Larsen, Brian Arthur

    2014-03-12

    This is a presentation in PDF format which is a quick spacecraft charging primer, meant to be used for program training. It goes into detail about charging physics, RBSP examples, and how to identify charging.

  20. Isolated 3-methylcrotonyl-CoA carboxylase deficiency: evidence for an allele-specific dominant negative effect and responsiveness to biotin therapy.

    PubMed

    Baumgartner, Matthias R; Dantas, M Fernanda; Suormala, Terttu; Almashanu, Shlomo; Giunta, Cecilia; Friebel, Dolores; Gebhardt, Boris; Fowler, Brian; Hoffmann, Georg F; Baumgartner, E Regula; Valle, David

    2004-11-01

    Deficiency of 3-methylcrotonyl-CoA carboxylase (MCC) results in elevated excretion of 3-methylcrotonylglycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA). MCC is a heteromeric mitochondrial enzyme comprising biotin-containing alpha subunits and smaller beta subunits, encoded by MCCA and MCCB, respectively. Mutations in these genes cause isolated MCC deficiency, an autosomal recessive disorder with a variable phenotype that ranges from severe neonatal to asymptomatic adult forms. No reported patients have responded to biotin therapy. Here, we describe two patients with a biochemical and, in one case, clinical phenotype of MCC deficiency, both of whom were responsive to biotin. The first patient presented at 3 months with seizures and progressive psychomotor retardation. Metabolic investigation at 2 years revealed elevated excretion of 3-MCG and 3-HIVA, suggesting MCC deficiency. High-dose biotin therapy was associated with a dramatic reduction in seizures, normalization of the electroencephalogram, and correction of the organic aciduria, within 4 weeks. MCC activity in fibroblasts was 25% of normal levels. The second patient, a newborn detected by tandem-mass-spectrometry newborn screening, displayed the same biochemical phenotype and remained asymptomatic with biotin up to the age of 18 months. In both patients, sequence analysis of the complete open reading frames of MCCA and MCCB revealed heterozygosity for MCCA-R385S and for the known polymorphic variant MCCA-P464H but revealed no other coding alterations. MCCA-R385S is unusual, in that it has a normal amount of MCC alpha protein but confers no MCC activity. We show that MCCA-R385S, but not other MCCA missense alleles, reduces the MCC activity of cotransfected MCCA-wild-type allele. Our results suggest that MCCA-R385S is a dominant negative allele and is biotin responsive in vivo.

  1. Characterization of polybacterial clinical samples using a set of group-specific broad-range primers targeting the 16S rRNA gene followed by DNA sequencing and RipSeq analysis

    PubMed Central

    Lekang, Katrine; Langeland, Nina; Wiker, Harald G.

    2011-01-01

    The standard use of a single universal broad-range PCR in direct 16S rDNA sequencing from polybacterial samples leaves the minor constituents at risk of remaining undetected because all bacterial DNA will be competing for the same reagents. In this article we introduce a set of three broad-range group-specific 16S rDNA PCRs that together cover the clinically relevant bacteria and apply them in the investigation of 25 polybacterial clinical samples. Mixed DNA chromatograms from samples containing more than one species per primer group were analysed using RipSeq Mixed (iSentio, Norway), a web-based application for the interpretation of chromatograms containing up to three different species. The group-specific PCRs reduced complexity in the resulting DNA chromatograms and made the assay more sensitive in situations with unequal species concentrations. Together this allowed for identification of a significantly higher number of bacterial species than did standard direct sequencing with a single universal primer pair and RipSeq analysis (95 vs 51). The method could improve microbiological diagnostics for important groups of patients and can be established in any laboratory with experience in direct 16S rDNA sequencing. PMID:21436365

  2. The -5 A/G single-nucleotide polymorphism in the core promoter region of MT2A and its effect on allele-specific gene expression and Cd, Zn and Cu levels in laryngeal cancer.

    PubMed

    Starska, Katarzyna; Krześlak, Anna; Forma, Ewa; Olszewski, Jurek; Morawiec-Sztandera, Alina; Aleksandrowicz, Paweł; Lewy-Trenda, Iwona; Bryś, Magdalena

    2014-10-15

    Metallothioneins (MTs) are low molecular weight, cysteine-rich heavy metal-binding proteins which participate in the mechanisms of Zn homeostasis, and protect against toxic metals. MTs contain metal-thiolate cluster groups and suppress metal toxicity by binding to them. The aim of this study was to determine the -5 A/G (rs28366003) single-nucleotide polymorphism (SNP) in the core promoter region of the MT2A gene and to investigate its effect on allele-specific gene expression and Cd, Zn and Cu content in squamous cell laryngeal cancer (SCC) and non-cancerous laryngeal mucosa (NCM) as a control. The MT2A promoter region -5 A/G SNP was determined by restriction fragment length polymorphism using 323 SCC and 116 NCM. MT2A gene analysis was performed by quantitative real-time PCR. The frequency of A allele carriage was 94.2% and 91.8% in SCC and NCM, respectively, while G allele carriage was detected in 5.8% and 8.2% of SCC and NCM samples, respectively. As a result, a significant association was identified between the -5 A/G SNP in the MT2A gene with mRNA expression in both groups. Metal levels were analyzed by flame atomic absorption spectrometry. The significant differences were identified between A/A and both the A/G and G/G genotypes, with regard to the concentration of the contaminating metal. The Spearman rank correlation results showed that the MT2A expression and Cd, Zn, Cu levels were negatively correlated. Results obtained in this study suggest that -5 A/G SNP in MT2A gene may have an effect on allele-specific gene expression and accumulation of metal levels in laryngeal cancer.

  3. Impact of HLA-B alleles, epitope binding affinity, functional avidity, and viral coinfection on the immunodominance of virus-specific CTL responses.

    PubMed

    Bihl, Florian; Frahm, Nicole; Di Giammarino, Loriana; Sidney, John; John, Mina; Yusim, Karina; Woodberry, Tonia; Sango, Kaori; Hewitt, Hannah S; Henry, Leah; Linde, Caitlyn H; Chisholm, John V; Zaman, Tauheed M; Pae, Eunice; Mallal, Simon; Walker, Bruce D; Sette, Alessandro; Korber, Bette T; Heckerman, David; Brander, Christian

    2006-04-01

    Immunodominance is variably used to describe either the most frequently detectable response among tested individuals or the strongest response within a single individual, yet factors determining either inter- or intraindividual immunodominance are still poorly understood. More than 90 individuals were tested against 184 HIV- and 92 EBV-derived, previously defined CTL epitopes. The data show that HLA-B-restricted epitopes were significantly more frequently recognized than HLA-A- or HLA-C-restricted epitopes. HLA-B-restricted epitopes also induced responses of higher magnitude than did either HLA-A- or HLA-C-restricted epitopes, although this comparison only reached statistical significance for EBV epitopes. For both viruses, the magnitude and frequency of recognition were correlated with each other, but not with the epitope binding affinity to the restricting HLA allele. The presence or absence of HIV coinfection did not impact EBV epitope immunodominance patterns significantly. Peptide titration studies showed that the magnitude of responses was associated with high functional avidity, requiring low concentration of cognate peptide to respond in in vitro assays. The data support the important role of HLA-B alleles in antiviral immunity and afford a better understanding of the factors contributing to inter- and intraindividual immunodominance.

  4. Detection of new HLA-DPB1 alleles generated by interallelic gene conversion using PCR amplification of DPB1 second exon sequences from sperm

    SciTech Connect

    Erlich, H.; Zangenberg, G.; Bugawan, T.

    1994-09-01

    The rate at which allelic diversity at the HLA class I and class II loci evolves has been the subject of considerable controversy as have the mechanisms which generate new alleles. The patchwork pattern of polymorphism, particularly within the second exon of the HLA-DPB1 locus where the polymorphic sequence motifs are localized to 6 discrete regions, is consistent with the hypothesis that much of the allelic sequence variation may have been generated by segmental exchange (gene conversion). To measure the rate of new DPB1 variant generation, we have developed a strategy in which DPB1 second exon sequences are amplified from pools of FACS-sorted sperm (n=50) from a heterozygous sperm donor. Pools of sperm from these heterozygous individuals are amplified with an allele-specific primer for one allele and analyzed with sequence-specific oligonucleotide probes (SSOP) complementary to the other allele. This screening procedure, which is capable of detecting a single variant molecule in a pool of parental alleles, allows the identification of new variants that have been generated by recombination and/or gene conversion between the two parental alleles. To control for potential PCR artifacts, the same screening procedure was carried out with mixtures of sperm from DPB1 *0301/*0301 and DPB1 *0401/ 0401 individuals. Pools containing putative new variants DPB1 alleles were analyzed further by cloning into M13 and sequencing the M13 clones. Our current estimate is that about 1/10,000 sperm from these heterozygous individuals represents a new DPB1 allele generated by micro-gene conversion within the second exon.

  5. Comparison of a multiplexed MassARRAY system with real-time allele-specific PCR technology for genotyping of methicillin-resistant Staphylococcus aureus.

    PubMed

    Syrmis, M W; Moser, R J; Whiley, D M; Vaska, V; Coombs, G W; Nissen, M D; Sloots, T P; Nimmo, G R

    2011-12-01

    The Sequenom MassARRAY iPLEX single-nucleotide polymorphism (SNP) typing platform uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with single-base extension PCR for high-throughput multiplex SNP detection. In this study, we investigated the use of iPLEX MassARRAY technology for methicillin-resistant Staphylococcus aureus (MRSA) genotyping. A 16-plex MassARRAY iPLEX GOLD assay (MRSA-iPLEX) was developed that targets a set of informative SNPs and binary genes for MRSA characterization. The method was evaluated with 147 MRSA isolates, and the results were compared with those of an established SYBR Green-based real-time PCR system utilizing the same SNP-binary markers. A total of 2352 markers belonging to 44 SNP-binary profiles were analysed by both real-time PCR and MRSA-iPLEX. With real-time PCR as the reference standard, MRSA-iPLEX correctly assigned 2298 of the 2352 (97.7%) markers. Sequence variation in the MRSA-iPLEX primer targets accounted for the majority of MRSA-iPLEX erroneous results, highlighting the importance of primer target selection. MRSA-iPLEX provided optimal throughput for MRSA genotyping, and was, on a reagent basis, more cost-effective than the real-time PCR methods. The 16-plex MRSA-iPLEX is a suitable alternative to SYBR Green-based real-time PCR typing of major sequence types and clonal complexes of MRSA.

  6. Patho‐Genes.org: a website dedicated to gene sequences of potential bioterror bacteria and PCR primers used to amplify them

    PubMed Central

    Gardès, Julien; Bachar, Dipankar; Croce, Olivier; Christen, Richard

    2012-01-01

    Summary Pathogenic agents can be very hard to detect, and usually they do not cause illness for several hours or days. To improve the speed and the accuracy of detection tests and satisfy the needs of early diagnosis, molecular biology methods such as PCR are now used. However, selecting a proper target gene and designing good primers is often not easy. We present a dedicated website, http://patho‐genes.org, where we provide every sequence, functional annotation, published primer and relevant article for every annotated gene of major pathogenic bacterial species listed as key agents to be used for a bioterrorism attack. Each published primer was analysed to determine its melting temperature, its specificity and its coverage (i.e. its sensitivity against every allele of its target gene). Data generated have been organized in the form of data sheet for each gene, which are available through multiple browser panels and query systems. PMID:22681780

  7. The Septic Shock-associated IL-10 -1082 A>G Polymorphism Mediates Allele-specific Transcription via Poly ADP-ribose Polymerase 1 in Macrophages Engulfing Apoptotic Cells

    PubMed Central

    Kang, Xiaoyan; Kim, Ha-Jeong; Ramirez, Michelle; Salameh, Sarah; Ma, Xiaojing

    2013-01-01

    The biallelic Interleukin-10 single nucleotide polymorphism (SNP) at -1082 of the promoter region linked to individual variation in cytokine inducibility has been strongly implicated in several pathological conditions including the development of, and outcomes in, septic shock during pneumococcal infection, acute respiratory distress syndrome, and cardiac dysfunction. However, the molecular basis of the SNP-mediated variable IL-10 production levels has not been explored. Here we report that the -1082G>A alleles in the promoter region of the human IL-10 gene physically interact with a nuclear protein in an allele-specific manner that results in different levels of IL-10 transcription. This protein has been identified as poly ADP-ribose polymerase 1 (PARP-1). We show that PARP-1 acts as a transcription repressor, and its DNA-binding activity is strongly regulated in macrophages that engulf apoptotic cells but not stimulated with lippopolysaccharides. These findings unveil a novel role of PARP-1 in the regulation of IL-10 production in an allele-dependent way, which determines individual susceptibility to sepsis-induced inflammatory pathology and the immunological sequelae in a physiological process where clearance of infection-induced apoptotic cells by professional phagocytes triggers the cytokine synthesis. PMID:20181890

  8. Multi-allele genotyping platform for the simultaneous detection of mutations in the Wilson disease related ATP7B gene.

    PubMed

    Amvrosiadou, Maria; Petropoulou, Margarita; Poulou, Myrto; Tzetis, Maria; Kanavakis, Emmanuel; Christopoulos, Theodore K; Ioannou, Penelope C

    2015-12-01

    Wilson's disease is an inherited disorder of copper transport in the hepatocytes with a wide range of genotype and phenotype characteristics. Mutations in the ATP7B gene are responsible for the disease. Approximately, over 500 mutations in the ATP7B gene have been described to date. We report a method for the simultaneous detection of the ten most common ATP7B gene mutations in Greek patients. The method comprises 3 simple steps: (i) multiplex PCR amplification of fragments in the ATP7B gene flanking the mutations (ii) multiplex primer extension reaction of the unpurified amplification products using allele-specific primers and (iii) visual detection of the primer extension reaction products within minutes by means of dry-reagent multi-allele dipstick assay using anti-biotin conjugated gold nanoparticles. Optimization studies on the efficiency and specificity of the PEXT reaction were performed. The method was evaluated by genotyping 46 DNA samples of known genotype and 34 blind samples. The results were fully concordant with those obtained by reference methods. The method is simple, rapid, cost-effective and it does not require specialized instrumentation or highly qualified personnel. PMID:26580967

  9. Multi-allele genotyping platform for the simultaneous detection of mutations in the Wilson disease related ATP7B gene.

    PubMed

    Amvrosiadou, Maria; Petropoulou, Margarita; Poulou, Myrto; Tzetis, Maria; Kanavakis, Emmanuel; Christopoulos, Theodore K; Ioannou, Penelope C

    2015-12-01

    Wilson's disease is an inherited disorder of copper transport in the hepatocytes with a wide range of genotype and phenotype characteristics. Mutations in the ATP7B gene are responsible for the disease. Approximately, over 500 mutations in the ATP7B gene have been described to date. We report a method for the simultaneous detection of the ten most common ATP7B gene mutations in Greek patients. The method comprises 3 simple steps: (i) multiplex PCR amplification of fragments in the ATP7B gene flanking the mutations (ii) multiplex primer extension reaction of the unpurified amplification products using allele-specific primers and (iii) visual detection of the primer extension reaction products within minutes by means of dry-reagent multi-allele dipstick assay using anti-biotin conjugated gold nanoparticles. Optimization studies on the efficiency and specificity of the PEXT reaction were performed. The method was evaluated by genotyping 46 DNA samples of known genotype and 34 blind samples. The results were fully concordant with those obtained by reference methods. The method is simple, rapid, cost-effective and it does not require specialized instrumentation or highly qualified personnel.

  10. Detecting the H3F3A mutant allele found in high-grade pediatric glioma by real-time PCR.

    PubMed

    Zhang, Ray; Han, Jing; Daniels, David; Huang, Haojie; Zhang, Zhiguo

    2016-01-01

    Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brain tumor with a median survival of 1 year after diagnosis. It has been reported recently that about 80% of DIPG cases and 70% of midline glioblastomas contain a mutation at one allele of the H3F3A gene (encoding histone H3 variant H3.3), replacing the lysine 27 with methionine (K27M). In order to facilitate diagnosis of DIPG patients, a quick and reliable method to identify the H3F3A K27M mutation is needed. Here, we describe a real-time PCR-based procedure involving a mutant-specific primer, a blocker oligonucleotide, and a reverse primer that can differentiate samples with H3F3A K27M mutation from those that do not. We first tested four different mutant-specific primers for their ability to selectively amplify H3F3A K27M-mutant allele and found that one primer amplified the mutant allele more efficiently than the rest. We then determined the optimal concentration of blocker oligo that significantly improved amplification of the H3F3A K27M-mutant allele. Using this optimized real-time PCR assay, we analyzed eleven samples, two of which containing H3F3A K27M mutation, and found that these two samples were differentially amplified from the nine others. In addition, we were able to discern the H3F3A K27M mutation in a newly obtained pediatric brainstem glioblastoma sample whose H3.3 status was not known previously, and in three other DIPG samples as well as paraffin embedded samples. These results demonstrate that we have developed a new reliable procedure for detecting the H3F3A K27M mutation in pediatric glioblastoma patient samples. PMID:26376656

  11. Evaluation of a new efficient procedure for single-nucleotide polymorphism genotyping: tetra-primer amplification refractory mutation system-polymerase chain reaction.

    PubMed

    Okayama, Naoko; Fujimura, Kozue; Nakamura, Junji; Suehiro, Yutaka; Hamanaka, Yuichiro; Hinoda, Yuji

    2004-01-01

    Tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) is a new efficient method for single-nucleotide polymorphism (SNP) genotyping. To determine the optimal conditions for ARMS-PCR we attempted to genotype ten SNPs. DNA was extracted from the peripheral blood of 168 unrelated healthy Japanese volunteers. Two problems inhibited uniform efficiency of the amplification of three bands. The first problem was the lower amplification efficiency of the shorter and allele-specific products compared with the largest product. This phenomenon was overcome by increasing the relative concentration of the inner primers. The second problem was non-specific amplification of the shorter products. To reduce the amplification of these non-specific bands, adjusting any one of the following PCR conditions was effective: i) reducing the ratio of the inner primer concentration relative to that of the outer primers; ii) increasing the annealing temperature for the initial 5-10 cycles; iii) hot start PCR. With these procedures all ten of the SNPs were successfully genotyped. Our present data may be useful in the further application of tetra-primer ARMS-PCR to SNP genotyping.

  12. PrimerIdent: A web based tool for conserved primer design

    PubMed Central

    Pessoa, Alberto M; Pereira, Susana; Teixeira, Jorge

    2010-01-01

    Conserved primers across multiple species and simultaneously specific for a certain isozyme can be rare and difficult to find. PrimerIdent was developed aiming to automate this primer design and selection process in a given nucleotide sequence alignment, providing an intuitive, easy to interpret graphical result, which offers a list of all possible primers that meet the user criteria, with a colour-code identity to each sequence in the alignment. The software here presented is a simple and intuitive web based tool that is suitable for distinguishing very similar nucleotide sequences, such as isozymes­coding sequences, to enable the conserved primer design across multiple species, necessary for approaches that rely on knowing if a primer is suitable for a certain set of pre-aligned sequences, to design a specific primer to a certain sequence variation, or a combination thereof. This extremely useful software can, therefore, be used as a tool for the specific amplification of individual members of multigenic families across related species and also to evaluate the differential expression of isogenes for a given species. Availability http://primerident.up.pt PMID:21346862

  13. Relationship between HLA-DRB1 allele polymorphisms and familial aggregations of hepatocellular carcinoma

    PubMed Central

    Ma, S.; Wu, J.; Wu, J.; Wei, Y.; Zhang, L.; Ning, Q.; Hu, D.

    2016-01-01

    Objective We explored the relationship between HLA-DRB1 allele polymorphisms and familial aggregation of hepatocellular carcinoma (fhcc). Methods Polymerase chain reaction sequence-specific primers were used to determine HLA-DRB1 genotypes for 130 members of families with 2 or more liver cancer patients and for 130 members of families without any diagnosed cancers. The genotype profiles were then compared to explore the relationship between HLA-DRB1 gene polymorphism and fhcc. Result Of 11 selected alleles, the frequencies of DRB1*11 and DRB1*12 were significantly lower in the fhcc group than in no-cancer group (p < 0.05; odds ratio: 0.286; 95% confidence interval: 0.091 to 0.901; and odds ratio: 0.493; 95% confidence interval: 0.292 to 0.893). Differences in the frequencies of the other 9 alleles were not statistically significant in the two groups (p > 0.05). Conclusions Our research suggests that if genetic factors play a role in fhcc, the deficiency in the DRB1*11 and DRB1*12 alleles might be the risk factor at work in Guangxi Zhuang Autonomous Region, P.R.C. PMID:26966407

  14. Allelic Imbalance of mRNA Associated with α2-HS Glycoprotein (Fetuin-A) Polymorphism

    PubMed Central

    Inaoka, Yoshihiko; Osawa, Motoki; Mukasa, Nahoko; Miyashita, Keiko; Satoh, Fumiko; Kakimoto, Yu

    2015-01-01

    Alpha 2-HS glycoprotein (AHSG), also designated as fetuin-A, exhibits polymorphism in population genetics consisting of two major alleles of AHSG∗1 and AHSG∗2. The serum level in the AHSG∗1 homozygote is significantly higher than that of the AHSG∗2 homozygote. This study examined the molecular mechanism for the cis-regulatory expression. To quantitate allele-specific mRNA in intra-assays of the heterozygote, RT-PCR method employing primers that were incorporated to the two closely located SNPs was developed. The respective magnitudes of AHSG∗1 to AHSG∗2 in the liver tissues and hepatic culture cells of PLC/PRF/5 were determined quantitatively as 2.5-fold and 6.2-fold. The mRNA expressional difference of two major alleles was observed, which is consistent with that in the serum level. The culture cells carried heterozygous genotypes in rs4917 and rs4918, but homozygous one in rs2248690. It was unlikely that the imbalance was derived from the SNP located in the promotor site. Furthermore, to investigate the effect of mRNA degradation, RNA synthesis in the cell culture was inhibited potently by the addition of actinomycin-D. No marked change was apparent between the two alleles. The results indicated that the cis-regulatory expressional difference is expected to occur at the level of transcription or splicing of mRNA. PMID:26549924

  15. China Energy Primer

    SciTech Connect

    Ni, Chun Chun

    2009-11-16

    Based on extensive analysis of the 'China Energy Databook Version 7' (October 2008) this Primer for China's Energy Industry draws a broad picture of China's energy industry with the two goals of helping users read and interpret the data presented in the 'China Energy Databook' and understand the historical evolution of China's energy inustry. Primer provides comprehensive historical reviews of China's energy industry including its supply and demand, exports and imports, investments, environment, and most importantly, its complicated pricing system, a key element in the analysis of China's energy sector.

  16. Allele-Specific PCR Method Based on pncA and oxyR Sequences for Distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: Intraspecific M. bovis pncA Sequence Polymorphism

    PubMed Central

    de los Monteros, Luz Elena Espinosa; Galán, Juan Carlos; Gutiérrez, Montserrat; Samper, Sofía; García Marín, Juan F.; Martín, Carlos; Domínguez, Lucas; de Rafael, Luis; Baquero, Fernando; Gómez-Mampaso, Enrique; Blázquez, Jesús

    1998-01-01

    An allele-specific amplification method based on two genetic polymorphisms to differentiate Mycobacterium tuberculosis from Mycobacterium bovis was tested. Based on the differences found at position 169 in the pncA genes from M. tuberculosis and M. bovis, a PCR system which was able to differentiate most of the 237 M. tuberculosis complex isolates tested in one of the two species was developed. All 121 M. tuberculosis strains showed the expected base (cytosine) at position 169. Most of the M. bovis isolates had a guanine at the cited position. Nevertheless, 18 of the 116 M. bovis isolates, all of them goat isolates, showed the pncA polymorphism specific to M. tuberculosis. These results suggest that goat M. bovis may be the nicotinamidase-missing link at the origin of the M. tuberculosis species. Based on the polymorphism found at position 285 in the oxyR gene, the same system was used to differentiate M. tuberculosis from M. bovis. In this case, DNAs from all 121 M. tuberculosis isolates had the expected base (guanine) at this position. In addition, all 116 M. bovis isolates, including those from goats, showed the identical polymorphism (adenine). The oxyR allele-specific amplification method can differentiate M. bovis from M. tuberculosis, is rapid (results can be obtained in less than 3 h), and is easy to perform. PMID:9431955

  17. Allele-specific suppressors of lin-1(R175Opal) identify functions of MOC-3 and DPH-3 in tRNA modification complexes in Caenorhabditis elegans.

    PubMed

    Kim, Sunhong; Johnson, Wade; Chen, Changchun; Sewell, Aileen K; Byström, Anders S; Han, Min

    2010-08-01

    The elongator (ELP) complex consisting of Elp1-6p has been indicated to play roles in multiple cellular processes. In yeast, the ELP complex has been shown to genetically interact with Uba4p/Urm1p and Kti11-13p for a function in tRNA modification. Through a Caenorhabditis elegans genetic suppressor screen and positional cloning, we discovered that loss-of-function mutations of moc-3 and dph-3, orthologs of the yeast UBA4 and KTI11, respectively, effectively suppress the Multivulva (Muv) phenotype of the lin-1(e1275, R175Opal) mutation. These mutations do not suppress the Muv phenotype caused by other lin-1 alleles or by gain-of-function alleles of ras or raf that act upstream of lin-1. The suppression can also be reverted by RNA interference of lin-1. Furthermore, we showed that dph-3(lf) also suppressed the defect of lin-1(e1275) in promoting the expression of a downstream target (egl-17). These results indicate that suppression by the moc-3 and dph-3 mutations is due to the elevated activity of lin-1(e1275) itself rather than the altered activity of a factor downstream of lin-1. We further showed that loss-of-function mutations of urm-1 and elpc-1-4, the worm counterparts of URM1 and ELP complex components in yeast, also suppressed lin-1(e1275). We also confirmed that moc-3(lf) and dph-3(lf) have defects in tRNA modifications as do the mutants of their yeast orthologs. These results, together with the observation of a likely readthrough product from a lin-1(e1275)::gfp fusion transgene indicate that the aberrant tRNA modification led to failed recognition of a premature stop codon in lin-1(e1275). Our genetic data suggest that the functional interaction of moc-3/urm-1 and dph-3 with the ELP complex is an evolutionarily conserved mechanism involved in tRNA functions that are important for accurate translation. PMID:20479142

  18. Association of human leukocyte antigen DQB1 and DRB1 alleles with chronic hepatitis B

    PubMed Central

    Doganay, Levent; Fejzullahu, Arta; Katrinli, Seyma; Yilmaz Enc, Feruze; Ozturk, Oguzhan; Colak, Yasar; Ulasoglu, Celal; Tuncer, Ilyas; Dinler Doganay, Gizem

    2014-01-01

    AIM: To investigate the effect of human leukocyte antigen (HLA) DRB1 and DQB1 alleles on the inactive and advanced stages of chronic hepatitis B. METHODS: Patient records at a single institution’s hepatology clinic were reviewed. Demographic data, laboratory results, endoscopy results, virological parameters, biopsy scores and treatment statuses were recorded. In total, 355 patients were eligible for the study, of whom 226 (63.7%) were male. Overall, 82 (23.1%) were hepatitis B early antigen (HBeAg) positive, 87 (24.5%) had cirrhosis, and 66 (18.6%) had inactive disease. The presence of DQB1 and DRB1 alleles was determined by polymerase chain reaction with sequence-specific primers. The distribution of the genotyped alleles among patients with cirrhosis and patients with chronic active hepatitis was analyzed. RESULTS: The most frequent HLA DQB1 allele was DQB1*03:01 (48.2%), and the most frequent HLA DRB1 allele was DRB1*13/14 (51.8%). DQB1*05:01 was more frequent in patients with active disease than in inactive patients (27% vs 9.1%; P = 0.002, Pc = 0.026). DRB1*07 was rare in patients with cirrhosis compared with non-cirrhotics (3.4% vs 16%; P = 0.002, Pc = 0.022). Older age (P < 0.001) and male gender (P = 0.008) were the other factors that affected the presence of cirrhosis. In a multivariate logistic regression analysis, DRB1*07 remained a significant negative predictor of cirrhosis (P = 0.015). A bioinformatics analysis revealed that a polymorphic amino acid sequence in DRB1*07 may alter interaction with the T-cell recognition site. CONCLUSION: This study demonstrates that HLA alleles may influence cirrhosis development and disease activity in Turkish chronic hepatitis B patients. PMID:25009391

  19. Development of primer sets for loop-mediated isothermal amplification that enables rapid and specific detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three ...

  20. Primer on Social Economics.

    ERIC Educational Resources Information Center

    Darcy, Robert L.

    An elaboration of the author's booklet entitled "First Steps Toward Economic Understanding," this primer is designed to help the reader develop a functional understanding of the economic process so that he can make wiser decisions on issues of social policy and on matters affecting his economic well-being. The document is not "economics in one…

  1. An SAT® Validity Primer

    ERIC Educational Resources Information Center

    Shaw, Emily J.

    2015-01-01

    This primer should provide the reader with a deeper understanding of the concept of test validity and will present the recent available validity evidence on the relationship between SAT® scores and important college outcomes. In addition, the content examined on the SAT will be discussed as well as the fundamental attention paid to the fairness of…

  2. ANL supplement to the UNICOS primer

    SciTech Connect

    Wiley, M.S.; Karlovsky, S.R.

    1991-06-01

    The ANL Supplement to the UNICOS Primer (ANL/TM 460) introduces the Cray X-MP interactive and batch services available at Argonne National Laboratory. It serves as a companion to the UNICOS Primer (Cray publication SG-2010 6.0). Whereas the UNICOS Primer discusses standard Unix issues of Cray computing, this manual discusses those issues specific to Cray computing at ANL. If this is your first experience on a Unix-based system, we assume that you have read at least Chapters 1 through 3 of the UNICOS Primer. The Glossary at the back of the UNICOS Primer will also be useful to you. If you are already familiar with a Unix system, it should suffice to keep the UNICOS Primer handy as you use this document. To learn about Unix programming in greater detail, we recommend A Practical Guide to the Unix System, by Mark G. Sobell. This manual and all other sources referred to in this document are available for purchase at the Document Distribution Counter in Building 221, Room A-134. We assume that you have already read the Guide to Computing at ANL (ANL/TM 336) to get an overview of all the computing facilities and services available at Argonne National Laboratory. You should also refer to Recommended Documentation for Computer Users at ANL (ANL/TM 379) for additional guidance in selecting available documentation that will best fill your particular computing needs.

  3. State-of-the-art monitoring of cytomegalovirus-specific cell-mediated immunity after organ transplant: a primer for the clinician.

    PubMed

    Egli, Adrian; Humar, Atul; Kumar, Deepali

    2012-12-01

    Cytomegalovirus (CMV) is one of the most common infections after solid organ transplantation. Improved assays to predict viral replication and disease would help refine current preventive strategies. Monitoring of CMV-specific T-cell responses may help guide clinical decision making. Several techniques are now available to quantify CMV-specific T-cell responses, including flow cytometry, enzyme-linked immunosorbent spot assay, and enzyme-linked immunosorbent assay. Standardization and validation of these assays have the potential to significantly change the monitoring and treatment of CMV and further personalize CMV prevention strategies. In this review, we discuss the measurement of CMV-specific T-cell responses and their clinical impact on the management of CMV after organ transplantation.

  4. Creating State-Specific Resources: A Technical Assistance Model That Works. Primers on Implementing Special Education in Charter Schools. Special Report

    ERIC Educational Resources Information Center

    Lange, Cheryl M.

    2008-01-01

    A model developed by the Technical Assistance Customizer (TAC) Project offers state education agencies and other organizations an approach to assist them with converting national findings and resources to state-specific materials in a timely and cost-effective manner. Moreover, the approach recognizes the importance of collaboration with and the…

  5. The − 5 A/G single-nucleotide polymorphism in the core promoter region of MT2A and its effect on allele-specific gene expression and Cd, Zn and Cu levels in laryngeal cancer

    SciTech Connect

    Starska, Katarzyna; Krześlak, Anna; Forma, Ewa; Morawiec-Sztandera, Alina; Aleksandrowicz, Paweł; Lewy-Trenda, Iwona; and others

    2014-10-15

    Metallothioneins (MTs) are low molecular weight, cysteine-rich heavy metal-binding proteins which participate in the mechanisms of Zn homeostasis, and protect against toxic metals. MTs contain metal-thiolate cluster groups and suppress metal toxicity by binding to them. The aim of this study was to determine the − 5 A/G (rs28366003) single-nucleotide polymorphism (SNP) in the core promoter region of the MT2A gene and to investigate its effect on allele-specific gene expression and Cd, Zn and Cu content in squamous cell laryngeal cancer (SCC) and non-cancerous laryngeal mucosa (NCM) as a control. The MT2A promoter region − 5 A/G SNP was determined by restriction fragment length polymorphism using 323 SCC and 116 NCM. MT2A gene analysis was performed by quantitative real-time PCR. The frequency of A allele carriage was 94.2% and 91.8% in SCC and NCM, respectively, while G allele carriage was detected in 5.8% and 8.2% of SCC and NCM samples, respectively. As a result, a significant association was identified between the − 5 A/G SNP in the MT2A gene with mRNA expression in both groups. Metal levels were analyzed by flame atomic absorption spectrometry. The significant differences were identified between A/A and both the A/G and G/G genotypes, with regard to the concentration of the contaminating metal. The Spearman rank correlation results showed that the MT2A expression and Cd, Zn, Cu levels were negatively correlated. Results obtained in this study suggest that − 5 A/G SNP in MT2A gene may have an effect on allele-specific gene expression and accumulation of metal levels in laryngeal cancer. - Highlights: • MT2A gene expression and metal content in laryngeal cancer tissues • Association between SNP (rs28366003) and expression of MT2A • Significant associations between the SNP and Cd, Zn and Cu levels • Negative correlation between MT2A gene expression and Cd, Zn and Cu levels.

  6. Transferability of microsatellite primers developed for stingless bees to four other species of the genus Melipona.

    PubMed

    Viana, M V C; Miranda, E A; de Francisco, A K; Carvalho, C A L; Waldschmidt, A M

    2011-11-22

    Microsatellite markers are a useful tool for ecological monitoring of natural and managed populations. A technical limitation is the necessity for investment in the development of primers. Heterologous primers can provide an alternative to searching for new loci. In bees, these markers have been used in populational and intracolonial genetic analyses. The genus Melipona has the largest number of species among bee genera, about 70, occurring throughout the Neotropical region. However, only five species of the genus Melipona have specific microsatellite markers. Given the great diversity of this genus, this number is not representative. We analyzed the transferability of 49 microsatellite loci to four other species of the genus Melipona (M. scutellaris, M. mondury, M. mandacaia, and M. quadrifasciata). Four individuals of each species, from different localities, were used in amplification tests. Primer pairs described for five Melipona species and for Trigona carbonaria were tested. Among the 49 loci, 22 gave amplification products for all four species, while three gave nonspecific bands and five showed no amplification products. The remaining loci varied in the pattern of amplification, according to the species examined. The number of alleles ranged from 1 to 6. The results demonstrate the possibility of using these heterologous markers in other Melipona species, increasing the number of loci that can be analyzed and contributing to further genetic analyses of intra- and intercolonial structure, which is required for conservation measure planning, genetic improvement and resolution of taxonomic problems.

  7. Maternal and paternal genomes function independently in mouse ova in establishing expression of the imprinted genes Snrpn and Igf2r: no evidence for allelic trans-sensing and counting mechanisms.

    PubMed Central

    Szabó, P E; Mann, J R

    1996-01-01

    It has often been suggested that the parental-specific expression of mammalian imprinted genes might be dependent on maternal-paternal intergenomic or interallelic interactions. Using quantitative allele-specific RT-PCR single nucleotide primer extension assays developed for two imprinted genes, Snrpn and Igf2r, we demonstrate: (i) No role for maternal-paternal allelic interactions: the modes of parental-specific expression of Snrpn and Igf2r in normal ova were unchanged in gynogenetic and androgenetic ova; the latter contain two maternal and two paternal genomes respectively, and cannot undergo maternal-paternal interactions. (ii) No role for allelic counting or exclusion mechanisms: in individual blastomeres of androgenetic ova, both paternal Snrpn alleles were active (Snrpn was not expressed in gynogenetic ova), and in individual gynogenetic and androgenetic blastomeres, both maternal and paternal Igf2r alleles, respectively, were active. (iii) No role for ploidy: the mode of parental-specific expression of Snrpn and Igf2r in normal diploid ova was unchanged in individual blastomeres of triploid and tetraploid ova. Thus, the maternal and paternal genomes function independently in establishing the pre-implantation mode of parental-specific expression of Snrpn and Igf2r, with no role for trans-allelic/genomic interaction phenomena. In addition, the results show that inactive and biallelic modes of expression of imprinted genes are potential mechanisms for the death of gynogenones and androgenones at the peri-implantation stage. Images PMID:8947024

  8. Linkage disequilibrium between a 5q13 microsatellite null allele and SMA indicates complex but stable deletions/duplications

    SciTech Connect

    Surhk, L.C.; Aubry, H.L.; MacKenzie, A.E.

    1994-09-01

    SMA is a recessively inherited degeneration of alpha motor neurons presenting in three distinct types with diverse onsets ranging from infancy (type I) to childhood (types II and III). All forms have been mapped to chromosome 5q13.1. We have developed PCR primers for two distinct microsatellite repeats (MSR), CATT-40G1 and CATT-192F7. These subloci are constituents of a previously reported 5q13.1 complex CA dinucleotide MSR, CATT-1, with four or more copies per chromosome. Type 1 SMA families (n=35) were examined because stricter clinical criteria permitted greater diagnostic accuracy and, possibly, greater mutational homogeneity. One allele per chromosome is amplified with the 40G1 and 192F7 subloci-specific primers out of a total of 3 and 7 possible alleles, respectively. Significant linkage disequilibrium was observed between the disease phenotype and the 40G1 null allele ({chi}{sup 2}=38.23) and 192F7 allele with 19 CA repeats ({chi}{sup 2}=6.62) in contrast to two markers D5S435 ({chi}{sup 2}=.42) and D5S351 ({chi}{sup 2}=.16), which have been shown to flank the SMA locus at less than 1% recombination. The null alleles present in both subloci appeared to be due to stable deletions rather than isolated DNA sequence polymorphisms. In contrast to recent reports, no evidence of de novo rearrangements were observed. Furthermore, duplications of these subloci have been observed in three normal chromosomes. Our data are consistent with unequal cross-overs generating deletions/duplications which not only account for the complexity being observed in the genomic walk within the critical SMA region, but could also be causally related SMA mutations. Results of similar linkage disequilibrium analyses employing 3 additional 5q13.1 microsatellites will also be presented.

  9. A Screen for Modifiers of Cilia Phenotypes Reveals Novel MKS Alleles and Uncovers a Specific Genetic Interaction between osm-3 and nphp-4

    PubMed Central

    Williams, Corey L.; Pieczynski, Jay N.; Roszczynialski, Kelly N.; Covington, Jannese E.; Malarkey, Erik B.; Yoder, Bradley K.

    2016-01-01

    Nephronophthisis (NPHP) is a ciliopathy in which genetic modifiers may underlie the variable penetrance of clinical features. To identify modifiers, a screen was conducted on C. elegans nphp-4(tm925) mutants. Mutations in ten loci exacerbating nphp-4(tm925) ciliary defects were obtained. Four loci have been identified, three of which are established ciliopathy genes mks-1, mks-2, and mks-5. The fourth allele (yhw66) is a missense mutation (S316F) in OSM-3, a kinesin required for cilia distal segment assembly. While osm-3(yhw66) mutants alone have no overt cilia phenotype, nphp-4(tm925);osm-3(yhw66) double mutants lack distal segments and are dye-filling (Dyf) and osmotic avoidance (Osm) defective, similar to osm-3(mn357) null mutants. In osm-3(yhw66) mutants anterograde intraflagellar transport (IFT) velocity is reduced. Furthermore, expression of OSM-3(S316F)::GFP reduced IFT velocities in nphp-4(tm925) mutants, but not in wild type animals. In silico analysis indicates the S316F mutation may affect a phosphorylation site. Putative phospho-null OSM-3(S316F) and phospho-mimetic OSM-3(S316D) proteins accumulate at the cilia base and tip respectively. FRAP analysis indicates that the cilia entry rate of OSM-3(S316F) is slower than OSM-3 and that in the presence of OSM-3(S316F), OSM-3 and OSM-3(S316D) rates decrease. In the presence OSM-3::GFP or OSM-3(S316D)::GFP, OSM-3(S316F)::tdTomato redistributes along the cilium and accumulates in the cilia tip. OSM-3(S316F) and OSM-3(S316D) are functional as they restore cilia distal segment formation in osm-3(mn357) null mutants; however, only OSM-3(S316F) rescues the osm-3(mn357) null Dyf phenotype. Despite rescue of cilia length in osm-3(mn357) null mutants, neither OSM-3(S316F) nor OSM-3(S316D) restores ciliary defects in nphp-4(tm925);osm-3(yhw66) double mutants. Thus, these OSM-3 mutations cause NPHP-4 dependent and independent phenotypes. These data indicate that in addition to regulating cilia protein entry or exit

  10. Crystalline Silica Primer

    USGS Publications Warehouse

    ,

    1992-01-01

    substance and will present a nontechnical overview of the techniques used to measure crystalline silica. Because this primer is meant to be a starting point for anyone interested in learning more about crystalline silica, a list of selected readings and other resources is included. The detailed glossary, which defines many terms that are beyond the scope of this publication, is designed to help the reader move from this presentation to a more technical one, the inevitable next step.

  11. Coal Bed Methane Primer

    SciTech Connect

    Dan Arthur; Bruce Langhus; Jon Seekins

    2005-05-25

    During the second half of the 1990's Coal Bed Methane (CBM) production increased dramatically nationwide to represent a significant new source of income and natural gas for many independent and established producers. Matching these soaring production rates during this period was a heightened public awareness of environmental concerns. These concerns left unexplained and under-addressed have created a significant growth in public involvement generating literally thousands of unfocused project comments for various regional NEPA efforts resulting in the delayed development of public and fee lands. The accelerating interest in CBM development coupled to the growth in public involvement has prompted the conceptualization of this project for the development of a CBM Primer. The Primer is designed to serve as a summary document, which introduces and encapsulates information pertinent to the development of Coal Bed Methane (CBM), including focused discussions of coal deposits, methane as a natural formed gas, split mineral estates, development techniques, operational issues, producing methods, applicable regulatory frameworks, land and resource management, mitigation measures, preparation of project plans, data availability, Indian Trust issues and relevant environmental technologies. An important aspect of gaining access to federal, state, tribal, or fee lands involves education of a broad array of stakeholders, including land and mineral owners, regulators, conservationists, tribal governments, special interest groups, and numerous others that could be impacted by the development of coal bed methane. Perhaps the most crucial aspect of successfully developing CBM resources is stakeholder education. Currently, an inconsistent picture of CBM exists. There is a significant lack of understanding on the parts of nearly all stakeholders, including industry, government, special interest groups, and land owners. It is envisioned the Primer would being used by a variety of

  12. Primer on molecular genetics

    SciTech Connect

    Not Available

    1992-04-01

    This report is taken from the April 1992 draft of the DOE Human Genome 1991--1992 Program Report, which is expected to be published in May 1992. The primer is intended to be an introduction to basic principles of molecular genetics pertaining to the genome project. The material contained herein is not final and may be incomplete. Techniques of genetic mapping and DNA sequencing are described.

  13. Cloning and characterization of genomic DNA sequences of four self-incompatibility alleles in sweet cherry ( Prunus avium L.).

    PubMed

    Wünsch, A; Hormaza, J I

    2004-01-01

    Gametophytic self-incompatibility (GSI) in sweet cherry is determined by a locus S with multiple alleles. In the style, the S-locus codifies for an allele-specific ribonuclease ( S-RNase) that is involved in the rejection of pollen that carries the same S allele. In this work we report the cloning and genomic DNA sequence analysis including the 5' flanking regions of four S-RNases of sweet cherry ( Prunus avium L., Rosaceae). DNA from the cultivars Ferrovia, Pico Colorado, Taleguera Brillante and Vittoria was amplified through PCR using primers designed in the conserved sequences of sweet cherry S-RNases. Two alleles were amplified for each cultivar and three of them correspond to three new S-alleles named S23, S24 and S25 present in 'Pico Colorado', 'Vittoria' and 'Taleguera Brillante' respectively. To confirm the identity of the amplified fragments, the genomic DNA of these three putative S-RNases and the allele S12 amplified in the cultivar Ferrovia were cloned and sequenced. The nucleotide and deduced amino-acid sequences obtained contained the structural features of rosaceous S-RNases. The isolation of the 5'-flanking sequences of these four S-RNases revealed a conserved putative TATA box and high similarity among them downstream from that sequence. However, similarity was low compared with the 5'-flanking regions of S-RNases from the Maloideae. S6- and S24-RNase sequences are highly similar, and most amino-acid substitutions among these two RNases occur outside the rosaceous hypervariable region (RHV), but within another highly variable region. The confirmation of the different specificity of these two S-RNases would help elucidate which regions of the S-RNase sequences play a role in S-pollen specific recognition.

  14. PCR Strategies for Complete Allele Calling in Multigene Families Using High-Throughput Sequencing Approaches.

    PubMed

    Marmesat, Elena; Soriano, Laura; Mazzoni, Camila J; Sommer, Simone; Godoy, José A

    2016-01-01

    The characterization of multigene families with high copy number variation is often approached through PCR amplification with highly degenerate primers to account for all expected variants flanking the region of interest. Such an approach often introduces PCR biases that result in an unbalanced representation of targets in high-throughput sequencing libraries that eventually results in incomplete detection of the targeted alleles. Here we confirm this result and propose two different amplification strategies to alleviate this problem. The first strategy (called pooled-PCRs) targets different subsets of alleles in multiple independent PCRs using different moderately degenerate primer pairs, whereas the second approach (called pooled-primers) uses a custom-made pool of non-degenerate primers in a single PCR. We compare their pe