Science.gov

Sample records for allele-specific pcr as-pcr

  1. Direct Fluorescence Detection of Allele-Specific PCR Products Using Novel Energy-Transfer Labeled Primers.

    PubMed

    Winn-Deen

    1998-12-01

    Background: Currently analysis of point mutations can be done by allele-specific polymerase chain reaction (PCR) followed by gel analysis or by gene-specific PCR followed by hybridization with an allele-specific probe. Both of these mutation detection methods require post-PCR laboratory time and run the risk of contaminating subsequent experiments with the PCR product liberated during the detection step. The author has combined the PCR amplification and detection steps into a single procedure suitable for closed-tube analysis. Methods and Results: Allele-specific PCR primers were designed as Sunrise energy-transfer primers and contained a 3' terminal mismatch to distinguish between normal and mutant DNA. Cloned normal (W64) and mutant (R64) templates of the beta3-adrenergic receptor gene were tested to verify amplification specificity and yield. A no-target negative control was also run with each reaction. After PCR, each reaction was tested for fluorescence yield by measuring fluorescence on a spectrofluorimeter or fluorescent microtitreplate reader. The cloned controls and 24 patient samples were tested for the W64R mutation by two methods. The direct fluorescence results with the Sunrise allele-specific PCR method gave comparable genotypes to those obtained with the PCR/ restriction digest/gel electrophoresis control method. No PCR artifacts were observed in the negative controls or in the PCR reactions run with the mismatched target. Conclusions: The results of this pilot study indicate good PCR product and fluorescence yield from allele-specific energy-transfer labeled primers, and the capability of distinguishing between normal and mutant alleles based on fluorescence alone, without the need for restriction digestion, gel electrophoresis, or hybridization with an allele-specific probe. PMID:10089280

  2. 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

  3. 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

  4. Nested Allele-Specific PCR Primers Distinguish Genetic Groups of Uncinula necator

    PubMed Central

    Délye, Christophe; Ronchi, Valérie; Laigret, Frédéric; Corio-Costet, Marie-France

    1999-01-01

    Isolates of the obligately biotrophic fungus Uncinula necator cluster in three distinct genetic groups (groups I, II, and III). We designed PCR primers specific for these groups in order to monitor field populations of U. necator. We used the nucleotide sequences of the gene that encodes eburicol 14α-demethylase (CYP51) and of the ribosomal DNA internal transcribed spacer 1 (ITS1), ITS2, and 5.8S regions. We identified four point mutations (three in CYP51 and one in ITS1) that distinguished groups I and II from group III based on a sample of 132 single-spore isolates originating from Europe, Tunisia, Israel, India, and Australia. We developed a nested allele-specific PCR assay in which the CYP51 point mutations were used to detect and distinguish groups I and II from group III in crude mildewed samples from vineyards. In a preliminary study performed with samples from French vineyards in which isolates belonging to genetic groups I and III were present, we found that a shift from a population composed primarily of group I isolates to a population composed primarily of group III isolates occurred during the grapevine growing season. PMID:10473400

  5. 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

  6. Comparison of immunohistochemistry, DNA sequencing and allele-specific PCR for the detection of IDH1 mutations in gliomas.

    PubMed

    Loussouarn, Delphine; Le Loupp, Anne-Gaëlle; Frenel, Jean-Sébastien; Leclair, François; Von Deimling, Andreas; Aumont, Maud; Martin, Stéphane; Campone, Mario; Denis, Marc G

    2012-06-01

    Previous studies have identified mutations of the isocitrate dehydrogenase 1 (IDH1) gene in more than 70% of World Health Organization (WHO) grade II and III gliomas. The most frequent mutation leads to a specific amino acid change from arginine to histidine at codon 132 (c.395G>A, p.R132H). IDH1 mutated tumors have a better prognosis than IDH1 non-mutated tumors. The aim of our study was to evaluate and compare the methods of mIDH1 R132H immunohistochemistry, allele-specific PCR and DNA sequencing for determination of IDH1 status. We performed a retrospective study of 91 patients with WHO grade II (n=43) and III (n=48) oligodendrogliomas. A fragment of exon 4 spanning the sequence encoding the catalytic domain of IDH1, including codon 132, was amplified and sequenced using standard conditions. Allele-specific amplification was performed using two forward primers with variations in their 3' nucleotides such that each was specific for the wild-type or the mutated variant, and one reverse primer. Immunohistochemistry was performed with mouse monoclonal mIDH1 R132H. DNA was extracted from FFPE sections following macrodissection. IDH1 mutations were found in 55/90 patients (61.1%) by direct sequencing. R132H mutations were found in 47/55 patients (85.4%). The results of the allele-specific PCR positively correlated with those from DNA sequencing. Other mutations (p.R132C, p.R132S and pR132G) were found by DNA sequencing in 3, 3 and 2 tumors, respectively (8/55 patients, 14.6%). mIDH1 R132H immunostaining was found in the 47 patients presenting the R132H mutation (sensitivity 47/47, 100% for this mutation). None of the tumors presenting a wild-type IDH1 gene were stained (specificity 35/35, 100%). Our results demonstrate that immunohistochemistry using the mIDH1 R132H antibody and allele-specific amplification are highly sensitive techniques to detect the most frequent mutation of the IDH1 gene. PMID:22447191

  7. Citrus (Rutaceae) SNP markers based on Competitive Allele-Specific PCR; transferability across the Aurantioideae subfamily1

    PubMed Central

    Garcia-Lor, Andres; Ancillo, Gema; Navarro, Luis; Ollitrault, Patrick

    2013-01-01

    • Premise of the study: Single nucleotide polymorphism (SNP) markers based on Competitive Allele-Specific PCR (KASPar) were developed from sequences of three Citrus species. Their transferability was tested in 63 Citrus genotypes and 19 relative genera of the subfamily Aurantioideae to estimate the potential of SNP markers, selected from a limited intrageneric discovery panel, for ongoing broader diversity analysis at the intra- and intergeneric levels and systematic germplasm bank characterization. • Methods and Results: Forty-two SNP markers were developed using KASPar technology. Forty-one were successfully genotyped in all of the Citrus germplasm, where intra- and interspecific polymorphisms were observed. The transferability and diversity decreased with increasing taxonomic distance. • Conclusions: SNP markers based on the KASPar method developed from sequence data of a limited intrageneric discovery panel provide a valuable molecular resource for genetic diversity analysis of germplasm within a genus and should be useful for germplasm fingerprinting at a much broader diversity level. PMID:25202535

  8. 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

  9. Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model.

    PubMed

    Monteiro, L; Bonnemaison, D; Vekris, A; Petry, K G; Bonnet, J; Vidal, R; Cabrita, J; Mégraud, F

    1997-04-01

    A model was developed to study inhibitors present in feces which prevent the use of PCR for the detection of Helicobacter pylori. A DNA fragment amplified with the same primers as H. pylori was used to spike samples before extraction by a modified QIAamp tissue method. Inhibitors, separated on an Ultrogel AcA44 column, were characterized. Inhibitors in feces are complex polysaccharides possibly originating from vegetable material in the diet. PMID:9157172

  10. 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

  11. 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 Central

    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

  12. Detection of EGFR Mutations by TaqMan Mutation Detection Assays Powered by Competitive Allele-Specific TaqMan PCR Technology

    PubMed Central

    Roma, Cristin; Esposito, Claudia; Rachiglio, Anna Maria; Pasquale, Raffaella; Chicchinelli, Nicoletta; Mancini, Rita; Pisconti, Salvatore; Botti, Gerardo; Morabito, Alessandro

    2013-01-01

    Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR) is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA) based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC) samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2); the maximum cycle threshold (Ct = 37); and the cut-off ΔCt value (7) for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples. PMID:24364033

  13. 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. PMID:22154270

  14. 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

  15. 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

  16. 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

  17. Development of an allele-specific PCR assay for simultaneous sero-typing of avian pathogenic Escherichia coli predominant O1, O2, O18 and O78 strains.

    PubMed

    Wang, Shaohui; Meng, Qingmei; Dai, Jianjun; Han, Xiangan; Han, Yue; Ding, Chan; Liu, Haiwen; Yu, Shengqing

    2014-01-01

    Systemic infections by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. E. coli strains belonging to serotypes O1, O2, O18 and O78 are preferentially associated with avian colibacillosis. The rfb gene cluster controlling O antigen synthesis is usually various among different E. coli serotypes. In present study, the rfb gene clusters of E. coli serotypes O1, O2, O18 and O78 were characterized and compared. Based on the serotype-specific genes in rfb gene cluster, an allele-specific polymerase chain reaction (PCR) assay was developed. This PCR assay was highly specific and reliable for sero-typing of APEC O1, O2, O18 and O78 strains. The sensitivity of the assay was determined as 10 pg DNA or 10 colony forming units (CFUs) bacteria for serotypes O2 and O18 strains, and 500 pg DNA or 1,000 CFUs bacteria for serotypes O1 and O78 strains. Using this PCR system, APEC isolates and the infected tissue samples were categorized successfully. Furthermore, it was able to differentiate the serotypes for the samples with multi-agglutination in the traditional serum agglutination assay. Therefore, the allele-specific PCR is more simple, rapid and accurate assay for APEC diagnosis, epidemiologic study and vaccine development. PMID:24805368

  18. Improved allele-specific PCR assays for detection of clarithromycin and fluoroquinolone resistant of Helicobacter pylori in gastric biopsies: identification of N87I mutation in GyrA.

    PubMed

    Trespalacios, Alba A; Rimbara, Emiko; Otero, William; Reddy, Rita; Graham, David Y

    2015-04-01

    Molecular testing can rapidly detect Helicobacter pylori susceptibility using gastric biopsies. Allele-specific polymerase chain reaction (ASP-PCR) was used to identify H. pylori 23S rRNA and gyrA mutation using gastric biopsies from Colombian patients and confirmed by PCR and sequencing of the 23S rRNA and gyrA genes. The sensitivity and specificity of ASP-PCR were compared with susceptibilities measured by agar dilution. Samples included gastric biopsies from 107 biopsies with H. pylori infections and 20 H. pylori negative. The sensitivity and specificity of ASP-PCR for the 23S rRNA gene were both 100%. The sensitivity and specificity of ASP-PCR for the gyrA gene, published in 2007 by Nishizawa et al., were 52% and 92.7%, respectively; the lower sensitivity was due to the presence of mutation N87I in our samples, which were not detected by the test. In this study, we designed new primers to detect the mutation N87I in GyrA. The ASP-PCR was performed with the original primers plus the new primers. The molecular test with the new primers improved the sensitivity to 100%. In conclusion, ASP-PCR provides a specific and rapid means of predicting resistance to clarithromycin and levofloxacin in gastric biopsies. PMID:25600075

  19. 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

  20. 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

  1. 454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples

    PubMed Central

    Altimari, Annalisa; de Biase, Dario; De Maglio, Giovanna; Gruppioni, Elisa; Capizzi, Elisa; Degiovanni, Alessio; D’Errico, Antonia; Pession, Annalisa; Pizzolitto, Stefano; Fiorentino, Michelangelo; Tallini, Giovanni

    2013-01-01

    Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues. PMID

  2. Hybrid Capture 2 is as Effective as PCR Testing for High Risk Human Papillomavirus in Head and Neck Cancers

    PubMed Central

    Hooper, Jody E.; Hebert, Jessica F.; Schilling, Amy; Gross, Neil D.; Schindler, Joshua S.; Lagowski, James P.; Kulesz-Martin, Molly; Corless, Christopher L.; Morgan, Terry K.

    2014-01-01

    High risk human papillomavirus (HPV) infection is a common cause of oropharyngeal squamous cell carcinoma, especially in young male nonsmokers. Accurately diagnosing HPV-associated oral cancers is important, because they have a better prognosis and may be treated differently than smoking-related oral carcinomas. Various methods have been validated to test for high risk HPV in cervical tissue samples and they are in routine clinical use to detect dysplasia before it progresses to invasive disease. Similarly, future screening for HPV-mediated oropharyngeal dysplasia may identify patients before it progresses. Our objective was to compare four of these methods in a retrospective series of 87 oral and oropharyngeal squamous cell carcinomas that had archived fresh-frozen and paraffin-embedded tissue for evaluation. Patient age, gender, smoking history, and tumor location were also recorded. DNA prepared from fresh-frozen tissue was tested for HPV genotypes by multiplex PCR analysis (Diatherix), and high risk HPV screening was done with Hybrid Capture 2 (Qiagen hc2) and Cervista (Hologic). Histologic sections were immunostained for p16 (mtm/Roche). HPV positive outcome was defined as agreement between at least two of the three genetic tests and used for X2 analysis and calculations of diagnostic predictive value. As expected, high risk HPV-positive oral cancers were most common in the tonsil and base of tongue (oropharynx) of younger male (55 years vs 65 years) (p=0.0002) non-smokers (p=0.01). Most positive cases were HPV16 (33/36, 92%). Hybrid Capture 2 and Cervista were as sensitive as PCR and had fewer false positives than p16 immunohistochemistry. PMID:25839700

  3. Allele-specific expression assays using Solexa

    PubMed Central

    Main, Bradley J; Bickel, Ryan D; McIntyre, Lauren M; Graze, Rita M; Calabrese, Peter P; Nuzhdin, Sergey V

    2009-01-01

    Background Allele-specific expression (ASE) assays can be used to identify cis, trans, and cis-by-trans regulatory variation. Understanding the source of expression variation has important implications for disease susceptibility, phenotypic diversity, and adaptation. While ASE is commonly measured via relative fluorescence at a SNP, next generation sequencing provides an opportunity to measure ASE in an accurate and high-throughput manner using read counts. Results We introduce a Solexa-based method to perform large numbers of ASE assays using only a single lane of a Solexa flowcell. In brief, transcripts of interest, which contain a known SNP, are PCR enriched and barcoded to enable multiplexing. Then high-throughput sequencing is used to estimate allele-specific expression using sequencing counts. To validate this method, we measured the allelic bias in a dilution series and found high correlations between measured and expected values (r>0.9, p < 0.001). We applied this method to a set of 5 genes in a Drosophila simulans parental mix, F1 and introgression and found that for these genes the majority of expression divergence can be explained by cis-regulatory variation. Conclusion We present a new method with the capacity to measure ASE for large numbers of assays using as little as one lane of a Solexa flowcell. This will be a valuable technique for molecular and population genetic studies, as well as for verification of genome-wide data sets. PMID:19740431

  4. Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission

    PubMed Central

    Hauser, Andrea; Kuecherer, Claudia; Kunz, Andrea; Dabrowski, Piotr Wojtek; Radonić, Aleksandar; Nitsche, Andreas; Theuring, Stefanie; Bannert, Norbert; Sewangi, Julius; Mbezi, Paulina; Dugange, Festo; Harms, Gundel; Meixenberger, Karolin

    2015-01-01

    Background Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT). Methods Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F). In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System. Results Drug-resistant HIV-variants were identified in 69% (20/29) of women by UDS and in 45% (13/29) by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24). By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41). The proportions of variants quantified by UDS were approximately 2–3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml), resulting in missing or insufficient sequence coverage. Conclusions Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in

  5. Detection of the BRAF V600E mutation in serous ovarian tumors: a comparative analysis of immunohistochemistry with a mutation-specific monoclonal antibody and allele-specific PCR.

    PubMed

    Bösmüller, Hans; Fischer, Anna; Pham, Deborah L; Fehm, Tanja; Capper, David; von Deimling, Andreas; Bonzheim, Irina; Staebler, Annette; Fend, Falko

    2013-03-01

    Mutations of components of the mitogen-activated protein kinase pathway, mainly BRAF, are common in serous ovarian borderline tumors, whereas high-grade serous ovarian carcinomas rarely show this feature. With the advent of specific kinase inhibitors active against BRAF-mutated cancers, rapid and sensitive detection of the BRAF V600E, by far the most common mutation of this gene, is of great practical relevance. Currently, BRAF mutations are detected by DNA-based techniques. Recently, a monoclonal antibody (VE1) specific for the BRAF V600E protein suitable for archival tissues has been described. In this study, we compared detection of the V600E mutation in serous ovarian tumors by VE1 immunostaining and by allele-specific polymerase chain reaction. All 141 cases of high-grade serous ovarian cancer showed negative or rarely weak, diffuse background VE1 immunostaining, and BRAF wild type was confirmed by molecular analysis in all tested cases. In contrast, 1 (14%) of 7 low-grade serous carcinomas and 22 (71%) of 31 serous borderline tumors revealed moderate to strong VE1 positivity. Immunostaining was clearly evaluable in all cases with sufficient tumor cells, and only rare cases with narrow cytoplasm were difficult to interpret. The V600E mutation was confirmed by allele-specific polymerase chain reaction and sequencing in all VE1-positive cases. Two VE1-positive cases with low epithelial cell content required repeat microdissection to confirm the presence of the mutation. Immunohistochemistry with the VE1 antibody is a specific and sensitive tool for detection of the BRAF V600E mutation in serous ovarian tumors and may provide a practical screening test, especially in tumor samples with low epithelial content. PMID:23089489

  6. Allele Specific p53 Mutant Reactivation

    PubMed Central

    Yu, Xin; Vazquez, Alexei; Levine, Arnold J.; Carpizo, Darren R.

    2012-01-01

    Summary Rescuing the function of mutant p53 protein is an attractive cancer therapeutic strategy. Using the NCI anticancer drug screen data, we identified two compounds from the thiosemicarbazone family that manifest increased growth inhibitory activity in mutant p53 cells, particularly for the p53R175 mutant. Mechanistic studies reveal that NSC319726 restores WT structure and function to the p53R175 mutant. This compound kills p53R172H knock-in mice with extensive apoptosis and inhibits xenograft tumor growth in a 175-allele specific mutant p53 dependent manner. This activity depends upon the zinc ion chelating properties of the compound as well as redox changes. These data identify NSC319726 as a p53R175 mutant reactivator and as a lead compound for p53 targeted drug development. PMID:22624712

  7. Probe-free allele-specific copy number detection and analysis of tumors.

    PubMed

    Zhu, Ailin; Guan, Xiaowei; Gu, Xinbin; Xie, Guiqin

    2016-03-15

    Cancer development and progression frequently involve nucleotide mutations as well as amplifications and deletions of genomic segments. Quantification of allele-specific copy number is an important step in characterizing tumor genomes for precision medicine. Despite advances in approaches to high-throughput genomic DNA analysis, inexpensive and simple methods for analyzing complex nucleotide and copy number variants are still needed. Real-time polymerase chain reaction (PCR) methods for discovering and genotyping single nucleotide polymorphisms are becoming increasingly important in genetic analysis. In this study, we describe a simple, single-tube, probe-free method that combines SYBR Green I-based quantitative real-time PCR and quantitative melting curve analysis both to detect specific nucleotide variants and to quantify allele-specific copy number variants of tumors. The approach is based on the quantification of the targets of interest and the relative abundance of two alleles in a single tube. The specificity, sensitivity, and utility of the assay were demonstrated in detecting allele-specific copy number changes critical for carcinogenesis and therapeutic intervention. Our approach would be useful for allele-specific copy number analysis or precise genotyping. PMID:26743720

  8. 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

  9. Multiplex Allele-Specific Amplification from Whole Blood for Detecting Multiple Polymorphisms Simultaneously

    PubMed Central

    Zhu, Jianjie; Chen, Lanxin; Mao, Yong; Zhou, Huan

    2013-01-01

    Allele-specific amplification on the basis of polymerase chain reaction (PCR) has been widely used for single-nucleotide polymorphism (SNP) genotyping. However, the extraction of PCR-compatible genomic DNA from whole blood is usually required. This process is complicated and tedious, and is prone to cause cross-contamination between samples. To facilitate direct PCR amplification from whole blood without the extraction of genomic DNA, we optimized the pH value of PCR solution and the concentrations of magnesium ions and facilitator glycerol. Then, we developed multiplex allele-specific amplifications from whole blood and applied them to a case–control study. In this study, we successfully established triplex, five-plex, and eight-plex allele-specific amplifications from whole blood for determining the distribution of genotypes and alleles of 14 polymorphisms in 97 gastric cancer patients and 141 healthy controls. Statistical analysis results showed significant association of SNPs rs9344, rs1799931, and rs1800629 with the risk of gastric cancer. This method is accurate, time-saving, cost-effective, and easy-to-do, especially suitable for clinical prediction of disease susceptibility. PMID:23072573

  10. 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.

  11. Allele-Specific Amplification in Cancer Revealed by SNP Array Analysis

    PubMed Central

    2005-01-01

    Amplification, deletion, and loss of heterozygosity of genomic DNA are hallmarks of cancer. In recent years a variety of studies have emerged measuring total chromosomal copy number at increasingly high resolution. Similarly, loss-of-heterozygosity events have been finely mapped using high-throughput genotyping technologies. We have developed a probe-level allele-specific quantitation procedure that extracts both copy number and allelotype information from single nucleotide polymorphism (SNP) array data to arrive at allele-specific copy number across the genome. Our approach applies an expectation-maximization algorithm to a model derived from a novel classification of SNP array probes. This method is the first to our knowledge that is able to (a) determine the generalized genotype of aberrant samples at each SNP site (e.g., CCCCT at an amplified site), and (b) infer the copy number of each parental chromosome across the genome. With this method, we are able to determine not just where amplifications and deletions occur, but also the haplotype of the region being amplified or deleted. The merit of our model and general approach is demonstrated by very precise genotyping of normal samples, and our allele-specific copy number inferences are validated using PCR experiments. Applying our method to a collection of lung cancer samples, we are able to conclude that amplification is essentially monoallelic, as would be expected under the mechanisms currently believed responsible for gene amplification. This suggests that a specific parental chromosome may be targeted for amplification, whether because of germ line or somatic variation. An R software package containing the methods described in this paper is freely available at http://genome.dfci.harvard.edu/~tlaframb/PLASQ. PMID:16322765

  12. MYD88 L265P in Waldenström macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative allele-specific polymerase chain reaction

    PubMed Central

    Xu, Lian; Hunter, Zachary R.; Yang, Guang; Zhou, Yangsheng; Cao, Yang; Liu, Xia; Morra, Enrica; Trojani, Alessandra; Greco, Antonino; Arcaini, Luca; Varettoni, Maria; Brown, Jennifer R.; Tai, Yu-Tzu; Anderson, Kenneth C.; Munshi, Nikhil C.; Patterson, Christopher J.; Manning, Robert J.; Tripsas, Christina K.; Lindeman, Neal I.

    2013-01-01

    By whole-genome and/or Sanger sequencing, we recently identified a somatic mutation (MYD88 L265P) that stimulates nuclear factor κB activity and is present in >90% of Waldenström macroglobulinemia (WM) patients. MYD88 L265P was absent in 90% of immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS) patients. We therefore developed conventional and real-time allele-specific polymerase chain reaction (AS-PCR) assays for more sensitive detection and quantification of MYD88 L265P. Using either assay, MYD88 L265P was detected in 97 of 104 (93%) WM and 13 of 24 (54%) IgM MGUS patients and was either absent or rarely expressed in samples from splenic marginal zone lymphoma (2/20; 10%), CLL (1/26; 4%), multiple myeloma (including IgM cases, 0/14), and immunoglobulin G MGUS (0/9) patients as well as healthy donors (0/40; P < 1.5 × 10−5 for WM vs other cohorts). Real-time AS-PCR identified IgM MGUS patients progressing to WM and showed a high rate of concordance between MYD88 L265P ΔCT and BM disease involvement (r = 0.89, P = .008) in WM patients undergoing treatment. These studies identify MYD88 L265P as a widely present mutation in WM and IgM MGUS patients using highly sensitive and specific AS-PCR assays with potential use in diagnostic discrimination and/or response assessment. The finding of this mutation in many IgM MGUS patients suggests that MYD88 L265P may be an early oncogenic event in WM pathogenesis. PMID:23321251

  13. Allele-specific tumor spectrum in pten knockin mice.

    PubMed

    Wang, Hui; Karikomi, Matt; Naidu, Shan; Rajmohan, Ravi; Caserta, Enrico; Chen, Hui-Zi; Rawahneh, Maysoon; Moffitt, Julie; Stephens, Julie A; Fernandez, Soledad A; Weinstein, Michael; Wang, Danxin; Sadee, Wolfgang; La Perle, Krista; Stromberg, Paul; Rosol, Thomas J; Eng, Charis; Ostrowski, Michael C; Leone, Gustavo

    2010-03-16

    Germline mutations in the tumor suppressor gene PTEN (phosphatase and tensin homology deleted on chromosome 10) cause Cowden and Bannayan-Riley-Ruvalcaba (BRR) syndromes, two dominantly inherited disorders characterized by mental retardation, multiple hamartomas, and variable cancer risk. Here, we modeled three sentinel mutant alleles of PTEN identified in patients with Cowden syndrome and show that the nonsense Pten(4-5) and missense Pten(C124R) and Pten(G129E) alleles lacking lipid phosphatase activity cause similar developmental abnormalities but distinct tumor spectra with varying severity and age of onset. Allele-specific differences may be accounted for by loss of function for Pten(4-5), hypomorphic function for Pten(C124R), and gain of function for Pten(G129E). These data demonstrate that the variable tumor phenotypes observed in patients with Cowden and BRR syndromes can be attributed to specific mutations in PTEN that alter protein function through distinct mechanisms. PMID:20194734

  14. 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

  15. Comparative Anatomy of Chromosomal Domains with Imprinted and Non-Imprinted Allele-Specific DNA Methylation

    PubMed Central

    Kerkel, Kristi; Yale, Alexander; Yotova, Iveta; Drost, Natalia; Lax, Simon; Nhan-Chang, Chia-Ling; Powell, Charles; Borczuk, Alain; Aviv, Abraham; Wapner, Ronald; Chen, Xiaowei; Nagy, Peter L.; Schork, Nicholas; Do, Catherine; Torkamani, Ali; Tycko, Benjamin

    2013-01-01

    Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM. PMID:24009515

  16. Comparative anatomy of chromosomal domains with imprinted and non-imprinted allele-specific DNA methylation.

    PubMed

    Paliwal, Anupam; Temkin, Alexis M; Kerkel, Kristi; Yale, Alexander; Yotova, Iveta; Drost, Natalia; Lax, Simon; Nhan-Chang, Chia-Ling; Powell, Charles; Borczuk, Alain; Aviv, Abraham; Wapner, Ronald; Chen, Xiaowei; Nagy, Peter L; Schork, Nicholas; Do, Catherine; Torkamani, Ali; Tycko, Benjamin

    2013-08-01

    Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM. PMID:24009515

  17. Allelic diversity of a beer haze active protein gene in cultivated and Tibetan wild barley and development of allelic specific markers.

    PubMed

    Ye, Lingzhen; Dai, Fei; Qiu, Long; Sun, Dongfa; Zhang, Guoping

    2011-07-13

    The formation of haze is a serious quality problem in beer production. It has been shown that the use of silica elute (SE)-ve malt (absence of molecular weight (MW) ∼14000 Da) for brewing can improve haze stability in the resultant beer, and the protein was identified as a barley trypsin inhibitor of the chloroform/methanol type (BTI-CMe). The objectives of this study were to determine (1) the allelic diversity of the gene controlling BTI-CMe in cultivated and Tibetan wild barley and (2) allele-specific (AS) markers for screening SE protein type. A survey of 172 Tibetan annual wild barley accessions and 71 cultivated barley genotypes was conducted, and 104 wild accessions and 35 cultivated genotypes were identified as SE+ve and 68 wild accessions and 36 cultivated genotypes as SE-ve. The allelic diversity of the gene controlling BTI-CMe was investigated by cloning, alignment, and association analysis. It was found that there were significant differences between the SE+ve and SE-ve types in single-nucleotide polymorphisms at 234 (SNP(234)), SNP(313), and SNP(385.) Furthermore, two sets of AS markers were developed to screen SE protein type based on SNP(313). AS-PCR had results very similar to those obtained by immunoblot method. Mapping analysis showed that the gene controlling the MW∼14 kDa band was located on the short arm of chromosome 3H, at the position of marker BPB-0527 (33.302 cM) in the Franklin/Yerong DH population. PMID:21608526

  18. 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.

  19. 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

  20. Allele-Specific Gene Expression Is Widespread Across the Genome and Biological Processes

    PubMed Central

    Goñi, Joaquín; Piedrafita, Gabriel; Fernando, Olga; Navarro, Arcadi; Villoslada, Pablo

    2009-01-01

    Allelic specific gene expression (ASGE) appears to be an important factor in human phenotypic variability and as a consequence, for the development of complex traits and diseases. In order to study ASGE across the human genome, we have performed a study in which genotyping was coupled with an analysis of ASGE by screening 11,500 SNPs using the Mapping 10 K Array to identify differential allelic expression. We found that from the 5,133 SNPs that were suitable for analysis (heterozygous in our sample and expressed in peripheral blood mononuclear cells), 2,934 (57%) SNPs had differential allelic expression. Such SNPs were equally distributed along human chromosomes and biological processes. We validated the presence or absence of ASGE in 18 out 20 SNPs (90%) randomly selected by real time PCR in 48 human subjects. In addition, we observed that SNPs close to -but not included in- segmental duplications had increased levels of ASGE. Finally, we found that transcripts of unknown function or non-coding RNAs, also display ASGE: from a total of 2,308 intronic SNPs, 1510 (65%) SNPs underwent differential allelic expression. In summary, ASGE is a widespread mechanism in the human genome whose regulation seems to be far more complex than expected. PMID:19127300

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

    PubMed Central

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

    2015-01-01

    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

  2. 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

  3. Known unknowns for allele-specific expression and genomic imprinting effects

    PubMed Central

    2014-01-01

    Recent studies have provided evidence for non-canonical imprinting effects that are associated with allele-specific expression biases at the tissue level in mice. These imprinting effects have features that are distinct from canonical imprinting effects that involve allele silencing. Here, I discuss some of the evidence for non-canonical imprinting effects in the context of random X-inactivation and epigenetic allele-specific expression effects on the autosomes. I propose several mechanisms that may underlie non-canonical imprinting effects and outline future directions and approaches to study these effects at the cellular level in vivo. The growing evidence for complex allele-specific expression effects that are cell- and developmental stage-specific has opened a new frontier for study. Currently, the function of these effects and the underlying regulatory mechanisms are largely unknown. PMID:25343032

  4. Allele-specific polymerase chain reaction for the detection of Alzheimer’s disease-related single nucleotide polymorphisms

    PubMed Central

    2013-01-01

    Background The incidence of Alzheimer’s disease, particularly in developing countries, is expected to increase exponentially as the population ages. Continuing research in this area is essential in order to better understand this disease and develop strategies for treatment and prevention. Genome-wide association studies have identified several loci as genetic risk factors of AD aside from apolipoprotein E such as bridging integrator (BIN1), clusterin (CLU), ATP-binding cassette sub-family A member 7 (ABCA7), complement receptor 1 (CR1) and phosphatidylinositol binding clathrin assembly protein (PICALM). However genetic research in developing countries is often limited by lack of funding and expertise. This study therefore developed and validated a simple, cost effective polymerase chain reaction based technique to determine these single nucleotide polymorphisms. Methods An allele-specific PCR method was developed to detect single nucleotide polymorphisms of BIN1 rs744373, CLU rs11136000, ABCA7 rs3764650, CR1 rs3818361 and PICALM rs3851179 in human DNA samples. Allele-specific primers were designed by using appropriate software to permit the PCR amplification only if the nucleotide at the 3’-end of the primer complemented the base at the wild-type or variant-type DNA sample. The primers were then searched for uniqueness using the Basic Local Alignment Search Tool search engine. Results The assay was tested on a hundred samples and accurately detected the homozygous wild-type, homozygous variant-type and heterozygous of each SNP. Validation was by direct DNA sequencing. Conclusion This method will enable researchers to carry out genetic polymorphism studies for genetic risk factors associated with late-onset Alzheimer’s disease (BIN1, CLU, ABCA7, CR1 and PICALM) without the use of expensive instrumentation and reagents. PMID:23419238

  5. 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

  6. 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

  7. Allele-specific copy number profiling by next-generation DNA sequencing.

    PubMed

    Chen, Hao; Bell, John M; Zavala, Nicolas A; Ji, Hanlee P; Zhang, Nancy R

    2015-02-27

    The progression and clonal development of tumors often involve amplifications and deletions of genomic DNA. Estimation of allele-specific copy number, which quantifies the number of copies of each allele at each variant loci rather than the total number of chromosome copies, is an important step in the characterization of tumor genomes and the inference of their clonal history. We describe a new method, falcon, for finding somatic allele-specific copy number changes by next generation sequencing of tumors with matched normals. falcon is based on a change-point model on a bivariate mixed Binomial process, which explicitly models the copy numbers of the two chromosome haplotypes and corrects for local allele-specific coverage biases. By using the Binomial distribution rather than a normal approximation, falcon more effectively pools evidence from sites with low coverage. A modified Bayesian information criterion is used to guide model selection for determining the number of copy number events. Falcon is evaluated on in silico spike-in data and applied to the analysis of a pre-malignant colon tumor sample and late-stage colorectal adenocarcinoma from the same individual. The allele-specific copy number estimates obtained by falcon allows us to draw detailed conclusions regarding the clonal history of the individual's colon cancer. PMID:25477383

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

    2016-01-01

    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

  13. 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

  14. 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.

  15. 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

  16. Correction of hair shaft defects through allele-specific silencing of mutant Krt75

    PubMed Central

    Liu, Ying; Snedecor, Elizabeth R.; Zhang, Xu; Xu, Yan-Feng; Huang, Lan; Jones, Evan; Zhang, Lianfeng; Clark, Richard A.; Roop, Dennis R.; Qin, Chuan; Chen, Jiang

    2015-01-01

    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 siRNAs 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 shRNA persistently suppressed this phenotype. The phenotypic correction was associated with 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 the mutant keratin genes. PMID:26763422

  17. 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

  18. 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

  19. 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. PMID:24667781

  20. Allelic Specificity of Ube3a Expression in the Mouse Brain during Postnatal Development

    PubMed Central

    JUDSON, MATTHEW C.; SOSA-PAGAN, JASON O.; DEL CID, WILMER A.; HAN, JI EUN; PHILPOT, BENJAMIN D.

    2014-01-01

    Genetic alterations of the maternal UBE3A allele result in Angelman syndrome (AS), a neurodevelopmental disorder characterized by severe developmental delay, lack of speech, and difficulty with movement and balance. The combined effects of maternal UBE3A mutation and cell type-specific epigenetic silencing of paternal UBE3A are hypothesized to result in a complete loss of functional UBE3A protein in neurons. However, the allelic specificity of UBE3A expression in neurons and other cell types in the brain has yet to be characterized throughout development, including the early postnatal period when AS phenotypes emerge. Here we define maternal and paternal allele-specific Ube3a protein expression throughout postnatal brain development in the mouse, a species which exhibits orthologous epigenetic silencing of paternal Ube3a in neurons and AS-like behavioral phenotypes subsequent to maternal Ube3a deletion. We find that neurons downregulate paternal Ube3a protein expression as they mature and, with the exception of neurons born from postnatal stem cell niches, do not express detectable paternal Ube3a beyond the first postnatal week. By contrast, neurons express maternal Ube3a throughout postnatal development, during which time localization of the protein becomes increasingly nuclear. Unlike neurons, astrocytes and oligodendrotyes biallelically express Ube3a. Notably, mature oligodendrocytes emerge as the predominant Ube3a-expressing glial cell type in the cortex and white matter tracts during postnatal development. These findings demonstrate the spatiotemporal characteristics of allele-specific Ube3a expression in key brain cell types, thereby improving our understanding of the developmental parameters of paternal Ube3a silencing and the cellular basis of AS. PMID:24254964

  1. Rapid Origin Determination of the Northern Mauxia Shrimp (Acetes chinensis) Based on Allele Specific Polymerase Chain Reaction of Partial Mitochondrial 16S rRNA Gene

    PubMed Central

    Kang, Jung-Ha; Noh, Eun-Soo; Park, Jung-Youn; An, Chel-Min; Choi, Jung-Hwa; Kim, Jin-Koo

    2015-01-01

    Acetes chinensis is an economically important shrimp that belongs to the Sergestidae family; following fermentation, A. chinensis′ economic value, however, is low in China, and much of the catch in China is exported to Korea at a low price, thus leading to potential false labeling. For this reason, we developed a simple method to identify A. chinensis′ origin using allele-specific polymerase chain reaction (PCR). Ten single nucleotide polymorphisms (SNPs) were identified from partial (i.e., 570 bp) DNA sequence analysis of the mitochondrial 16s rRNA gene in 96 Korean and 96 Chinese individual shrimp. Among 10 SNP sites, four sites were observed in populations from both countries, and two sites located in the middle with SNP sites at their 3′-ends were used to design allele-specific primers. Among the eight internal primers, the C220F primer specific to the Chinese A. chinensis population amplified a DNA fragment of 364 bp only from that population. We were able to identify the A. chinensis population origin with 100% accuracy using multiplex PCR performed with two external primers and C220F primers. These results show that the 16S rRNA gene that is generally used for the identification of species can be used for the identification of the origin within species of A. chinensis, which is an important finding for the fair trade of the species between Korea and China. PMID:25656197

  2. Rapid Origin Determination of the Northern Mauxia Shrimp (Acetes chinensis) Based on Allele Specific Polymerase Chain Reaction of Partial Mitochondrial 16S rRNA Gene.

    PubMed

    Kang, Jung-Ha; Noh, Eun-Soo; Park, Jung-Youn; An, Chel-Min; Choi, Jung-Hwa; Kim, Jin-Koo

    2015-04-01

    Acetes chinensis is an economically important shrimp that belongs to the Sergestidae family; following fermentation, A. chinensis' economic value, however, is low in China, and much of the catch in China is exported to Korea at a low price, thus leading to potential false labeling. For this reason, we developed a simple method to identify A. chinensis' origin using allele-specific polymerase chain reaction (PCR). Ten single nucleotide polymorphisms (SNPs) were identified from partial (i.e., 570 bp) DNA sequence analysis of the mitochondrial 16s rRNA gene in 96 Korean and 96 Chinese individual shrimp. Among 10 SNP sites, four sites were observed in populations from both countries, and two sites located in the middle with SNP sites at their 3'-ends were used to design allele-specific primers. Among the eight internal primers, the C220F primer specific to the Chinese A. chinensis population amplified a DNA fragment of 364 bp only from that population. We were able to identify the A. chinensis population origin with 100% accuracy using multiplex PCR performed with two external primers and C220F primers. These results show that the 16S rRNA gene that is generally used for the identification of species can be used for the identification of the origin within species of A. chinensis, which is an important finding for the fair trade of the species between Korea and China. PMID:25656197

  3. 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

  4. A rapid and efficient strategy to generate allele-specific anti-HLA monoclonal antibodies.

    PubMed

    Yamazaki, Satoshi; Suzuki, Nao; Saito, Tsuneyoshi; Ishii, Yumiko; Takiguchi, Masafumi; Nakauchi, Hiromitsu; Watanabe, Nobukazu

    2009-03-31

    That generation of allele-specific anti-human leukocyte antigen (HLA) monoclonal antibodies (ASHmAb) is very difficult is well known. This is thought to be due to the unique epitope structure, an assemblage of amino acid residues that lie separately in the amino acid sequence of human HLA, and to its low antigenicity compared with that of common epitopes recognized as xenogeneic determinants by mice. Here we report a rapid and efficient strategy to generate ASHmAb. Different from usual immunization methods is that we suppressed the production of non-allele-specific anti-HLA antibodies against xenogeneic determinants of HLA molecules by immunizing human HLA-B51 transgenic mice against non-HLA-B51 HLA tetramers. In addition, HLA-coated beads enabled rapid and efficient screening for ASHmAb. ASHmAb generated by this strategy will be useful for HLA typing and for clinical diagnosis, such as flow cytometry-based chimerism analysis for early detection of graft failure and relapse of leukemia after HLA-mismatched hematopoietic stem cell transplantation. PMID:19187783

  5. Hypervariable Domains of Self-Incompatibility RNases Mediate Allele-Specific Pollen Recognition.

    PubMed Central

    Matton, D. P.; Maes, O.; Laublin, G.; Xike, Q.; Bertrand, C.; Morse, D.; Cappadocia, M.

    1997-01-01

    Self-incompatibility (SI) in angiosperms is a genetic mechanism that promotes outcrossing through rejection of self-pollen. In the Solanaceae, SI is determined by a multiallelic S locus whose only known product is an S RNase. S RNases show a characteristic pattern of five conserved and two hypervariable regions. These are thought to be involved in the catalytic function and in allelic specificity, respectively. When the Solanum chacoense S12S14 genotype is transformed with an S11 RNase, the styles of plants expressing significant levels of the transgene reject S11 pollen. A previously characterized S RNase, S13, differs from the S11 RNase by only 10 amino acids, four of which are located in the hypervariable regions. When S12S14 plants were transformed with a chimeric S11 gene in which these four residues were substituted with those present in the S13 RNase, the transgenic plants acquired the S13 phenotype. This result demonstrates that the S RNase hypervariable regions control allelic specificity. PMID:12237346

  6. 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

  7. 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

  8. 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

  9. Molecular characterization and a multiplex allele-specific PCR method for detection of thiabendazole resistance in Penicillium expansum from apple

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thiabendazole (TBZ) is commonly used as a postharvest treatment for control of blue mold in apples caused by Penicillium expansum. Different point mutations in the ß-tubulin gene conferring benzimidazole resistance have been reported in plant pathogens, but molecular mechanisms of TBZ resistance in ...

  10. Quantitative genotyping of single-nucleotide polymorphisms by allele-specific oligonucleotide hybridization on DNA microarrays.

    PubMed

    Rickert, Andreas M; Ballvora, Agim; Matzner, Ulrich; Klemm, Manfred; Gebhardt, Christiane

    2005-08-01

    Genotyping of SNPs (single-nucleotide polymorphisms) has challenged the development of several novel techniques. Most of these methods have been introduced to discriminate binary SNPs in diploid species. In the present study, the quantitative genotyping of SNPs in natural DNA pools of a polyploid organism via DNA microarrays was analysed. Three randomly selected SNP loci were genotyped in the tetraploid species potato (Solanum tuberosum). For each SNP, 24 oligomers were designed, 12 with forward and 12 with reverse orientation. They contained the polymorphic site at one of the positions 11, 14 and 17. Several steps of optimizations were performed, including the 'materials' used and the establishment of hybridization conditions. Glass surfaces were either epoxy- or aldehyde-modified, and allele-specific oligonucleotides contained either SH or NH2 groups. Hybridization stringency conditions were established by varying the concentration of formamide in the hybridization buffer. For SNP BA213c14t7/403, the quantitative discrimination between all four different naturally occurring genotypes could be demonstrated. PMID:15847606

  11. Allele-Specific Behavior of Molecular Networks: Understanding Small-Molecule Drug Response in Yeast

    PubMed Central

    Li, Chunquan; Hao, Dapeng; Zhang, Shaojun; Zhou, Meng; Su, Fei; Chen, Xi; Zhi, Hui; Li, Xia

    2013-01-01

    The study of systems genetics is changing the way the genetic and molecular basis of phenotypic variation, such as disease susceptibility and drug response, is being analyzed. Moreover, systems genetics aids in the translation of insights from systems biology into genetics. The use of systems genetics enables greater attention to be focused on the potential impact of genetic perturbations on the molecular states of networks that in turn affects complex traits. In this study, we developed models to detect allele-specific perturbations on interactions, in which a genetic locus with alternative alleles exerted a differing influence on an interaction. We utilized the models to investigate the dynamic behavior of an integrated molecular network undergoing genetic perturbations in yeast. Our results revealed the complexity of regulatory relationships between genetic loci and networks, in which different genetic loci perturb specific network modules. In addition, significant within-module functional coherence was found. We then used the network perturbation model to elucidate the underlying molecular mechanisms of individual differences in response to 100 diverse small molecule drugs. As a result, we identified sub-networks in the integrated network that responded to variations in DNA associated with response to diverse compounds and were significantly enriched for known drug targets. Literature mining results provided strong independent evidence for the effectiveness of these genetic perturbing networks in the elucidation of small-molecule responses in yeast. PMID:23308257

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

    PubMed

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

    2015-01-01

    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. PMID:25987481

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

    PubMed Central

    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

    2014-01-01

    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

  14. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25987481

  16. Germline Allele-Specific Expression of DAPK1 in Chronic Lymphocytic Leukemia

    PubMed Central

    Hielscher, Thomas; Mertens, Daniel; Raval, Aparna; Oakes, Christopher C.; Tanner, Stephan M.; de la Chapelle, Albert; Byrd, John C.; Stilgenbauer, Stephan; Plass, Christoph

    2013-01-01

    We previously reported a rare germline variant (c.1-6531) that resulted in allele–specific expression (ASE) of death-associated protein kinase 1 (DAPK1) and predisposition to chronic lymphocytic leukemia (CLL). We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE) with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2%) CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5′ upstream regulatory region, within distinct exons or in the 3′-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL. PMID:23383130

  17. Dynamic variation in allele-specific gene expression of Paraoxonase-1 in murine and human tissues

    PubMed Central

    Parker-Katiraee, Layla; Bousiaki, Eleni; Monk, David; Moore, Gudrun E.; Nakabayashi, Kazuhiko; Scherer, Stephen W.

    2008-01-01

    Differential allelic expression has been shown to be common in mice, humans and maize, and variability in the expression of polymorphic alleles has been associated with human disease. Here, we describe the differential expression pattern of Paraoxonase-1, a gene involved in lipid metabolism and implicated in the formation of atherosclerotic lesions. We measured the expression of the murine Paraoxonase-1 gene (Pon1) in livers at different stages of embryonic development using F1 hybrid crosses and quantified the transcriptional level of both parental alleles. Using human foetal tissues, we analysed the expression of the human orthologue (PON1) and found monoallelic or preferential allelic expression in 6/7 and 4/4 samples from liver and pancreas, respectively. We observed that Pon1 does not show a parent-of-origin preference in its allelic expression, but has dramatic variations in allele-specific expression occurring throughout development. This study has important repercussions in the analysis of haplotypes at disease loci, since it implies that the expression of polymorphic alleles can be unequal and dynamic. PMID:18678600

  18. Allele-Specific Network Reveals Combinatorial Interaction That Transcends Small Effects in Psoriasis GWAS

    PubMed Central

    Climer, Sharlee; Templeton, Alan R.; Zhang, Weixiong

    2014-01-01

    Hundreds of genetic markers have shown associations with various complex diseases, yet the “missing heritability” remains alarmingly elusive. Combinatorial interactions may account for a substantial portion of this missing heritability, but their discoveries have been impeded by computational complexity and genetic heterogeneity. We present BlocBuster, a novel systems-level approach that efficiently constructs genome-wide, allele-specific networks that accurately segregate homogenous combinations of genetic factors, tests the associations of these combinations with the given phenotype, and rigorously validates the results using a series of unbiased validation methods. BlocBuster employs a correlation measure that is customized for single nucleotide polymorphisms and returns a multi-faceted collection of values that captures genetic heterogeneity. We applied BlocBuster to analyze psoriasis, discovering a combinatorial pattern with an odds ratio of 3.64 and Bonferroni-corrected p-value of 5.01×10−16. This pattern was replicated in independent data, reflecting robustness of the method. In addition to improving prediction of disease susceptibility and broadening our understanding of the pathogenesis underlying psoriasis, these results demonstrate BlocBuster's potential for discovering combinatorial genetic associations within heterogeneous genome-wide data, thereby transcending the limiting “small effects” produced by individual markers examined in isolation. PMID:25233071

  19. 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

  20. 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

  1. 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

  2. Allele-specific gene expression in a wild nonhuman primate population

    PubMed Central

    Tung, J.; Akinyi, M. Y.; Mutura, S.; Altmann, J.; Wray, G. A.; Alberts, S. C.

    2015-01-01

    Natural populations hold enormous potential for evolutionary genetic studies, especially when phenotypic, genetic and environmental data are all available on the same individuals. However, untangling the genotype-phenotype relationship in natural populations remains a major challenge. Here, we describe results of an investigation of one class of phenotype, allele-specific gene expression (ASGE), in the well-studied natural population of baboons of the Amboseli basin, Kenya. ASGE measurements identify cases in which one allele of a gene is overexpressed relative to the alternative allele of the same gene, within individuals, thus providing a control for background genetic and environmental effects. Here, we characterize the incidence of ASGE in the Amboseli baboon population, focusing on the genetic and environmental contributions to ASGE in a set of eleven genes involved in immunity and defence. Within this set, we identify evidence for common ASGE in four genes. We also present examples of two relationships between cis-regulatory genetic variants and the ASGE phenotype. Finally, we identify one case in which this relationship is influenced by a novel gene-environment interaction. Specifically, the dominance rank of an individual’s mother during its early life (an aspect of that individual’s social environment) influences the expression of the gene CCL5 via an interaction with cis-regulatory genetic variation. These results illustrate how environmental and ecological data can be integrated into evolutionary genetic studies of functional variation in natural populations. They also highlight the potential importance of early life environmental variation in shaping the genetic architecture of complex traits in wild mammals. PMID:21226779

  3. Molecular genetic mechanisms of allelic specific regulation of murine Comt expression.

    PubMed

    Segall, Samantha K; Shabalina, Svetlana A; Meloto, Carolina B; Wen, Xia; Cunningham, Danielle; Tarantino, Lisa M; Wiltshire, Tim; Gauthier, Josée; Tohyama, Sarasa; Martin, Loren J; Mogil, Jeffrey S; Diatchenko, Luda

    2015-10-01

    A functional allele of the mouse catechol-O-methyltransferase (Comt) gene is defined by the insertion of a B2 short interspersed repeat element in its 3'-untranslated region (UTR). This allele has been associated with a number of phenotypes, such as pain and anxiety. In comparison with mice carrying the ancestral allele (Comt+), Comt B2i mice show higher Comt mRNA and enzymatic activity levels. Here, we investigated the molecular genetic mechanisms underlying this allelic specific regulation of Comt expression. Insertion of the B2 element introduces an early polyadenylation signal generating a shorter Comt transcript, in addition to the longer ancestral mRNA. Comparative analysis and in silico prediction of Comt mRNA potential targets within the transcript 3' to the B2 element was performed and allowed choosing microRNA (miRNA) candidates for experimental screening: mmu-miR-3470a, mmu-miR-3470b, and mmu-miR-667. Cell transfection with each miRNA downregulated the expression of the ancestral transcript and COMT enzymatic activity. Our in vivo experiments showed that mmu-miR-667-3p is strongly correlated with decreasing amounts of Comt mRNA in the brain, and lentiviral injections of mmu-miR-3470a, mmu-miR-3470b, and mmu-miR-667 increase hypersensitivity in the mouse formalin model, consistent with reduced COMT activity. In summary, our data demonstrate that the Comt+ transcript contains regulatory miRNA signals in its 3'-untranslated region leading to mRNA degradation; these signals, however, are absent in the shorter transcript, resulting in higher mRNA expression and activity levels. PMID:26067582

  4. Mechanisms and Disease Associations of Haplotype-Dependent Allele-Specific DNA Methylation.

    PubMed

    Do, Catherine; Lang, Charles F; Lin, John; Darbary, Huferesh; Krupska, Izabela; Gaba, Aulona; Petukhova, Lynn; Vonsattel, Jean-Paul; Gallagher, Mary P; Goland, Robin S; Clynes, Raphael A; Dwork, Andrew; Kral, John G; Monk, Catherine; Christiano, Angela M; Tycko, Benjamin

    2016-05-01

    Haplotype-dependent allele-specific methylation (hap-ASM) can impact disease susceptibility, but maps of this phenomenon using stringent criteria in disease-relevant tissues remain sparse. Here we apply array-based and Methyl-Seq approaches to multiple human tissues and cell types, including brain, purified neurons and glia, T lymphocytes, and placenta, and identify 795 hap-ASM differentially methylated regions (DMRs) and 3,082 strong methylation quantitative trait loci (mQTLs), most not previously reported. More than half of these DMRs have cell type-restricted ASM, and among them are 188 hap-ASM DMRs and 933 mQTLs located near GWAS signals for immune and neurological disorders. Targeted bis-seq confirmed hap-ASM in 12/13 loci tested, including CCDC155, CD69, FRMD1, IRF1, KBTBD11, and S100A(∗)-ILF2, associated with immune phenotypes, MYT1L, PTPRN2, CMTM8 and CELF2, associated with neurological disorders, NGFR and HLA-DRB6, associated with both immunological and brain disorders, and ZFP57, a trans-acting regulator of genomic imprinting. Polymorphic CTCF and transcription factor (TF) binding sites were over-represented among hap-ASM DMRs and mQTLs, and analysis of the human data, supplemented by cross-species comparisons to macaques, indicated that CTCF and TF binding likelihood predicts the strength and direction of the allelic methylation asymmetry. These results show that hap-ASM is highly tissue specific; an important trans-acting regulator of genomic imprinting is regulated by this phenomenon; and variation in CTCF and TF binding sites is an underlying mechanism, and maps of hap-ASM and mQTLs reveal regulatory sequences underlying supra- and sub-threshold GWAS peaks in immunological and neurological disorders. PMID:27153397

  5. 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

  6. PAP-LMPCR for improved, allele-specific footprinting and automated chromatin fine structure analysis

    PubMed Central

    Ingram, R.; Gao, C.; LeBon, J.; Liu, Q.; Mayoral, R. J.; Sommer, S. S.; Hoogenkamp, M.; Riggs, A. D.; Bonifer, C.

    2008-01-01

    The analysis of chromatin fine structure and transcription factor occupancy of differentially expressed genes by in vivo footprinting and ligation-mediated-PCR (LMPCR) is a powerful tool to understand the impact of chromatin on gene expression. However, as with all PCR-based techniques, the accuracy of the experiments has often been reduced by sequence similarities and the presence of GC-rich or repeat sequences, and some sequences are completely refractory to analysis. Here we describe a novel method, pyrophosphorolysis activated polymerization LMPCR or PAP-LMPCR, which is capable of generating accurate and reproducible footprints specific for individual alleles and can read through sequences previously not accessible for analysis. In addition, we have adapted this technique for automation, thus enabling the simultaneous and rapid analysis of chromatin structure at many different genes. PMID:18208840

  7. [Rapid PCR authentication Lonicera japanica].

    PubMed

    Jiang, Chao; Hou, Jing-Yi; Huang, Lu-Qi; Yuan, Yuan; Chen, Min; Jin, Yan

    2014-10-01

    To simply and rapid authenticate Lonicera japanica. Rapid allele-specific PCR primer was designed base on trnL-trnF 625 G/T Single nucleotide polymorphism and the PCR reaction systems including annealing temperature was optimized; optimized results were performed to authenticate L. japanica and its 9 adulterants. When 100 x SYBR Green I was added in the PCR product of 87 degrees C initial denatured 1 min; 87 degrees C denatured 5 s, 68 degrees C annealing 5 s, 30 cycle; L. japanica visualize strong green fluorescence under 365 nm UV lamp whereas adulterants without. The results indicate rapid allele-specific PCR could authenticate L. japanica and its adulterants rapidly and simply. PMID:25612418

  8. 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

  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. Use of allele-specific FAIRE to determine functional regulatory polymorphism using large-scale genotyping arrays.

    PubMed

    Smith, Andrew J P; Howard, Philip; Shah, Sonia; Eriksson, Per; Stender, Stefan; Giambartolomei, Claudia; Folkersen, Lasse; Tybjærg-Hansen, Anne; Kumari, Meena; Palmen, Jutta; Hingorani, Aroon D; Talmud, Philippa J; Humphries, Steve E

    2012-01-01

    Following the widespread use of genome-wide association studies (GWAS), focus is turning towards identification of causal variants rather than simply genetic markers of diseases and traits. As a step towards a high-throughput method to identify genome-wide, non-coding, functional regulatory variants, we describe the technique of allele-specific FAIRE, utilising large-scale genotyping technology (FAIRE-gen) to determine allelic effects on chromatin accessibility and regulatory potential. FAIRE-gen was explored using lymphoblastoid cells and the 50,000 SNP Illumina CVD BeadChip. The technique identified an allele-specific regulatory polymorphism within NR1H3 (coding for LXR-α), rs7120118, coinciding with a previously GWAS-identified SNP for HDL-C levels. This finding was confirmed using FAIRE-gen with the 200,000 SNP Illumina Metabochip and verified with the established method of TaqMan allelic discrimination. Examination of this SNP in two prospective Caucasian cohorts comprising 15,000 individuals confirmed the association with HDL-C levels (combined beta = 0.016; p = 0.0006), and analysis of gene expression identified an allelic association with LXR-α expression in heart tissue. Using increasingly comprehensive genotyping chips and distinct tissues for examination, FAIRE-gen has the potential to aid the identification of many causal SNPs associated with disease from GWAS. PMID:22916038

  11. 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

  12. Overall and allele-specific expression of the SMC1A gene in female Cornelia de Lange syndrome patients and healthy controls

    PubMed Central

    Parenti, Ilaria; Rovina, Davide; Masciadri, Maura; Cereda, Anna; Azzollini, Jacopo; Picinelli, Chiara; Limongelli, Giuseppe; Finelli, Palma; Selicorni, Angelo; Russo, Silvia; Gervasini, Cristina; Larizza, Lidia

    2014-01-01

    Cornelia de Lange syndrome (CdLS) is a rare multisystem disorder characterized by facial dysmorphisms, limb anomalies, and growth and cognitive deficits. Mutations in genes encoding subunits (SMC1A, SMC3, RAD21) or regulators (NIPBL, HDAC8) of the cohesin complex account for approximately 65% of clinically diagnosed CdLS cases. The SMC1A gene (Xp11.22), responsible for 5% of CdLS cases, partially escapes X chromosome inactivation in humans and the allele on the inactive X chromosome is variably expressed. In this study, we evaluated overall and allele-specific SMC1A expression. Real-time PCR analysis conducted on 17 controls showed that SMC1A expression in females is 50% higher than in males. Immunoblotting experiments confirmed a 44% higher protein level in healthy females than in males, and showed no significant differences in SMC1A protein levels between controls and patients. Pyrosequencing was used to assess the reciprocal level of allelic expression in six female carriers of different SMC1A mutations and 15 controls who were heterozygous at a polymorphic transcribed SMC1A locus. The two alleles were expressed at a 1:1 ratio in the control group and at a 2:1 ratio in favor of the wild type allele in the test group. Since a dominant negative effect is considered the pathogenic mechanism in SMC1A-defective female patients, the level of allelic preferential expression might be one of the factors contributing to the wide phenotypic variability observed in these patients. An extension of this study to a larger cohort containing mild to borderline cases could enhance our understanding of the clinical spectrum of SMC1A-linked CdLS. PMID:24756084

  13. 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

  14. DNMT1 and AIM1 Imprinting in human placenta revealed through a genome-wide screen for allele-specific DNA methylation

    PubMed Central

    2013-01-01

    Background Genomic imprinting is an epigenetically regulated process wherein genes are expressed in a parent-of-origin specific manner. Many imprinted genes were initially identified in mice; some of these were subsequently shown not to be imprinted in humans. Such discrepancy reflects developmental, morphological and physiological differences between mouse and human tissues. This is particularly relevant for the placenta. Study of genomic imprinting thus needs to be carried out in a species and developmental stage-specific manner. We describe here a new strategy to study allele-specific DNA methylation in the human placenta for the discovery of novel imprinted genes. Results Using this methodology, we confirmed 16 differentially methylated regions (DMRs) associated with known imprinted genes. We chose 28 genomic regions for further testing and identified two imprinted genes (DNMT1 and AIM1). Both genes showed maternal allele-specific methylation and paternal allele-specific transcription. Imprinted expression for AIM1 was conserved in the cynomolgus macaque placenta, but not in other macaque tissues or in the mouse. Conclusions Our study indicates that while there are many genomic regions with allele-specific methylation in tissues like the placenta, only a small sub-set of them are associated with allele-specific transcription, suggesting alternative functions for such genomic regions. Nonetheless, novel tissue-specific imprinted genes remain to be discovered in humans. Their identification may help us better understand embryonic and fetal development. PMID:24094292

  15. Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients

    PubMed Central

    Seekhuntod, Sirirat; Thavarungkul, Paninee; Chaichanawongsaroj, Nuntaree

    2016-01-01

    Background Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations. Methods We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay. Results Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12. Conclusion The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues. PMID:26812617

  16. 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

  17. Allele-specific expression of mutated in colorectal cancer (MCC) gene and alternative susceptibility to colorectal cancer in schizophrenia

    PubMed Central

    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

  18. 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

  19. 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

  20. Ribosomal protein genes are highly enriched among genes with allele-specific expression in the interspecific F1 hybrid catfish.

    PubMed

    Chen, Ailu; Wang, Ruijia; Liu, Shikai; Peatman, Eric; Sun, Luyang; Bao, Lisui; Jiang, Chen; Li, Chao; Li, Yun; Zeng, Qifan; Liu, Zhanjiang

    2016-06-01

    Interspecific hybrids provide a rich source for the analysis of allele-specific expression (ASE). In this work, we analyzed ASE in F1 hybrid catfish using RNA-Seq datasets. While the vast majority of genes were expressed with both alleles, 7-8 % SNPs exhibited significant differences in allele ratios of expression. Of the 66,251 and 177,841 SNPs identified from the datasets of the liver and gill, 5420 (8.2 %) and 13,390 (7.5 %) SNPs were identified as significant ASE-SNPs, respectively. With these SNPs, a total of 1519 and 3075 ASE-genes were identified. Gene Ontology analysis revealed that genes encoding cytoplasmic ribosomal proteins (RP) were highly enriched among ASE genes. Parent-of-origin was determined for 27 and 30 ASE RP genes in the liver and gill, respectively. The results indicated that genes from both channel catfish and blue catfish were involved in ASE. However, each RP gene appeared to be almost exclusively expressed from only one parent, indicating that ribosomes in the hybrid catfish were in the "hybrid" form. Overall representation of RP transcripts among the transcriptome appeared lower in the F1 hybrid catfish than in channel catfish or blue catfish, suggesting that the "hybrid" ribosomes may work more efficiently for translation in the F1 hybrid catfish. PMID:26747053

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

    PubMed

    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

  2. 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

  3. 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

  4. Trans-species polymorphism and allele-specific expression in the CBF gene family of wild tomatoes.

    PubMed

    Mboup, Mamadou; Fischer, Iris; Lainer, Hilde; Stephan, Wolfgang

    2012-12-01

    Abiotic stresses such as drought, extreme temperatures, and salinity have a strong impact on plant adaptation. They act as selective forces on plant physiology and morphology. These selective pressures leave characteristic footprints that can be detected at the DNA sequence level using population genetic tools. On the basis of a candidate gene approach, we investigated signatures of adaptation in two wild tomato species, Solanum peruvianum and S. chilense. These species are native to western South America and constitute a model system for studying adaptation, due to their ability to colonize diverse habitats and the available genetic resources. We have determined the selective forces acting on the C-repeat binding factor (CBF) gene family, which consists of three genes, and is known to be involved in tolerance to abiotic stresses, in particular in cold tolerance. We also analyzed the expression pattern of these genes after drought and cold stresses. We found that CBF3 evolves under very strong purifying selection, CBF2 is under balancing selection in some populations of both species (S. peruvianum/Quicacha and S. chilense/Nazca) maintaining a trans-species polymorphism, and CBF1 is a pseudogene. In contrast to previous studies of cultivated tomatoes showing that only CBF1 was cold induced, we found that all three CBF genes are cold induced in wild tomatoes. All three genes are also drought induced. CBF2 exhibits an allele-specific expression pattern associated with the trans-species polymorphism. PMID:22787283

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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 ...

  10. 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...

  11. Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-Specific Assay

    PubMed Central

    Zhang, Guoqing; Cai, Fangping; de Rivera, Ivette Lorenzana; Zhou, Zhiyong; Zhang, Jing; Nkengasong, John

    2015-01-01

    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

  12. 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

  13. Comprehensively Evaluating cis-Regulatory Variation in the Human Prostate Transcriptome by Using Gene-Level Allele-Specific Expression

    PubMed Central

    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.

    2015-01-01

    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. PMID:25983244

  14. Recognition and Activation Domains Contribute to Allele-Specific Responses of an Arabidopsis NLR Receptor to an Oomycete Effector Protein

    PubMed Central

    Steinbrenner, Adam D.; Goritschnig, Sandra; Staskawicz, Brian J.

    2015-01-01

    In plants, specific recognition of pathogen effector proteins by nucleotide-binding leucine-rich repeat (NLR) receptors leads to activation of immune responses. RPP1, an NLR from Arabidopsis thaliana, recognizes the effector ATR1, from the oomycete pathogen Hyaloperonospora arabidopsidis, by direct association via C-terminal leucine-rich repeats (LRRs). Two RPP1 alleles, RPP1-NdA and RPP1-WsB, have narrow and broad recognition spectra, respectively, with RPP1-NdA recognizing a subset of the ATR1 variants recognized by RPP1-WsB. In this work, we further characterized direct effector recognition through random mutagenesis of an unrecognized ATR1 allele, ATR1-Cala2, screening for gain-of-recognition phenotypes in a tobacco hypersensitive response assay. We identified ATR1 mutants that a) confirm surface-exposed residues contribute to recognition by RPP1, and b) are recognized by and activate the narrow-spectrum allele RPP1-NdA, but not RPP1-WsB, in co-immunoprecipitation and bacterial growth inhibition assays. Thus, RPP1 alleles have distinct recognition specificities, rather than simply different sensitivity to activation. Using chimeric RPP1 constructs, we showed that RPP1-NdA LRRs were sufficient for allele-specific recognition (association with ATR1), but insufficient for receptor activation in the form of HR. Additional inclusion of the RPP1-NdA ARC2 subdomain, from the central NB-ARC domain, was required for a full range of activation specificity. Thus, cooperation between recognition and activation domains seems to be essential for NLR function. PMID:25671309

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

    PubMed

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

    2016-07-01

    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

  16. 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

  17. Hybrid sterility and evolution in Hawaiian Drosophila: differential gene and allele-specific expression analysis of backcross males.

    PubMed

    Brill, E; Kang, L; Michalak, K; Michalak, P; Price, D K

    2016-08-01

    The Hawaiian Drosophila are an iconic example of sequential colonization, adaptive radiation and speciation on islands. Genetic and phenotypic analysis of closely related species pairs that exhibit incomplete reproductive isolation can provide insights into the mechanisms of speciation. Drosophila silvestris from Hawai'i Island and Drosophila planitibia from Maui are two closely related allopatric Hawaiian picture-winged Drosophila that produce sterile F1 males but fertile F1 females, a pattern consistent with Haldane's rule. Backcrossing F1 hybrid females between these two species to parental species gives rise to recombinant males with three distinct sperm phenotypes despite a similar genomic background: motile sperm, no sperm (sterile), and immotile sperm. We found that these three reproductive morphologies of backcross hybrid males produce divergent gene expression profiles in testes, as measured with RNA sequencing. There were a total of 71 genes significantly differentially expressed between backcross males with no sperm compared with those backcross males with motile sperm and immotile sperm, but no significant differential gene expression between backcross males with motile sperm and backcross males with immotile sperm. All of these genes were underexpressed in males with no sperm, including a number of genes with previously known activities in adult testis. An allele-specific expression analysis showed overwhelmingly more cis-divergent than trans-divergent genes, with no significant difference in the ratio of cis- and trans-divergent genes among the sperm phenotypes. Overall, the results indicate that the regulation of gene expression involved in sperm production likely diverged relatively rapidly between these two closely related species. PMID:27220308

  18. Combined use of RFLP and PCR-ASO typing for HLA-DR-Dw and DQw typing.

    PubMed

    Bignon, J D; Bidwell, J L

    1991-01-01

    Due to some limitations of restriction fragment length polymorphism (RFLP) analysis in HLA-DR-DQ typing, we present a combined use of RFLP and polymerase chain reaction (PCR)-allele-specific oligonucleotide (ASO) typing. This scheme consists in selectively amplifying the few RFLP ill-defined genes (DR1/DR'Br' and DR4-Dw subsets) using PCR with allele specific primers to avoid cross-hybridization. PMID:1676910

  19. Development of three allele-specific codominant rice Waxy gene PCR markers suitable for marker assisted selection of amylose content and paste viscosity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Four Waxy haplotypes, which were identified previously as having different combinations of these three single-nucleotide polymorphisms (SNPs) of the Waxy gene, were highly correlated to the apparent amylose content and pasting properties, the important grain quality traits for predicting cooked rice...

  20. Development of three allele-specific co-Dominant PCR markers suitable for marker-assisted selection of amylose class and paste viscosity of rice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most rice is consumed as whole kernel cooked rice, and the consumer preferences for cooked rice texture and other sensory properties differ among regions of the world. Rice is also used as an ingredient in a multitude of foods by food-processing companies across the globe. These sensory and function...

  1. Dissection of expression-quantitative trait locus and allele specificity using a haploid/diploid plant system - insights into compensatory evolution of transcriptional regulation within populations.

    PubMed

    Verta, Jukka-Pekka; Landry, Christian R; MacKay, John

    2016-07-01

    Regulation of gene expression plays a central role in translating genotypic variation into phenotypic variation. Dissection of the genetic basis of expression variation is key to understanding how expression regulation evolves. Such analyses remain challenging in contexts where organisms are outbreeding, highly heterozygous and long-lived such as in the case of conifer trees. We developed an RNA sequencing (RNA-seq)-based approach for both expression-quantitative trait locus (eQTL) mapping and the detection of cis-acting (allele-specific) vs trans-acting (non-allele-specific) eQTLs. This method can be potentially applied to many conifers. We used haploid and diploid meiotic seed tissues of a single self-fertilized white spruce (Picea glauca) individual to dissect eQTLs according to linkage and allele specificity. The genetic architecture of local eQTLs linked to the expressed genes was particularly complex, consisting of cis-acting, trans-acting and, surprisingly, compensatory cis-trans effects. These compensatory effects influence expression in opposite directions and are neutral when combined in homozygotes. Nearly half of local eQTLs were under compensation, indicating that close linkage between compensatory cis-trans factors is common in spruce. Compensated genes were overrepresented in developmental and cell organization functions. Our haploid-diploid eQTL analysis in spruce revealed that compensatory cis-trans eQTLs segregate within populations and evolve in close genetic linkage. PMID:26891783

  2. Allele-specific chromatin remodeling in the ZPBP2/GSDMB/ORMDL3 locus associated with the risk of asthma and autoimmune disease.

    PubMed

    Verlaan, Dominique J; Berlivet, Soizik; Hunninghake, Gary M; Madore, Anne-Marie; Larivière, Mathieu; Moussette, Sanny; Grundberg, Elin; Kwan, Tony; Ouimet, Manon; Ge, Bing; Hoberman, Rose; Swiatek, Marcin; Dias, Joana; Lam, Kevin C L; Koka, Vonda; Harmsen, Eef; Soto-Quiros, Manuel; Avila, Lydiana; Celedón, Juan C; Weiss, Scott T; Dewar, Ken; Sinnett, Daniel; Laprise, Catherine; Raby, Benjamin A; Pastinen, Tomi; Naumova, Anna K

    2009-09-01

    Common SNPs in the chromosome 17q12-q21 region alter the risk for asthma, type 1 diabetes, primary biliary cirrhosis, and Crohn disease. Previous reports by us and others have linked the disease-associated genetic variants with changes in expression of GSDMB and ORMDL3 transcripts in human lymphoblastoid cell lines (LCLs). The variants also alter regulation of other transcripts, and this domain-wide cis-regulatory effect suggests a mechanism involving long-range chromatin interactions. Here, we further dissect the disease-linked haplotype and identify putative causal DNA variants via a combination of genetic and functional analyses. First, high-throughput resequencing of the region and genotyping of potential candidate variants were performed. Next, additional mapping of allelic expression differences in Yoruba HapMap LCLs allowed us to fine-map the basis of the cis-regulatory differences to a handful of candidate functional variants. Functional assays identified allele-specific differences in nucleosome distribution, an allele-specific association with the insulator protein CTCF, as well as a weak promoter activity for rs12936231. Overall, this study shows a common disease allele linked to changes in CTCF binding and nucleosome occupancy leading to altered domain-wide cis-regulation. Finally, a strong association between asthma and cis-regulatory haplotypes was observed in three independent family-based cohorts (p = 1.78 x 10(-8)). This study demonstrates the requirement of multiple parallel allele-specific tools for the investigation of noncoding disease variants and functional fine-mapping of human disease-associated haplotypes. PMID:19732864

  3. 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.

  4. A Unique Primer with an Inosine Chain at the 5′-Terminus Improves the Reliability of SNP Analysis Using the PCR-Amplified Product Length Polymorphism Method

    PubMed Central

    Shojo, Hideki; Tanaka, Mayumi; Takahashi, Ryohei; Kakuda, Tsuneo; Adachi, Noboru

    2015-01-01

    Polymerase chain reaction-amplified product length polymorphism (PCR-APLP) is one of the most convenient and reliable methods for single nucleotide polymorphism (SNP) analysis. This method is based on PCR, but uses allele-specific primers containing SNP sites at the 3′-terminus of each primer. To use this method at least two allele-specific primers and one “counter-primer”, which serves as a common forward or reverse primer of the allele-specific primers, are required. The allele-specific primers have SNP sites at the 3′-terminus, and another primer should have a few non-complementary flaps at the 5′-terminus to detect SNPs by determining the difference of amplicon length by PCR and subsequent electrophoresis. A major disadvantage of the addition of a non-complementary flap is the non-specific annealing of the primer with non-complementary flaps. However, a design principle for avoiding this undesired annealing has not been fully established, therefore, it is often difficult to design effective APLP primers. Here, we report allele-specific primers with an inosine chain at the 5′-terminus for PCR-APLP analysis. This unique design improves the competitiveness of allele-specific primers and the reliability of SNP analysis when using the PCR-APLP method. PMID:26381262

  5. Detection of BRAF Mutations Using a Fully Automated Platform and Comparison with High Resolution Melting, Real-Time Allele Specific Amplification, Immunohistochemistry and Next Generation Sequencing Assays, for Patients with Metastatic Melanoma

    PubMed Central

    Harlé, Alexandre; Salleron, Julia; Franczak, Claire; Dubois, Cindy; Filhine-Tressarieu, Pierre; Leroux, Agnès; Merlin, Jean-Louis

    2016-01-01

    Background Metastatic melanoma is a severe disease with one of the highest mortality rate in skin diseases. Overall survival has significantly improved with immunotherapy and targeted therapies. Kinase inhibitors targeting BRAF V600 showed promising results. BRAF genotyping is mandatory for the prescription of anti-BRAF therapies. Methods Fifty-nine formalin-fixed paraffin-embedded melanoma samples were assessed using High-Resolution-Melting (HRM) PCR, Real-time allele-specific amplification (RT-ASA) PCR, Next generation sequencing (NGS), immunohistochemistry (IHC) and the fully-automated molecular diagnostics platform IdyllaTM. Sensitivity, specificity, positive predictive value and negative predictive value were calculated using NGS as the reference standard to compare the different assays. Results BRAF mutations were found in 28(47.5%), 29(49.2%), 31(52.5%), 29(49.2%) and 27(45.8%) samples with HRM, RT-ASA, NGS, IdyllaTM and IHC respectively. Twenty-six (81.2%) samples were found bearing a c.1799T>A (p.Val600Glu) mutation, three (9.4%) with a c.1798_1799delinsAA (p.Val600Lys) mutation and one with c.1789_1790delinsTC (p.Leu597Ser) mutation. Two samples were found bearing complex mutations. Conclusions HRM appears the less sensitive assay for the detection of BRAF V600 mutations. The RT-ASA, IdyllaTM and IHC assays are suitable for routine molecular diagnostics aiming at the prescription of anti-BRAF therapies. IdyllaTM assay is fully-automated and requires less than 2 minutes for samples preparation and is the fastest of the tested assays. PMID:27111917

  6. Allele-specific extension allows base-pair neutral homozygotes to be discriminated by high-resolution melting of small amplicons.

    PubMed

    Cai, Yanning; Yuan, Yanpeng; Lin, Qingling; Chan, Piu

    2010-11-01

    Not all single-nucleotide polymorphisms (SNPs) can be determined using high-resolution melting (HRM) of small amplicons, especially class 3 and 4 SNPs. This is due mainly to the small shift in the melting temperature (Tm) between two types of homozygote. Choosing rs1869458 (a class 4 SNP) as a sample, we developed a modified small amplicon HRM assay. An allele-specific extension (ASE) primer, which ended at an SNP site and matched only one of the alleles, was added to the reaction as well as additional thermal steps for ASE. Following asymmetric polymerase chain reaction and melting curve analysis, heterozygotes were easily identified. Two types of homozygote were also distinguishable, indicating that extension primers 11 to 13 bases in length worked efficiently in an allele-specific way. Modification of the limiting amplification primer with locked nucleic acid increased the Tm difference between extension and amplification peaks and facilitated subsequent genotyping. In addition, 194 human genomic DNA samples were genotyped with the developed assay and by direct sequencing, with the different methods providing identical genotyping results. In conclusion, ASE-HRM is a simple, inexpensive, closed-tube genotyping method that can be used to examine all types of SNP. PMID:20599636

  7. Identification of Stmm3 locus Conferring Resistance to Late-stage Chemically Induced Skin Papillomas on Mouse Chromosome 4 by Congenic Mappingand Allele-specific Alteration Analysis

    PubMed Central

    Saito, Megumi; Okumura, Kazuhiro; Miura, Ikuo; Wakana, Shigeharu; Kominami, Ryo; Wakabayashi, Yuichi

    2014-01-01

    Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to chemically induced skin papillomas on chromosome 4 and 7 with a large number of [(FVB/N × MSM/Ms) F1 × FVB/N] backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 4. We used linkage analysis and a congenic mouse strain, FVB.MSM-Stmm3 to refine the location of Stmm3 (Skin tumor modifier of MSM 3) locus within a physical interval of about 34 Mb on distal chromosome 4. In addition, we used patterns of allele-specific imbalances in tumors from N2 and N10 congenic mice to narrow down further the region of Stmm3 locus to a physical distance of about 25 Mb. Furthermore, immunohistochemical analysis showed papillomas from congenic mice had less proliferative activity. These results suggest that Stmm3 responsible genes may have an influence on papilloma formation in the two-stage skin carcinogenesis by regulating papilloma growth rather than development. PMID:25077764

  8. 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.

  9. 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

  10. Allele-specific malE mutations that restore interactions between maltose-binding protein and the inner-membrane components of the maltose transport system.

    PubMed

    Treptow, N A; Shuman, H A

    1988-08-20

    Active accumulation of maltose and maltodextrins by Escherichia coli depends on an outer-membrane protein. LamB, a periplasmic maltose-binding protein (MalE, MBP) and three inner-membrane proteins, MalF, MalG and MalK. MalF and MalG are integral transmembrane proteins, while MalK is associated with the inner aspect of the cytoplasmic membrane via an interaction with MalG. Previously we have shown that MBP is essential for movement of maltose across the inner membrane. We have taken advantage of malF and malG mutants in which MBP interacts improperly with the membrane proteins. We describe the properties of malE mutations in which a proper interaction between MBP and defective MalF and MalG proteins has been restored. We found that these malE suppressor mutations are able to restore transport activity in an allele-specific manner. That is, a given malE mutation restores transport activity to different extents in different malF and malG mutants. Since both malF and malG mutations could be suppressed by allele-specific malE suppressors, we propose that, in wild-type bacteria, MBP interacts with sites on both MalF and MalG during active transport. The locations of different malE suppressor mutations indicate specific regions on MBP that are important for interacting with MalF and MalG. PMID:3050132

  11. Direct Detection of Erythromycin-Resistant Bordetella pertussis in Clinical Specimens by PCR.

    PubMed

    Wang, Zengguo; Han, Ruijun; Liu, Ying; Du, Quanli; Liu, Jifeng; Ma, Chaofeng; Li, Hengxin; He, Qiushui; Yan, Yongping

    2015-11-01

    Resistance of Bordetella pertussis to erythromycin has been increasingly reported. We developed an allele-specific PCR method for rapid detection of erythromycin-resistant B. pertussis directly from nasopharyngeal (NP) swab samples submitted for diagnostic PCR. Based on the proven association of erythromycin resistance with the A2047G mutation in the 23S rRNA of B. pertussis, four primers, two of which were designed to be specific for either the wild-type or the mutant allele, were used in two different versions of the allele-specific PCR assay. The methods were verified with results obtained by PCR-based sequencing of 16 recent B. pertussis isolates and 100 NP swab samples submitted for diagnostic PCR. The detection limits of the two PCR assays ranged from 10 to 100 fg per reaction for both erythromycin-susceptible and -resistant B. pertussis. Two amplified fragments of each PCR, of 286 and 112 bp, respectively, were obtained from a mutant allele of the isolates and/or NP swab samples containing B. pertussis DNAs. For the wild-type allele, only a 286-bp fragment was visible when the allele-specific PCR assay 1 was performed. No amplification was found when a number of non-Bordetella bacterial pathogens and NP swab samples that did not contain the DNAs of B. pertussis were examined. This assay can serve as an alternative for PCR-based sequencing, especially for local laboratories in resource-poor countries. PMID:26224847

  12. Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody

    DOE PAGESBeta

    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

  13. 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.

  14. Bulk segregant RNA-seq reveals expression and positional candidate genes and allele-specific expression for disease resistance against enteric septicemia of catfish

    PubMed Central

    2013-01-01

    Background The application of RNA-seq has accelerated gene expression profiling and identification of gene-associated SNPs in many species. However, the integrated studies of gene expression along with SNP mapping have been lacking. Coupling of RNA-seq with bulked segregant analysis (BSA) should allow correlation of expression patterns and associated SNPs with the phenotypes. Results In this study, we demonstrated the use of bulked segregant RNA-seq (BSR-Seq) for the analysis of differentially expressed genes and associated SNPs with disease resistance against enteric septicemia of catfish (ESC). A total of 1,255 differentially expressed genes were found between resistant and susceptible fish. In addition, 56,419 SNPs residing on 4,304 unique genes were identified as significant SNPs between susceptible and resistant fish. Detailed analysis of these significant SNPs allowed differentiation of significant SNPs caused by genetic segregation and those caused by allele-specific expression. Mapping of the significant SNPs, along with analysis of differentially expressed genes, allowed identification of candidate genes underlining disease resistance against ESC disease. Conclusions This study demonstrated the use of BSR-Seq for the identification of genes involved in disease resistance against ESC through expression profiling and mapping of significantly associated SNPs. BSR-Seq is applicable to analysis of genes underlining various performance and production traits without significant investment in the development of large genotyping platforms such as SNP arrays. PMID:24373586

  15. 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

  16. 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. PMID:25366802

  17. 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.

  18. 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

  19. 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

  20. Hybrid Capture 2 is as effective as PCR testing for high-risk human papillomavirus in head and neck cancers.

    PubMed

    Hooper, Jody E; Hebert, Jessica F; Schilling, Amy; Gross, Neil D; Schindler, Joshua S; Lagowski, James P; Kulesz-Martin, Molly; Corless, Christopher L; Morgan, Terry K

    2015-04-01

    High-risk human papillomavirus (HPV) infection is a common cause of oropharyngeal squamous cell carcinoma, especially in young male nonsmokers. Accurately diagnosing HPV-associated oral cancers is important, because they have a better prognosis and may be treated differently than smoking-related oral carcinomas. Various methods have been validated to test for high-risk HPV in cervical tissue samples, and they are in routine clinical use to detect dysplasia before it progresses to invasive disease. Similarly, future screening for HPV-mediated oropharyngeal dysplasia may identify patients before it progresses. Our objective was to compare 4 of these methods in a retrospective series of 87 oral and oropharyngeal squamous cell carcinomas that had archived fresh-frozen and paraffin-embedded tissue for evaluation. Patient age, sex, smoking history, and tumor location were also recorded. DNA prepared from fresh-frozen tissue was tested for HPV genotypes by multiplex polymerase chain reaction analysis, and high-risk HPV screening was carried out using Hybrid Capture 2 and Cervista. Histologic sections were immunostained for p16. HPV-positive outcome was defined as agreement between at least 2 of the 3 genetic tests and used for χ analysis and calculations of diagnostic predictive value. As expected, high-risk HPV-positive oral cancers were most common in the tonsil and base of the tongue (oropharynx) of younger male (55 vs. 65 y) (P=0.0002) nonsmokers (P=0.01). Most positive cases were HPV16 (33/36, 92%). Hybrid Capture 2 and Cervista were as sensitive as polymerase chain reaction and had fewer false positives than p16 immunohistochemical staining. PMID:25839700

  1. 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...

  2. Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function.

    PubMed

    Barbaro, Vanessa; Nasti, Annamaria A; Del Vecchio, Claudia; Ferrari, Stefano; Migliorati, Angelo; Raffa, Paolo; Lariccia, Vincenzo; Nespeca, Patrizia; Biasolo, Mariangela; Willoughby, Colin E; Ponzin, Diego; Palù, Giorgio; Parolin, Cristina; Di Iorio, Enzo

    2016-06-01

    Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease caused by heterozygous mutations in the p63 gene and characterized by limb defects, orofacial clefting, ectodermal dysplasia, and ocular defects. Patients develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. Medical and surgical treatments are ineffective and of limited benefit. Oral mucosa epithelial stem cells (OMESCs) represent an alternative source of stem cells capable of regenerating the corneal epithelium and, combined with gene therapy, could provide an attractive therapeutic avenue. OMESCs from EEC patients carrying the most severe p63 mutations (p.R279H and p.R304Q) were characterized and the genetic defect of p.R279H silenced using allele-specific (AS) small interfering RNAs (siRNAs). Systematic screening of locked nucleic acid (LNA)-siRNAs against R279H-p63 allele in (i) stable WT-ΔNp63α-RFP and R279H-ΔNp63α-EGFP cell lines, (ii) transient doubly transfected cell lines, and (iii) p.R279H OMESCs, identified a number of potent siRNA inhibitors for the mutant allele, which had no effect on wild-type p63. In addition, siRNA treatment led to longer acquired life span of mutated stem cells compared to controls, less accelerated stem cell differentiation in vitro, reduced proliferation properties, and effective ability in correcting the epithelial hypoplasia, thus giving rise to full thickness stratified and differentiated epithelia. This study demonstrates the phenotypic correction of mutant stem cells (OMESCs) in EEC syndrome by means of siRNA mediated AS silencing with restoration of function. The application of siRNA, alone or in combination with cell-based therapies, offers a therapeutic strategy for corneal blindness in EEC syndrome. Stem Cells 2016;34:1588-1600. PMID:26891374

  3. Detection and molecular characterization of two FAD3 genes controlling linolenic acid content and development of allele-specific markers in yellow mustard (Sinapis alba).

    PubMed

    Tian, Entang; Zeng, Fangqin; MacKay, Kimberly; Roslinsky, Vicky; Cheng, Bifang

    2014-01-01

    Development of yellow mustard (Sinapis alba L.) with superior quality traits (low erucic and linolenic acid contents, and low glucosinolate content) can make this species as a potential oilseed crop. We have recently isolated three inbred lines Y1127, Y514 and Y1035 with low (3.8%), medium (12.3%) and high (20.8%) linolenic acid (C18∶3) content, respectively, in this species. Inheritance studies detected two fatty acid desaturase 3 (FAD3) gene loci controlling the variation of C18∶3 content. QTL mapping revealed that the two FAD3 gene loci responsible for 73.0% and 23.4% of the total variation and were located on the linkage groups Sal02 and Sal10, respectively. The FAD3 gene on Sal02 was referred to as SalFAD3.LA1 and that on Sal10 as SalFAD3.LA2. The dominant and recessive alleles were designated as LA1 and la1 for SalFAD3.LA1, and LA2 and la2 for SalFAD3.LA2. Cloning and alignment of the coding and genomic DNA sequences revealed that the SalFAD3.LA1 and SalFAD3.LA2 genes each contained 8 exons and 7 introns. LA1 had a coding DNA sequence (CDS) of 1143 bp encoding a polypeptide of 380 amino acids, whereas la1 was a loss-of-function allele due to an insertion of 584 bp in exon 3. Both LA2 and la2 had a CDS of 1152 bp encoding a polypeptide of 383 amino acids. Allele-specific markers for LA1, la1, LA2 and la2 co-segregated with the C18∶3 content in the F2 populations and will be useful for improving fatty acid composition through marker assisted selection in yellow mustard breeding. PMID:24823372

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. Inter- and Intra-Individual Variation in Allele-Specific DNA Methylation and Gene Expression in Children Conceived using Assisted Reproductive Technology

    PubMed Central

    Turan, Nahid; Katari, Sunita; Gerson, Leigh F.; Chalian, Raffi; Foster, Michael W.; Gaughan, John P.; Coutifaris, Christos; Sapienza, Carmen

    2010-01-01

    Epidemiological studies have reported a higher incidence of rare disorders involving imprinted genes among children conceived using assisted reproductive technology (ART), suggesting that ART procedures may be disruptive to imprinted gene methylation patterns. We examined intra- and inter-individual variation in DNA methylation at the differentially methylated regions (DMRs) of the IGF2/H19 and IGF2R loci in a population of children conceived in vitro or in vivo. We found substantial variation in allele-specific methylation at both loci in both groups. Aberrant methylation of the maternal IGF2/H19 DMR was more common in the in vitro group, and the overall variance was also significantly greater in the in vitro group. We estimated the number of trophoblast stem cells in each group based on approximation of the variance of the binomial distribution of IGF2/H19 methylation ratios, as well as the distribution of X chromosome inactivation scores in placenta. Both of these independent measures indicated that placentas of the in vitro group were derived from fewer stem cells than the in vivo conceived group. Both IGF2 and H19 mRNAs were significantly lower in placenta from the in vitro group. Although average birth weight was lower in the in vitro group, we found no correlation between birth weight and IGF2 or IGF2R transcript levels or the ratio of IGF2/IGF2R transcript levels. Our results show that in vitro conception is associated with aberrant methylation patterns at the IGF2/H19 locus. However, very little of the inter- or intra-individual variation in H19 or IGF2 mRNA levels can be explained by differences in maternal DMR DNA methylation, in contrast to the expectations of current transcriptional imprinting models. Extraembryonic tissues of embryos cultured in vitro appear to be derived from fewer trophoblast stem cells. It is possible that this developmental difference has an effect on placental and fetal growth. PMID:20661447

  10. [Application of the PCR-APLP method to determine ABO genotypes in forensic samples].

    PubMed

    Watanabe, G; Umetsu, K; Osawa, M

    2000-08-01

    We carried out ABO genotyping of forensic samples by the amplified product length polymorphism (APLP) technique. We present two novel systems. One is termed as eight primers system, in which eight allele-specific primers are added into a single PCR reaction. Another is termed as six primers system. In both APLP systems, all alleles were clearly detected using DNA purified from forensic samples. In PCR amplification with direct addition of specimen, ABO genotyping was also possible to blood stain, seminal stain, blood, saliva and urine. Furthermore, ABO genotyping worked only to chimpanzee. This PCR-APLP method should be convepffnt and valuable for forensic practice. PMID:11060991

  11. 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.

  12. Virtual PCR

    SciTech Connect

    Gardner, S N; Clague, D S; Vandersall, J A; Hon, G; Williams, P L

    2006-02-23

    The polymerase chain reaction (PCR) stands among the keystone technologies for analysis of biological sequence data. PCR is used to amplify DNA, to generate many copies from as little as a single template. This is essential, for example, in processing forensic DNA samples, pathogen detection in clinical or biothreat surveillance applications, and medical genotyping for diagnosis and treatment of disease. It is used in virtually every laboratory doing molecular, cellular, genetic, ecologic, forensic, or medical research. Despite its ubiquity, we lack the precise predictive capability that would enable detailed optimization of PCR reaction dynamics. In this LDRD, we proposed to develop Virtual PCR (VPCR) software, a computational method to model the kinetic, thermodynamic, and biological processes of PCR reactions. Given a successful completion, these tools will allow us to predict both the sequences and concentrations of all species that are amplified during PCR. The ability to answer the following questions will allow us both to optimize the PCR process and interpret the PCR results: What products are amplified when sequence mixtures are present, containing multiple, closely related targets and multiplexed primers, which may hybridize with sequence mismatches? What are the effects of time, temperature, and DNA concentrations on the concentrations of products? A better understanding of these issues will improve the design and interpretation of PCR reactions. The status of the VPCR project after 1.5 years of funding is consistent with the goals of the overall project which was scoped for 3 years of funding. At half way through the projected timeline of the project we have an early beta version of the VPCR code. We have begun investigating means to improve the robustness of the code, performed preliminary experiments to test the code and begun drafting manuscripts for publication. Although an experimental protocol for testing the code was developed, the preliminary

  13. Allele-specific disparity in breast cancer

    PubMed Central

    2011-01-01

    Background In a cancer cell the number of copies of a locus may vary due to amplification and deletion and these variations are denoted as copy number alterations (CNAs). We focus on the disparity of CNAs in tumour samples, which were compared to those in blood in order to identify the directional loss of heterozygosity. Methods We propose a numerical algorithm and apply it to data from the Illumina 109K-SNP array on 112 samples from breast cancer patients. B-allele frequency (BAF) and log R ratio (LRR) of Illumina were used to estimate Euclidian distances. For each locus, we compared genotypes in blood and tumour for subset of samples being heterozygous in blood. We identified loci showing preferential disparity from heterozygous toward either the A/B-allele homozygous (allelic disparity). The chi-squared and Cochran-Armitage trend tests were used to examine whether there is an association between high levels of disparity in single nucleotide polymorphisms (SNPs) and molecular, clinical and tumour-related parameters. To identify pathways and network functions over-represented within the resulting gene sets, we used Ingenuity Pathway Analysis (IPA). Results To identify loci with a high level of disparity, we selected SNPs 1) with a substantial degree of disparity and 2) with substantial frequency (at least 50% of the samples heterozygous for the respective locus). We report the overall difference in disparity in high-grade tumours compared to low-grade tumours (p-value < 0.001) and significant associations between disparity in multiple single loci and clinical parameters. The most significantly associated network functions within the genes represented in the loci of disparity were identified, including lipid metabolism, small-molecule biochemistry, and nervous system development and function. No evidence for over-representation of directional disparity in a list of stem cell genes was obtained, however genes appeared to be more often altered by deletion than by amplification. Conclusions Our data suggest that directional loss and amplification exist in breast cancer. These are highly associated with grade, which may indicate that they are enforced with increasing number of cell divisions. Whether there is selective pressure for some loci to be preferentially amplified or deleted remains to be confirmed. PMID:22188678

  14. PCR thermocycler

    DOEpatents

    Benett, William J.; Richards, James B.

    2003-01-01

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  15. PCR thermocycler

    DOEpatents

    Benett, William J.; Richards, James B.

    2005-05-17

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  16. 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

  17. 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

  18. Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

    PubMed Central

    Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun

    2016-01-01

    AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher

  19. Selective alleviation of Mitomycin C sensitivity in lexA3 strains of Escherichia coli demands allele specificity of rif-nal mutations: a pivotal role for rpoB87-gyrA87 mutations.

    PubMed

    Shanmughapriya, Vinod; Meenakshi, Shanmugaraja; Munavar, M Hussain

    2014-01-01

    Very recently, we have reported about an unconventional mode of elicitation of Mitomycin C (MMC) specific resistance in lexA3 (SOS repair deficient) mutants due to a combination of Rif-Nal mutations (rpoB87-gyrA87). We have clearly shown that UvrB is mandatory for this unconventional MMC resistance in rpoB87-gyrA87-lexA3 strains and uvrB is expressed more even without DNA damage induction from its LexA dependent promoter despite the uncleavable LexA3 repressor. The rpoB87 allele is same as the rpoB3595 which is known to give rise to a fast moving RNA Polymerase and gyrA87 is a hitherto unreported Nal(R) allele. Thus, it is proposed that the RNA Polymerase with higher elongation rate with the mutant DNA Gyrase is able to overcome the repressional hurdle posed by LexA3 to express uvrB. In this study we have systematically analysed the effect of three other rpoB (rif) mutations-two known to give rise to fast moving RNAP (rpoB2 and rpoB111) and one to a slow moving RNAP (rpoB8) and four different alleles of gyrA Nal(R) mutations (gyrA199, gyrA247, gyrA250, gyrA259) isolated spontaneously, on elicitation of MMC resistance in lexA3 strains. Our results indicate that in order to acquire resistance to 0.5 µg/ml MMC cells require both rpoB87 and gyrA87 but resistance to 0.25 µg/ml of MMC can be brought about by either rpoB87, gyrA87, fast moving rpoB mutations or other nal mutations also. We have also depicted increased constitutive uvrB expression in strains carrying fast moving RNAP (rpoB2 and rpoB111) with gyrA87 and another nal mutation with rpoB87 and expression level in these strains is lesser than rpoB87-gyrA87 strain. These results evidently suggest an allele specific role for the rif-nal mutations to acquire MMC resistance in lexA3 strains via increased constitutive uvrB expression and a pivotal role for rpoB87-gyrA87 combination to elicit higher levels of resistance. PMID:24498357

  20. Comparison and contrast of genes and biological pathways responding to Marek’s disease virus infection using allele-specific expression and differential expression in broiler and layer chickens

    PubMed Central

    2013-01-01

    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. Enhancing MD genetic resistance is desirable to augment current vaccines and other MD control measures. High throughput sequencing was used to profile splenic transcriptomes from individual F1 progeny infected with MDV at 4 days of age from both outbred broilers (meat-type) and inbred layer (egg-type) chicken lines that differed in MD genetic resistance. The resulting information was used to identify SNPs, genes, and biological pathways exhibiting allele-specific expression (ASE) in response to MDV infection in each type of chicken. In addition, we compared and contrasted the results of pathway analyses (ASE and differential expression (DE)) between chicken types to help inform on the biological response to MDV infection. Results With 7 individuals per line and treatment group providing high power, we identified 6,132 single nucleotide polymorphisms (SNPs) in 4,768 genes and 4,528 SNPs in 3,718 genes in broilers and layers, respectively, that exhibited ASE in response to MDV infection. Furthermore, 548 and 434 genes in broilers and layers, respectively, were found to show DE following MDV infection. Comparing the datasets, only 72 SNPs and 850 genes for ASE and 20 genes for DE were common between the two bird types. Although the chicken types used in this study were genetically different, at the pathway level, both TLR receptor and JAK/STAT signaling pathways were enriched as well as exhibiting a high proportion of ASE genes, especially at the beginning of both above mentioned regulatory pathways. Conclusions RNA sequencing with adequate biological replicates is a powerful approach to identify high confidence SNPs, genes, and pathways that are associated with transcriptional response to MDV infection. In addition, the SNPs exhibiting ASE in response to MDV infection provide a

  1. Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR

    PubMed Central

    Liu, Jia; Song, Hongshuo; Liu, Donglai; Zuo, Tao; Lu, Fengmin; Zhuang, Hui; Gao, Feng

    2014-01-01

    Artificial recombinants can be generated during PCR when more than two genetically distinct templates coexist in a single PCR reaction. These recombinant amplicons can lead to the false interpretation of genetic diversity and incorrect identification of biological phenotypes that do not exist in vivo. We investigated how recombination between 2 or 35 genetically distinct HIV-1 genomes was affected by different PCR conditions using the parallel allele-specific sequencing (PASS) assay and the next generation sequencing method. In a standard PCR condition, about 40% of amplicons in a PCR reaction were recombinants. The high recombination frequency could be significantly reduced if the number of amplicons in a PCR reaction was below a threshold of 1013–1014 using low thermal cycles, fewer input templates, and longer extension time. Heteroduplexes (each DNA strand from a distinct template) were present at a large proportion in the PCR products when more thermal cycles, more templates, and shorter extension time were used. Importantly, the majority of recombinants were identified in heteroduplexes, indicating that the recombinants were mainly generated through heteroduplexes. Since prematurely terminated extension fragments can form heteroduplexes by annealing to different templates during PCR amplification, recombination has a better chance to occur with samples containing different genomes when the number of amplicons accumulate over the threshold. New technologies are warranted to accurately characterize complex quasispecies gene populations. PMID:25211143

  2. Sporulation properties and antimicrobial susceptibility in endemic and rare Clostridium difficile PCR ribotypes.

    PubMed

    Zidaric, Valerija; Rupnik, Maja

    2016-06-01

    Increased sporulation and antibiotic resistance have been proposed to be associated with certain Clostridium difficile epidemic strains such as PCR ribotype 027. In this study we examined these properties in another widespread PCR ribotype, 014/020, in comparison to prevalent PCR ribotype 002 and a group of rarely represented PCR ribotypes. Highest sporulation was observed in 014/020 strains at 24 h, while after 72 h PCR ribotype 002 and rare PCR ribotypes formed higher total number of spores. PCR ribotype 014/020 strains exhibited slightly higher resistance to tested antimicrobials, followed by group of rare PCR ribotypes and less common PCR ribotype 002. Neither sporulation properties nor antibiotic resistance clearly differed in endemic and rare strains. PMID:27095618

  3. Sex Determination Using PCR

    ERIC Educational Resources Information Center

    Kima, Peter E.; Rasche, Madeline E.

    2004-01-01

    PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on the pattern of PCR products, students can…

  4. Km typing with PCR: application to population screening.

    PubMed Central

    Kurth, J H; Bowcock, A M; Erlich, H A; Nevo, S; Cavalli-Sforza, L L

    1991-01-01

    The immunoglobulin kappa light chain (IgK) locus may play a significant role in the pathology of both infectious and autoimmune diseases. Most of the work on IgK genetics has been conducted using immunological techniques for allelic typing and sequence analysis. This is restricted by availability of reagents and can be both expensive and time-consuming. PCR primers were designed to amplify the kappa constant gene (Ck), and four allele-specific oligonucleotides (ASOs) were used to distinguish the alleles in the amplified PCR products. Direct sequencing of PCR products was performed to confirm that the primers specifically amplified the Ck region and the ASOs differentiated the Km alleles. Sequencing of an average of 209 nucleotides of DNA from 50 individuals revealed no variation except at codon 191, which is known to be involved in a frequent polymorphism. An analysis of 347 different individual DNAs from 10 human populations was conducted to determine Km allelic frequencies within these populations and to apply this type of data collection to population studies. Images Figure 4 Figure 5 PMID:1900145

  5. Pure chromosome-specific PCR libraries from single sorted chromosomes.

    PubMed Central

    VanDevanter, D R; Choongkittaworn, N M; Dyer, K A; Aten, J; Otto, P; Behler, C; Bryant, E M; Rabinovitch, P S

    1994-01-01

    Chromosome-specific DNA libraries can be very useful in molecular and cytogenetic genome mapping studies. We have developed a rapid and simple method for the generation of chromosome-specific DNA sequences that relies on polymerase chain reaction (PCR) amplification of a single flow-sorted chromosome or chromosome fragment. Previously reported methods for the development of chromosome libraries require larger numbers of chromosomes, with preparation of pure chromosomes sorted by flow cytometry, generation of somatic cell hybrids containing targeted chromosomes, or a combination of both procedures. These procedures are labor intensive, especially when hybrid cell lines are not already available, and this has limited the generation of chromosome-specific DNA libraries from nonhuman species. In contrast, a single sorted chromosome is a pure source of DNA for library production even when flow cytometric resolution of chromosome populations is poor. Furthermore, any sorting cytometer may be used with this technique. Using this approach, we demonstrate the generation of PCR libraries suitable for both molecular and fluorescence in situ hybridization studies from individual baboon and canine chromosomes, separate human homologues, and a rearranged marker chromosome from a transformed cell line. PCR libraries specific to subchromosomal regions have also been produced by sorting a small chromosome fragment. This simple and rapid technique will allow generation of nonhuman linkage maps and probes for fluorescence in situ hybridization and the characterization of marker chromosomes from solid tumors. In addition, allele-specific libraries generated by this strategy may also be useful for mapping genetic diseases. Images PMID:8016078

  6. The applicability of TaqMan-based quantitative real-time PCR assays for detecting and enumeratIng Cryptosporidium spp. oocysts in the environment

    EPA Science Inventory

    Molecular detection methods such as PCR have been extensively used to type Cryptosporidium oocysts detected in the environment. More recently, studies have developed quantitative real-time PCR assays for detection and quantification of microbial contaminants in water as well as ...

  7. Real-Time PCR

    NASA Astrophysics Data System (ADS)

    Evrard, A.; Boulle, N.; Lutfalla, G. S.

    Over the past few years there has been a considerable development of DNA amplification by polymerase chain reaction (PCR), and real-time PCR has now superseded conventional PCR techniques in many areas, e.g., the quantification of nucleic acids and genotyping. This new approach is based on the detection and quantification of a fluorescent signal proportional to the amount of amplicons generated by PCR. Real-time detection is achieved by coupling a thermocycler with a fluorimeter. This chapter discusses the general principles of quantitative real-time PCR, the different steps involved in implementing the technique, and some examples of applications in medicine. The polymerase chain reaction (PCR) provides a way of obtaining a large number of copies of a double-stranded DNA fragment of known sequence. This DNA amplification technique, developed in 1985 by K. Mullis (Cetus Corporation), saw a spectacular development over the space of a few years, revolutionising the methods used up to then in molecular biology. Indeed, PCR has many applications, such as the detection of small amounts of DNA, cloning, and quantitative analysis (assaying), each of which will be discussed further below.

  8. Overlap extension PCR cloning.

    PubMed

    Bryksin, Anton; Matsumura, Ichiro

    2013-01-01

    Rising demand for recombinant proteins has motivated the development of efficient and reliable cloning methods. Here we show how a beginner can clone virtually any DNA insert into a plasmid of choice without the use of restriction endonucleases or T4 DNA ligase. Chimeric primers encoding plasmid sequence at the 5' ends and insert sequence at the 3' ends are designed and synthesized. Phusion(®) DNA polymerase is utilized to amplify the desired insert by PCR. The double-stranded product is subsequently employed as a pair of mega-primers in a PCR-like reaction with circular plasmids. The original plasmids are then destroyed in restriction digests with Dpn I. The product of the overlap extension PCR is used to transform competent Escherichia coli cells. Phusion(®) DNA polymerase is used for both the amplification and fusion reactions, so both steps can be monitored and optimized in the same way. PMID:23996437

  9. A simple real-time polymerase chain reaction (PCR)-based assay for authentication of the Chinese Panax ginseng cultivar Damaya from a local ginseng population.

    PubMed

    Wang, H; Wang, J; Li, G

    2016-01-01

    Panax ginseng is one of the most important medicinal plants in the Orient. Owing to its increasing demand in the world market, cultivated ginseng has become the main source of medicinal material. Among the Chinese ginseng cultivars, Damaya commands higher prices and is grown in significant proportions among the local ginseng population. Due to the lack of rapid and accurate authentication methods, Damaya is distributed among different cultivars in the local ginseng population in China. Here, we identified a unique, Damaya-specific single nucleotide polymorphism (SNP) site present in the second intron of mitochondrial cytochrome c oxidase subunit 2 (cox2). Based on this SNP, a Damaya cultivar-specific primer was designed and an allele-specific polymerase chain reaction (PCR) was optimized for the effective molecular authentication of Damaya. We designed a method by combining a simple DNA isolation method with real-time allele-specific PCR using SYBR Green I fluorescent dye, and proved its efficacy in clearly discriminated Damaya cultivar from other Chinese ginseng cultivars according to the allelic discrimination analysis. Hence, this study provides a simple and rapid assay for the differentiation and conservation of Damaya from the local Chinese ginseng population. PMID:27420983

  10. Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

    PubMed Central

    Zonta, Eleonora; Garlan, Fanny; Pécuchet, Nicolas; Perez-Toralla, Karla; Caen, Ouriel; Milbury, Coren; Didelot, Audrey; Fabre, Elizabeth; Blons, Hélène; Laurent-Puig, Pierre; Taly, Valérie

    2016-01-01

    In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients. PMID:27416070

  11. QUALITY CONTROLS FOR PCR

    EPA Science Inventory

    The purpose of this presentation is to present an overview of the quality control (QC) sections of a draft EPA document entitled, "Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples." This document has been prepared by th...

  12. QUALITY ASSURANCE FOR PCR

    EPA Science Inventory

    The U.S. Environmental Protection Agency (EPA) held a workshop in January 2003 on the detection of viruses in water using polymerase chain reaction (PCR)-based methods. Speakers were asked to address a series of specific questions, including whether a single standard method coul...

  13. Identification of the Causative Organism of Tuberculous Lymphadenitis in Ethiopia by PCR

    PubMed Central

    Kidane, Dawit; Olobo, Joseph O.; Habte, Abebe; Negesse, Yohannes; Aseffa, Abraham; Abate, Getahun; Yassin, Mohammed A.; Bereda, Kiflu; Harboe, Morten

    2002-01-01

    Tuberculous lymphadenitis (TBLN) is a common form of extrapulmonary tuberculosis with multiple differential diagnoses. Demonstration of the etiologic agent by smear microscopy or culture of fine needle aspirate (FNA) specimens is often unsuccessful. FNA specimens from 40 patients presenting at a rural health center in South Ethiopia and diagnosed as positive for TBLN on the basis of clinical and cytological criteria were analyzed for mycobacterial DNA by PCR. Thirty (75%) had cervical lymphadenitis and 11 (27.5%) were seropositive for human immunodeficiency virus (HIV). Three primer sets were initially used to identify the causative agent at the genus (antigen 85 complex), complex (IS6110 insertion sequence), and species (pncA gene and allelic variation) levels. Among the forty TBLN cases, 35 (87.5%) were positive by PCR at the genus and complex levels. Based on PCR for detection of allelic variation at position 169, 24 (68.6%) of the 35 were positive for Mycobacterium tuberculosis and 6 (17.1%) were positive for M. bovis. These six were positive in additional PCR assays using the JB21-JB22 primer set, which is highly specific for M. bovis. Five (14.1%) showed amplification for both M. tuberculosis and M. bovis with the allele-specific primer set. Cooccurrence of pyrazinamide (PZA)-sensitive and -resistant M. tuberculosis in those five cases was indicated, since all were negative in assays with the JB21-JB22 primer set. This feature was seen in 3 of 11 HIV-positive and 2 of 29 HIV-negative individuals (P < 0.001). Conclusion: among 35 PCR-positive cases of TBLN from southern Ethiopia, 29 (82.9%) were caused by M. tuberculosis and six (17.1%) were caused by M. bovis. PMID:12409403

  14. DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays

    PubMed Central

    Nagamine, Kenjiro; Hung, Guo-Chiuan; Li, Bingjie; Lo, Shyh-Ching

    2015-01-01

    Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5–50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents. PMID:26279626

  15. DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays.

    PubMed

    Nagamine, Kenjiro; Hung, Guo-Chiuan; Li, Bingjie; Lo, Shyh-Ching

    2015-01-01

    Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5-50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents. PMID:26279626

  16. MAMMALIAN DNA IN PCR REAGENTS

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high- cycle PCR amplification t...

  17. Detection of HER2 Gene Polymorphism in Breast Cancer: PCR Optimization Study.

    PubMed

    Budiarto, Bugi Ratno; Desriani

    2016-01-01

    Cancers are the most deadly diseases in the world and their incidences continue to increase over time. Particularly, breast cancer in females places 1(st) rank among other types of cancers in term of cancer cases (23%) and death incidence (14%). Recent findings support the correlation between (Ile)655(Val) SNP in the HER2 gene with breast cancer risk. Moreover, the (Ile)655(Val) HER2 gene polymorphism could be a predictive factor in a neoadjuvant therapy setting. Precise detection of the (Ile)655(Val) HER2 gene SNP in early breast cancer patients will be beneficial in designing the most suitable treatment and in increasing the efficacy of anticancer drugs. Here we develop a rapid and inexpensive method for (Ile)655(Val) SNP detection in the HER2 gene based on allele-specific PCR technology. Two forward primers and one common reverse primer were designed to anneal specifically either on the HER2 gene fragment containing the GG genotype or to the HER2 gene fragment containing the AA genotype where one of these primers had been added with poly-GC at 5' upstream. Moreover, to increase discrimination level, mismatch bases at the SNP site and the 3(rd) base of each forward primers from 3'end were added. To test the performance of the designed primers in discriminating a polymorphism and its annealing temperature, breast cancer specimen-derived genomic DNA (with GG genotype) and pGEM_HER2/AA (with AA genotype) were used as templates in the PCR reaction. The optimal annealing temperature for SNP detection was at 51.5°C as showed by the appearance of a 150 base pair (bp) band as AA genotype (pGEM_HER2/AA template), 116bp band as GG genotype (genomic DNA template), and both types of bands as AG genotype (mix of pGEM_HER2/AA and genomic DNA template). Allelic types of breast cancer patients were also determined using this optimized method compared to sanger sequencing. The 100% accordance was shown for all types of genotypes in both methods. The allele-specific PCR in this

  18. Improved detection of episomal Banana streak viruses by multiplex immunocapture PCR.

    PubMed

    Le Provost, Grégoire; Iskra-Caruana, Marie-Line; Acina, Isabelle; Teycheney, Pierre-Yves

    2006-10-01

    Banana streak viruses (BSV) are currently the main viral constraint to Musa germplasm movement, genetic improvement and mass propagation. Therefore, it is necessary to develop and implement BSV detection strategies that are both reliable and sensitive, such as PCR-based techniques. Unfortunately, BSV endogenous pararetrovirus sequences (BSV EPRVs) are present in the genome of Musa balbisiana. They interfere with PCR-based detection of episomal BSV in infected banana and plantain, such as immunocapture PCR. Therefore, a multiplex, immunocapture PCR (M-IC-PCR) was developed for the detection of BSV. Musa sequence tagged microsatellite site (STMS) primers were selected and used in combination with BSV species-specific primers in order to monitor possible contamination by Musa genomic DNA, using multiplex PCR. Furthermore, immunocapture conditions were optimized in order to prevent Musa DNA from interfering with episomal BSV DNA during the PCR step. This improved detection method successfully allowed the accurate, specific and sensitive detection of episomal DNA only from distinct BSV species. Its implementation should benefit PCR-based detection of viruses for which homologous sequences are present in the genome of their hosts, including transgenic plants expressing viral sequences. PMID:16857272

  19. Rapid real-time PCR and high resolution melt analysis in a self-filling thermoplastic chip.

    PubMed

    Sposito, A; Hoang, V; DeVoe, D L

    2016-09-21

    A microfluidic platform designed for point-of-care PCR-based nucleic acid diagnostics is described. Compared to established microfluidic PCR technologies, the system is unique in its ability to achieve exceptionally rapid PCR amplification in a low cost thermoplastic format, together with high temperature accuracy enabling effective validation of reaction product by high resolution melt analysis performed in the same chamber as PCR. In addition, the system employs capillary pumping for automated loading of sample into the reaction chamber, combined with an integrated hydrophilic valve for precise self-metering of sample volumes into the device. Using the microfluidic system to target a mutation in the G6PC gene, efficient PCR from human genomic DNA template is achieved with cycle times as low as 14 s, full amplification in 8.5 min, and final melt analysis accurately identifying the desired amplicon. PMID:27460504

  20. Fluorescent detection of Southern blots and PCR-based genetic typing tests

    SciTech Connect

    Mansfield, E.S.; Worley, J.M.; Zimmerman, P.A.

    1994-09-01

    The Southern blot is used to study gene organization, to identify disease-causing genomic rearrangements, or for typing RFLP markers in forensic, paternity, or prenatal diagnostic testing. Fluorescence offers a much greater dynamic range and a more linear response than film used in radioactive or chemiluminescent detection of RFLPs. We therefore investigated using the Fluorimager{trademark} 575 (Molecular Dynamics, Inc.) for analyzing Southern blots. Using a single-locus probe to D2S44 (YNH24) (Promega Corp.), we detect as little as 100 ng (0.05 attomole) genomic DNA. The alkaline phosphatase-labeled probe is detected using AttoPhos (JBL Scientific), and the developed membrane is scanned with the Fluorimager. Biotinylated hybridization probes can also be developed using a streptavidin-alkaline phosphatase conjugate and AttoPhos. The instrument scan parameters can be adjusted to prevent overexposure and accompanying loss of resolution in images of blots, gels, or 96-well microplates. We have used these other sample formats in PCR-based genetic typing assays. We use FluorKit DQS (Molecular Dynamics) to accurately quantify PCR template DNA (1-500 ng) in 96-well microplates scanned using the same instrument. Mutation detection assays run include heteroduplex gels (5% polyacrylamide, 2.7 M urea), short tandem repeat (STR) markers, amplified fragment length polymorphisms (AmpFLP), competitive priming PCR, and allele-specific oligotyping. These assays are run using either 1- or 2-color labeling. We detect unlabeled PCR products, such as the AmpFLP marker D1S80 (Perkin-Elmer) by post-staining gels for 10 minutes with SYBR Green 1 (Molecular Probes) and scanning the wet gel. The Fluorimager scans a 20 x 25 cm sample within three minutes, allowing rapid optimization of fluorescent protocols and high sample throughput.

  1. TERT promoter mutations are associated with distant metastases in upper tract urothelial carcinomas and serve as urinary biomarkers detected by a sensitive castPCR.

    PubMed

    Wang, Kun; Liu, Tiantian; Ge, Nan; Liu, Li; Yuan, Xiaotian; Liu, Jikai; Kong, Feng; Wang, Chang; Ren, Hongbo; Yan, Keqiang; Hu, Sanyuan; Xu, Zhonghua; Björkholm, Magnus; Fan, Yidong; Zhao, Shengtian; Liu, Cheng; Xu, Dawei

    2014-12-15

    TERT promoter C228T and C250T mutations occur in various malignancies including bladder cancer (BC) and may serve as urinary tumor markers. However, the mutation association with clinical variables in upper tract urothelial carcinomas (UTUCs) is unclear. There is also a lack of sensitive tools to detect the minor mutant TERT promoter in bulk urinary DNA. Here we analyzed 220 UTUC patients [98 with renal pelvic carcinoma (RPC) and 122 with ureter carcinoma (UC)] and developed a Competitive Allele-Specific TaqMan PCR (castPCR) for urinary assay. We identified C228T or C250T mutations in 42 of 98 (43%) RPC and 23 of 122 (19%) UC tumors. Distant metastases were significantly correlated with UTUC patients harboring TERT promoter mutations (P = 0.001). C228T were detected in 6/10 and 9/10 of urine samples from patients with mutation-carrying tumors using Sanger sequencing and castPCR, respectively. When urine samples from 70 BC patients were analyzed together, the sensitivity of urinary C228T assay was 89% and 50% for castPCR and Sanger sequencing, respectively (P < 0.001). Collectively, TERT promoter mutations occur in UTUCs with a high frequency in RPCs and predict distant metastasis. castPCR assays of the mutation are a useful tool for urine-based diagnostics of urological malignancies. PMID:25474136

  2. Development of a multiplex real-time PCR assay for identification of members of the Anopheles gambiae species complex.

    PubMed

    Bass, Chris; Williamson, Martin S; Field, Linda M

    2008-07-01

    Two high-throughput assays for the identification of members of the Anopheles gambiae sensu lato species complex have recently been reported. These methods, are based on TaqMan single nucleotide polymorphism (SNP) genotyping that enables rapid scoring of mosquito DNA samples in real-time PCR reactions. Unfortunately, both assays are restricted in the number of species that they can identify and a combination of the two assays may be required to identify all possible species in certain regions. To overcome this limitation, and thereby further increase throughput while reducing costs, we have developed a new multiplex real-time PCR assay for identifying members of the An. gambiae complex. The new method uses three probes labelled with fluorophores with distinct emission and excitation spectra, allowing simultaneous detection of the two main malaria vectors from the non-vector sibling species, and can be used on single mosquito legs from silica-dried specimens. A genotyping trial of over 450 specimens collected from 13 countries in sub-Saharan Africa showed the multiplex assay to be highly specific and sensitive and it compared well against the two previously reported TaqMan assays and standard allele-specific PCR. PMID:18490000

  3. Digital droplet PCR on disk.

    PubMed

    Schuler, Friedrich; Trotter, Martin; Geltman, Marcel; Schwemmer, Frank; Wadle, Simon; Domínguez-Garrido, Elena; López, María; Cervera-Acedo, Cristina; Santibáñez, Paula; von Stetten, Felix; Zengerle, Roland; Paust, Nils

    2016-01-01

    Existing systems for digital droplet PCR (ddPCR) either suffer from low integration or are difficult to introduce to mass fabrication. Here we present an integrated system that is compatible to mass fabrication and combines emulsification, PCR, and fluorescence readout in a single chamber within a disposable cartridge (disk). Droplets are generated by injecting the sample into fluorinated oil via centrifugal step emulsification. The resulting emulsion is aligned in the PCR and readout zone by capillary action. During thermocycling, gas bubbles generated by degassing are removed by capillary driven transport through tapered regions in the PCR chamber. Thereby, the positioning of the emulsion within the readout zone of the PCR chamber is ensured at any time and no bubbles are present during readout. Manual handling of the disk solely requires pipetting of oil and PCR mix into the inlet structures, placing the disk into the thermocycler and subsequently into a microarray scanner. The functionality of the ddPCR process chain is demonstrated by quantitative detection of the cystic fibrosis causing mutation p.Phe508del, which is of interest for non-invasive prenatal testing (NIPT). The mutation was detected in a concentration range spanning four orders of magnitude. We envision that this work will lay the base for the development of highly integrated sample-to-digital-answer PCR systems that can be employed in routine clinical diagnosis. PMID:26610263

  4. Real-Time PCR and High-Resolution Melt Analysis for Rapid Detection of Mycobacterium leprae Drug Resistance Mutations and Strain Types

    PubMed Central

    Li, Wei; Matsuoka, Masanori; Kai, Masanori; Thapa, Pratibha; Khadge, Saraswoti; Hagge, Deanna A.; Brennan, Patrick J.

    2012-01-01

    Drug resistance surveillance and strain typing of Mycobacterium leprae are necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR–high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) of folP1, rpoB, and gyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included three folP1, two rpoB, and one gyrA mutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (<5%) could be attributed to low DNA concentration or PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations. PMID:22170923

  5. Development of a simple PCR-based assay for the identification of triazine resistance in the noxious plant common ragweed (Ambrosia artemisiifolia) and its applicability in higher plants.

    PubMed

    Mátyás, Kinga Klára; Taller, János; Cseh, András; Poczai, Péter; Cernák, István

    2011-12-01

    Bidirectional allele-specific PCR (Bi-PASA) was used to detect a point mutation causing triazine resistance in common ragweed (Ambrosia artemisiifolia). The external primers amplified a 278 bp standard DNA fragment in all genotypes. In the susceptible S264S genotypes, a 208 bp fragment was expected while in resistant S264G common ragweed genotypes a 109 bp band was expected. In resistant plants, both the wild and mutant type fragments were detected, indicating that the original triazine sensitive cpDNA is maintained in a heteroplasmic state in the resistant S264G genotypes. Additionally, in silico analysis confirmed the potential applicability of our diagnostic assay for other plant species. In 24 out of 74 taxa (32%), the assay could be used without any change, while in the others some of the primers should be redesigned before use. PMID:21809088

  6. A method for specific amplification and PCR sequencing of individual members of multigene families: application to the study of steroid 21-hydroxylase deficiency.

    PubMed

    Collier, S; Tassabehji, M; Strachan, T

    1992-02-01

    Mutations at the human HLA-linked CYP21B locus are responsible for 21-hydroxylase deficiency, a recessively inherited disorder of steroidogenesis. The scope for PCR-based analysis of the CYP21B gene has been restricted by the very high sequence homology between CYP21B and a closely related pseudogene, CYP21A. Here we describe a novel PCR sequencing strategy that allows the independent amplification of the entire CYP21B coding sequence and the subsequent enzyme-mediated conversion of the PCR product to a single-stranded form for dideoxy sequencing. We have used this approach to characterize the 21-hydroxylase deficiency allele associated with HLA-B55, the most frequent HLA marker associated with a CYP21B point mutation in the British population, and also an HLA-B35 associated allele of Asian origin. Allele-specific oligonucleotide (ASO) hybridization analyses have confirmed the selective amplification of CYP21B genes and the identity of the pathological mutations. The method can be adapted to permit selective amplification and PCR sequencing of individual closely related members of other multigene families and small-copy-number repetitive DNA families. PMID:1472941

  7. Real-time PCR genotyping and frequency of the myostatin F94L mutation in beef cattle breeds.

    PubMed

    Vankan, D M; Waine, D R; Fortes, M R S

    2010-04-01

    This research developed two real-time PCR assays, employing high-resolution melt and allele-specific analysis to accurately genotype the F94L mutation in cattle. This mutation (g.433C > A) in the growth differentiation factor 8 or myostatin gene has recently been shown to be functionally associated with increased muscle mass and carcass yield in cattle. The F94L mutation is not, like other myostatin mutations, associated with reduced fertility and dystocia. It is therefore a candidate for introgression into other breeds to improve retail beef yield and the development of a simple and accurate test to genotype this specific mutation is warranted. Variations in the efficiency of enzyme cleavage compromised the accuracy of genotyping by published methods, potentially resulting in an overestimation of the frequency of the mutant allele. The frequency of the F94L mutation was determined by real-time PCR in 1140 animals from 15 breeds of cattle in Australia. The mutation was present in Simmental (0.8%), Piedmontese (2%), Droughtmaster (4%) and Limousin (94.2%) but not found in Salers, Angus, Poll Hereford, Hereford, Gelbvieh, Charolais, Jersey, Brahman, Holstein, Shorthorn or Maine Anjou. The low prevalence of F94L in all beef breeds except Limousin indicates the significant potential for this mutation to improve retail yield in Australian beef cattle. PMID:22444040

  8. Identification of rifampin-resistant mycobacterium tuberculosis strains by hybridization, PCR, and ligase detaction reaction on oligonucleotide microchips.

    SciTech Connect

    Mikhailovich, V.; Lapa, S.; Gryadunov, D.; Sobolev, A.; Strizhkov, B.; Chernyh, N.; Skotnikova, O.; Irtuganova, O.; Moroz, A.; Litvinov, V.; Vladimirskii, M.; Perelman, M.; Chernousova, L.; Erokhin, V.; Mirzabekov, A.; Biochip Technology Center; Russian Academy of Sciences; Moscow Antituberculosis Center; Moscow Medical Academy; Russian Academy of Medical Sciences

    2001-07-01

    Three new molecular approaches were developed to identify drug-resistant strains of Mycobacterium tuberculosis using biochips with oligonucleotides immobilized in polyacrylamide gel pads. These approaches are significantly faster than traditional bacteriological methods. All three approaches -- hybridization, PCR, and ligase detection reaction -- were designed to analyze an 81-bp fragment of the gene rpoB encoding the {beta}-subunit of RNA polymerase, where most known mutations of rifampin resistance are located. The call set for hybridization analysis consisted of 42 immobilized oligonucleotides and enabled us to identify 30 mutant variants of the rpoB gene within 24 h. These variants are found in 95% of all mutants whose rifampin resistance is caused by mutations in the 81-bp fragment. Using the second approach, allele-specific on-chip PCR, it was possible to directly identify mutations in clinical samples within 1.5 h. The third approach, on-chip ligase detection reaction, was sensitive enough to reveal rifampin-resistant strains in a model mixture containing 1% of resistant and 99% of susceptible bacteria. This level of sensitivity is comparable to that from the determination of M. tuberculosis drug resistance by using standard bacteriological tests.

  9. Ligation-independent cloning of PCR products (LIC-PCR).

    PubMed Central

    Aslanidis, C; de Jong, P J

    1990-01-01

    A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5'-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3'-ends. The 3'-terminal sequence can be removed by the action of the (3'----5') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5'-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones. Images PMID:2235490

  10. A study of PCR inhibition mechanisms using real time PCR.

    PubMed

    Opel, Kerry L; Chung, Denise; McCord, Bruce R

    2010-01-01

    In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co-extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall we find that amplicon size and melting temperature are important in some inhibition mechanisms and not in others and the key issue in understanding PCR inhibition is determining the identity of the interfering substance. PMID:20015162

  11. Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

    PubMed Central

    Araújo, Cristina P.; Osório, Ana Luiza A.R.; Jorge, Klaudia S.G.; Ramos, Carlos A.N.; Souza Filho, Antonio F.; Vidal, Carlos E.S.; Vargas, Agueda P.C.; Roxo, Eliana; Rocha, Adalgiza S.; Suffys, Philip N.; Fonseca, Antônio A.; Silva, Marcio R.; Barbosa Neto, José D.; Cerqueira, Valíria D.; Araújo, Flábio R.

    2014-01-01

    Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis. PMID:25242951

  12. The PCR-SSP Manager computer program: a tool for maintaining sequence alignments and automatically updating the specificities of PCR-SSP primers and primer mixes.

    PubMed

    Bunce, M; Barnardo, M C; Welsh, K I

    1998-08-01

    An emerging problem of molecular typing methods such as PCR amplification using sequence-specific primers (PCR-SSP) is that they frequently require updating as new alleles are constantly being described which potentially affect the specificity of every PCR-SSP reaction. PCR-SSP uses pairs of primers to detect cis-linked polymorphisms and thus each new allele described must be compared to each individual primer pair. Furthermore, sequence homology between the various loci for class I and class II means that, for example, new HLA-A sequences have to be compared with HLA-B and HLA-C primer mixes to rule out cross-locus amplification. We have developed a computer program known as SSP Manager which is capable of aligning HLA class I and class II sequences obtained from Internet-accessible databases such as GenBank. The program then updates all individual primer specificities held in its database before updating the specificities of all primer mixes. Sets of primer mixes can then be combined from the primer mix directory to create PCR-SSP typing trays which are subsequently analysed by the program. A report is generated which stipulates whether all known sequences are amplified and the reason for apparent failure to test for individual alleles, e.g. a lack of relevant sequence information. SSP Manager has the flexibility to cope with unusual sequences (deletions and insertions), primers with internal mismatches and primers with a deliberate mismatch. The program also has many tools for developing new primer mixes, such as the facility to search for novel reactions using Boolean operators. The organisation and operational use of the SSP Manager program is described and its uses are illustrated with an updated allele list for our previously described Phototyping PCR-SSP class I and class II typing set. The SSP Manager is available on request from the authors. PMID:9756405

  13. Electrochemical DNA sensor for anthrax toxin activator gene atxA-detection of PCR amplicons.

    PubMed

    Das, Ritu; Goel, Ajay K; Sharma, Mukesh K; Upadhyay, Sanjay

    2015-12-15

    We report the DNA probe functionalized electrochemical genosensor for the detection of Bacillus anthracis, specific towards the regulatory gene atxA. The DNA sensor is fabricated on electrochemically deposited gold nanoparticle on self assembled layer of (3-Mercaptopropyl) trimethoxysilane (MPTS) on GC electrode. DNA hybridization is monitored by differential pulse voltammogram (DPV). The modified GC electrode is characterized by atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) method. We also quantified the DNA probe density on electrode surface by the chronocoulometric method. The detection is specific and selective for atxA gene by DNA probe on the electrode surface. No report is available for the detection of B. anthracis by using atxA an anthrax toxin activator gene. In the light of real and complex sample, we have studied the PCR amplicons of 303, 361 and 568 base pairs by using symmetric and asymmetric PCR approaches. The DNA probe of atxA gene efficiently hybridizes with different base pairs of PCR amplicons. The detection limit is found to be 1.0 pM (S/N ratio=3). The results indicate that the DNA sensor is able to detect synthetic target as well as PCR amplicons of different base pairs. PMID:26257186

  14. PCR amplfication on a microarray of gel-immobilized oligonucleotides : detection of bacterial toxin- and drug-resistent genes and their mutations.

    SciTech Connect

    Strizhkov, B. N.; Drobyshev, A. L.; Mikhailovich, V. M.; Mirzabekov, A. D.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology

    2000-10-01

    PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency, the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.

  15. [Detection and Serotyping of Streptococcus pneumoniae Carried in Healthy Adults with a Modified PCR Method].

    PubMed

    Ishihara, Yuka; Okamoto, Akira; Ohta, Michio

    2015-05-01

    Detection of Streptococcus pneumoniae colonized in the pharynx of healthy carriers currently relies on conventional culture methods of direct plating with pharyngeal swab specimens. The accurate measurement of the carriage of pneumococci, however, has not been necessarily achieved with these methods due to low density colonization and contamination of numerous oral streptococci that express α-hemolysis. A PCR-based detection method of pneumococci-specific for lytA as well as PCR serotyping of S. pneumoniae was recently developed and their effectiveness was confirmed. We modified the reaction conditions of these methods to improve the detection rate and applied them to the measurement of S. pneumoniae carried in healthy adults. Pharyngeal swab specimens obtained from 110 healthy volunteers over 40 and living in Nagoya were enriched for 5 hours with broth medium supplemented with rabbit serum and the template DNA for PCR was extracted from the mixed enriched culture. Of 110 specimens 36 (32.7%) were lytA-positive, the rate of which was much higher than the results of previous culture-based studies. The DNA template preparations were then used for PCR-based serotyping with primers specific for each of the types included in pneumococcal 23 valent vaccine (PPV23). We found that 28 out of 36 lytA-positive carriers were identified as being positive for the serotypes belonging to PPV23, although serotypes 6A and 6B were indistinguishable with the PCR method. The most frequent serotype was serotype 14, and serotypes 4, 18C, and 6A/B were also frequently identified. Five lytA-positive carriers were previously vaccinated with PPV23, and among them, 4 were positive for serotypes contained in PPV23. We recommend PCR-based identification and serotyping of S. pneumoniae in broth enrichment culture of pharyngeal swab specimens as a reliable method for the surveillance of healthy carriers with low density colonization. PMID:26552129

  16. PALATAL DYSMORPHOGENESIS: QUANTITATIVE RT-PCR

    EPA Science Inventory

    ABSTRACT

    Palatal Dysmorphogenesis : Quantitative RT-PCR

    Gary A. Held and Barbara D. Abbott

    Reverse transcription PCR (RT-PCR) is a very sensitive method for detecting mRNA in tissue samples. However, as it is usually performed it is does not yield quantitativ...

  17. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    ERIC Educational Resources Information Center

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  18. Real-time PCR in Food Science: PCR Diagnostics.

    PubMed

    Rodriguez-Lazaro, David; Cook, Nigel; Hernandez, Marta

    2013-01-01

    A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labelling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programmes of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control. PMID:23513039

  19. Electrothermal modeling of silicon PCR chips

    NASA Astrophysics Data System (ADS)

    Cui, Zheng; Zhao, Zhan; Xia, Shanhong

    2001-04-01

    Polymerase chain reaction (PCR) on a microchip has drawn considerable attention in recent years. Although a microchip can have must fast heating and cooling rate, the delicacy in its structure makes the PCR experiment difficult and cracks often occurs particularly for the thin membrane type of PCR chips. Electrothermal modeling of PCR chips is presented using commercial MEMS software tool IntelliSuiteTM, with the aim of identifying the problems encountered in experiment and finding an optimum chip structure. Heating characteristics of four different heater designs have been compared, so have the PCR chambers with fixed frame and with suspended frame. The thermal stress analysis has shown that the structure and heater design can make significant difference in heating characteristics and in reducing the failure of PCR chips. The computer simulation has confirmed what has been found in experiment the reason of membrane cracks. Improvement in PCR chip design has been proposed.

  20. Molecular Surveillance of True Nontypeable Haemophilus influenzae: An Evaluation of PCR Screening Assays

    PubMed Central

    Binks, Michael J.; Temple, Beth; Kirkham, Lea-Ann; Wiertsema, Selma P.; Dunne, Eileen M.; Richmond, Peter C.; Marsh, Robyn L.; Leach, Amanda J.; Smith-Vaughan, Heidi C.

    2012-01-01

    Background Unambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination. Methodology/Principal Findings Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22), Hh (n = 27) or equivocal (n = 11), were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3) and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction. Conclusions/Significance Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease. PMID:22470516

  1. Multiplexed Primer Prediction for PCR

    2007-07-23

    MPP predicts sets of multiplex-compatible primers for Polymerase Chain Reaction (PCR), finding a near minimal set of primers such that at least one amplicon will be generated from every target sequence in the input file. The code finds highly conserved oligos that are suitable as primers, according to user-specified desired primer characteristics such as length, melting temperature, and amplicon length. The primers are predicted not to form unwanted dimer or hairpin structures. The target sequencesmore » used as input can be diverse, since no multiple sequence alighment is required. The code is scalable, taking up to tens of thousands of sequences as input, and works, for example, to find a "universal primer set" for all viral genomes provided as a single input file. The code generates a periodic check-point file, thus in the event of premature execution termination, the application can be restarted from the last check-point file.« less

  2. NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions

    NASA Astrophysics Data System (ADS)

    Shen, Cenchao; Yang, Wenjuan; Ji, Qiaoli; Maki, Hisaji; Dong, Anjie; Zhang, Zhizhou

    2009-11-01

    Nanoparticle-assisted PCR (polymerase chain reaction) technology is getting more and more attention recently. It is believed that some of the DNA recombinant technologies will be upgraded by nanotechnology in the near future, among which DNA replication is one of the core manipulation techniques. So whether or not the DNA replication fidelity is compromised in nanoparticle-assisted PCR is a question. In this study, a total of 16 different metallic and non-metallic nanoparticles (NPs) were tested for their effects on DNA replication fidelity in vitro and in vivo. Sixteen types of nanomaterials were distinctly different in enhancing the PCR efficiency, and their relative capacity to retain DNA replication fidelity was largely different from each other based on rpsL gene mutation assay. Generally speaking, metallic nanoparticles induced larger error rates in DNA replication fidelity than non-metallic nanoparticles, and non-metallic nanomaterials such as carbon nanopowder or nanotubes were still safe as PCR enhancers because they did not compromise the DNA replication fidelity in the Taq DNA polymerase-based PCR system.

  3. Raising the shields: PCR in the presence of metallic surfaces protected by tailor-made coatings.

    PubMed

    Scherag, Frank D; Brandstetter, Thomas; Rühe, Jürgen

    2014-10-01

    The implementation of PCR reactions in the presence of metallic surfaces is interesting for the generation of novel bioanalytical devices, because metals exhibit high mechanical stability, good thermal conductivity, and flexibility during deformation. However, metallic substrates are usually non-compatible with enzymatic reactions such as PCR due to poisoning of the active center of the enzyme or nonspecific adsorption of the enzymeto the metal surface, which could result in protein denaturation. We present a method for the generation of polymer coatings on metallic surfaces which are designed to minimize protein adsorption and also prevent the release of metal ions. These coatings consist of three layers covalently linked to each other; a self-assembled monolayer to promote adhesion, a photochemically generated barrier layer and a photochemically generated hydrogel. The coatings can be deposited onto aluminum, stainless steel, gold and copper surfaces. We compare PCR efficiencies in the presence of bare metallic surfaces with those of surfaces treated with the novel coating system. PMID:25108478

  4. Highly-efficient colony PCR method for red yeasts and its application to identify mutations within two leucine auxotroph mutants.

    PubMed

    Lin, Xinping; Yang, Fan; Zhou, Yongjin; Zhu, Zhiwei; Jin, Guojie; Zhang, Sufang; Zhao, Zongbao Kent

    2012-11-01

    Red yeasts hold great promise in the production of microbial lipids and carotenoids. Genetic study of red yeasts has attracted much attention; however, rapid amplification of genes from red yeast samples remains technically challenging. Here a highly efficient method for the preparation of genomic DNA (gDNA) template, which could be directly used for PCR, was developed. Cells from colonies or liquid cultures were collected and sequentially treated by microwave, plMAN5C, proteinase K and boiling (MMPB) in a single tube to give cell lysates that were qualified as PCR templates. Single-copied gDNA fragments o up to 2.8 kb were successfully amplified. We also demonstrated successful application of this method for species in the Ascomycetes and Basidiomycetes and identification of two leucine auxotroph mutants of Rhodotorula glutinis. This method could be widely employed for the screening and genetic engineering of various yeasts. PMID:23065821

  5. Diagnosis of neonatal group B Streptococcus sepsis by nested-PCR of residual urine samples.

    PubMed

    Cezarino, Bruno Nicolino; Yamamoto, Lidia; Del Negro, Gilda Maria Barbaro; Rocha, Daisy; Okay, Thelma Suely

    2008-01-01

    Group B streptococcus (GBS) remains the most common cause of early-onset sepsis in newborns. Laboratory gold-standard, broth culture methods are highly specific, but lack sensitivity. The aim of this study was to validate a nested-PCR and to determine whether residue volumes of urine samples obtained by non invasive, non sterile methods could be used to confirm neonatal GBS sepsis. The nested-PCR was performed with primers of the major GBS surface antigen. Unavailability of biological samples to perform life supporting exams, as well as others to elucidate the etiology of infections is a frequent problem concerning newborn patients. Nevertheless, we decided to include cases according to strict criteria: newborns had to present with signs and symptoms compatible with GBS infection; at least one of the following biological samples had to be sent for culture: blood, urine, or cerebrospinal fluid; availability of residue volumes of the samples sent for cultures, or of others collected on the day of hospitalization, prior to antibiotic therapy prescription, to be analyzed by PCR; favorable outcome after GBS empiric treatment. In only one newborn GBS infection was confirmed by cultures, while infection was only presumptive in the other three patients (they fulfilled inclusion criteria but were GBS-culture negative). From a total of 12 biological samples (5 blood, 3 CSF and 4 urine specimen), eight were tested by culture methods (2/8 were positive), and 8 were tested by PCR (7/8 were positive), and only 4 samples were simultaneously tested by both methods (1 positive by culture and 3 by PCR). In conclusion, although based on a restricted number of neonates and samples, our results suggest that the proposed nested-PCR might be used to diagnose GBS sepsis as it has successfully amplified the three types of biological samples analyzed (blood, urine and cerebrospinal fluid), and was more sensitive than culture methods as PCR in urine confirmed diagnosis in all four patients

  6. Diagnosis of neonatal group B Streptococcus sepsis by nested-PCR of residual urine samples

    PubMed Central

    Cezarino, Bruno Nicolino; Yamamoto, Lidia; Del Negro, Gilda Maria Barbaro; Rocha, Daisy; Okay, Thelma Suely

    2008-01-01

    Group B streptococcus (GBS) remains the most common cause of early-onset sepsis in newborns. Laboratory gold-standard, broth culture methods are highly specific, but lack sensitivity. The aim of this study was to validate a nested-PCR and to determine whether residue volumes of urine samples obtained by non invasive, non sterile methods could be used to confirm neonatal GBS sepsis. The nested-PCR was performed with primers of the major GBS surface antigen. Unavailability of biological samples to perform life supporting exams, as well as others to elucidate the etiology of infections is a frequent problem concerning newborn patients. Nevertheless, we decided to include cases according to strict criteria: newborns had to present with signs and symptoms compatible with GBS infection; at least one of the following biological samples had to be sent for culture: blood, urine, or cerebrospinal fluid; availability of residue volumes of the samples sent for cultures, or of others collected on the day of hospitalization, prior to antibiotic therapy prescription, to be analyzed by PCR; favorable outcome after GBS empiric treatment. In only one newborn GBS infection was confirmed by cultures, while infection was only presumptive in the other three patients (they fulfilled inclusion criteria but were GBS-culture negative). From a total of 12 biological samples (5 blood, 3 CSF and 4 urine specimen), eight were tested by culture methods (2/8 were positive), and 8 were tested by PCR (7/8 were positive), and only 4 samples were simultaneously tested by both methods (1 positive by culture and 3 by PCR). In conclusion, although based on a restricted number of neonates and samples, our results suggest that the proposed nested-PCR might be used to diagnose GBS sepsis as it has successfully amplified the three types of biological samples analyzed (blood, urine and cerebrospinal fluid), and was more sensitive than culture methods as PCR in urine confirmed diagnosis in all four patients

  7. Analysis of the constitution of the beer yeast genome by PCR, sequencing and subtelomeric sequence hybridization.

    PubMed

    Casaregola, S; Nguyen, H V; Lapathitis, G; Kotyk, A; Gaillardin, C

    2001-07-01

    The lager brewing yeasts, Saccharomyces pastorianus (synonym Saccharomyces carlsbergensis), are allopolyploid, containing parts of two divergent genomes. Saccharomyces cerevisiae contributed to the formation of these hybrids, although the identity of the other species is still unclear. The presence of alleles specific to S. cerevisiae and S. pastorianus was tested for by PCR/RFLP in brewing yeasts of various origins and in members of the Saccharomyces sensu stricto complex. S. cerevisiae-type alleles of two genes, HIS4 and YCL008c, were identified in another brewing yeast, S. pastorianus CBS 1503 (Saccharomyces monacensis), thought to be the source of the other contributor to the lager hybrid. This is consistent with the hybridization of S. cerevisiae subtelomeric sequences X and Y' to the electrophoretic karyotype of this strain. S. pastorianus CBS 1503 (S. monacensis) is therefore probably not an ancestor of S. pastorianus, but a related hybrid. Saccharomyces bayanus, also thought to be one of the contributors to the lager yeast hybrid, is a heterogeneous taxon containing at least two subgroups, one close to the type strain, CBS 380T, the other close to CBS 395 (Saccharomyces uvarum). The partial sequences of several genes (HIS4, MET10, URA3) were shown to be identical or very similar (over 99%) in S. pastorianus CBS 1513 (S. carlsbergensis), S. bayanus CBS 380T and its close derivatives, showing that S. pastorianus and S. bayanus have a common ancestor. A distinction between two subgroups within S. bayanus was made on the basis of sequence analysis: the subgroup represented by S. bayanus CBS 395 (S. uvarum) has 6-8% sequence divergence within the genes HIS4, MET10 and MET2 from S. bayanus CBS 380T, indicating that the two S. bayanus subgroups diverged recently. The detection of specific alleles by PCR/RFLP and hybridization with S. cerevisiae subtelomeric sequences X and Y' to electrophoretic karyotypes of brewing yeasts and related species confirmed our

  8. Real-time PCR in microfluidic devices

    NASA Astrophysics Data System (ADS)

    Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Hansen-Hagge, Thomas; Gärtner, Claudia

    2014-03-01

    A central method in a standard biochemical laboratory is represented by the polymerase chain reaction (PCR), therefore many attempts have been performed so far to implement this technique in lab-on-a-chip (LOC) devices. PCR is an ideal candidate for miniaturization because of a reduction of assay time and decreased costs for expensive bio-chemicals. In case of the "classical" PCR, detection is done by identification of DNA fragments electrophoretically separated in agarose gels. This method is meanwhile frequently replaced by the so-called Real-Time-PCR because here the exponential increase of amplificates can be observed directly by measurement of DNA interacting fluorescent dyes. Two main methods for on-chip PCRs are available: traditional "batch" PCR in chambers on a chip using thermal cycling, requiring about 30 minutes for a typical PCR protocol and continuous-flow PCR, where the liquid is guided over stationary temperature zones. In the latter case, the PCR protocol can be as fast as 5 minutes. In the presented work, a proof of concept is demonstrated for a real-time-detection of PCR products in microfluidic systems.

  9. Propidium monoazide reverse transcriptase PCR and RT-qPCR for detecting infectious enterovirus and norovirus.

    PubMed

    Karim, Mohammad R; Fout, G Shay; Johnson, Clifford H; White, Karen M; Parshionikar, Sandhya U

    2015-07-01

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the public health significance of positive findings are limited. In this study, PMA RT-PCR and RT-qPCR assays were evaluated for selective detection of infectious poliovirus, murine norovirus (MNV-1), and Norwalk virus. Viruses were inactivated using heat, chlorine, and ultraviolet light (UV). Infectious and non-infectious viruses were treated with PMA before RT-PCR and RT-qPCR. PMA RT-PCR was able to differentiate selectively between infectious and heat and chlorine inactivated poliovirus. PMA RT-PCR was able to differentiate selectively between infectious and noninfectious murine norovirus only when inactivated by chlorine. However, PMA RT-PCR could not differentiate infectious Norwalk virus from virus particles rendered non-infectious by any treatment. PMA RT-PCR assay was not able to differentiate between infectious and UV inactivated viruses suggesting that viral capsid damage may be necessary for PMA to enter and bind to the viral genome. PMA RT-PCR on naked MNV-1 and Norwalk virus RNA suggest that PMA RT-PCR can be used to detect intact, potentially infectious MNV-1 and Norwalk viruses and can be used to exclude the detection of free viral RNA by PCR assay. PMID:25796356

  10. Comparative analysis of conventional PCR and real-time PCR to diagnose shrimp WSD

    PubMed Central

    Leal, C.A.G.; Carvalho-Castro, G.A.; Cottorello, A.C.; Leite, R.C.; Figueiredo, H.C.P.

    2013-01-01

    The aims of this study were to standard and optimize a qPCR protocol with FAM-BHQ1 probe, and to compare its sensitivity against TaqMan qPCR and PCR methods to diagnose shrimp WSD. The FAM-BHQ1 qPCR presented higher clinical sensitivity and showed to be a robust alternative to detect WSSV in clinical samples. PMID:24516428

  11. Testing for Genetically Modified Foods Using PCR

    ERIC Educational Resources Information Center

    Taylor, Ann; Sajan, Samin

    2005-01-01

    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  12. Methods for comparing multiple digital PCR experiments.

    PubMed

    Burdukiewicz, Michał; Rödiger, Stefan; Sobczyk, Piotr; Menschikowski, Mario; Schierack, Peter; Mackiewicz, Paweł

    2016-09-01

    The estimated mean copy per partition (λ) is the essential information from a digital PCR (dPCR) experiment because λ can be used to calculate the target concentration in a sample. However, little information is available how to statistically compare dPCR runs of multiple runs or reduplicates. The comparison of λ values from several runs is a multiple comparison problem, which can be solved using the binary structure of dPCR data. We propose and evaluate two novel methods based on Generalized Linear Models (GLM) and Multiple Ratio Tests (MRT) for comparison of digital PCR experiments. We enriched our MRT framework with computation of simultaneous confidence intervals suitable for comparing multiple dPCR runs. The evaluation of both statistical methods support that MRT is faster and more robust for dPCR experiments performed in large scale. Our theoretical results were confirmed by the analysis of dPCR measurements of dilution series. Both methods were implemented in the dpcR package (v. 0.2) for the open source R statistical computing environment. PMID:27551672

  13. Digital PCR for detection of citrus pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus trees are often infected with multiple pathogens of economic importance, especially those with insect or mite vectors. Real-time/quantitative PCR (qPCR) has been used for high-throughput detection and relative quantification of pathogens; however, target reference or standards are required. I...

  14. Single-tube, non-isotopic, multiplex PCR/OLA assay and sequence-coded separation for simultaneous screening of 31 cystic fibrosis mutations

    SciTech Connect

    Brinson, E.C.; Adriano, T.; Bloch, W.

    1994-09-01

    We have developed a rapid, single-tube, non-isotopic assay that screens a patient sample for the presence of 31 cystic fibrosis (CF) mutations. This assay can identify these mutations in a single reaction tube and a single electrophoresis run. Sample preparation is a simple, boil-and-go procedure, completed in less than an hour. The assay is composed of a 15-plex PCR, followed by a 61-plex oligonucleotide ligation assay (OLA), and incorporates a novel detection scheme, Sequence Coded Separation. Initially, the multiplex PCR amplifies 15 relevant segments of the CFTR gene, simultaneously. These PCR amplicons serve as templates for the multiplex OLA, which detects the normal or mutant allele at all loci, simultaneously. Each polymorphic site is interrogated by three oligonucleotide probes, a common probe and two allele-specific probes. Each common probe is tagged with a fluorescent dye, and the competing normal and mutant allelic probes incorporate different, non-nucleotide, mobility modifiers. These modifiers are composed of hexaethylene oxide (HEO) units, incorporated as HEO phosphoramidite monomers during automated DNA synthesis. The OLA is based on both probe hybridization and the ability of DNA ligase to discriminate single base mismatches at the junction between paired probes. Each single tube assay is electrophoresed in a single gel lane of a 4-color fluorescent DNA sequencer (Applied Biosystems, Model 373A). Each of the ligation products is identified by its unique combination of electrophoretic mobility and one of three colors. The fourth color is reserved for the in-lane size standard, used by GENESCAN{sup TM} software (Applied Biosystems) to size the OLA electrophoresis products. The Genotyper{sub TM} software (Applied Biosystems) decodes these Sequence-Coded-Separation data to create a patient summary report for all loci tested.

  15. Assessment of the real-time PCR and different digital PCR platforms for DNA quantification.

    PubMed

    Pavšič, Jernej; Žel, Jana; Milavec, Mojca

    2016-01-01

    Digital PCR (dPCR) is beginning to supersede real-time PCR (qPCR) for quantification of nucleic acids in many different applications. Several analytical properties of the two most commonly used dPCR platforms, namely the QX100 system (Bio-Rad) and the 12.765 array of the Biomark system (Fluidigm), have already been evaluated and compared with those of qPCR. However, to the best of our knowledge, direct comparison between the three of these platforms using the same DNA material has not been done, and the 37 K array on the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity and limit of quantification. Here, a first assessment of qPCR, the QX100 system and both arrays of the Biomark system was performed with plasmid and genomic DNA from human cytomegalovirus. With use of PCR components that alter the efficiency of qPCR, each dPCR platform demonstrated consistent copy-number estimations, which indicates the high resilience of dPCR. Two approaches, one considering the total reaction volume and the other considering the effective reaction size, were used to assess linearity, analytical sensitivity and variability. When the total reaction volume was considered, the best performance was observed with qPCR, followed by the QX100 system and the Biomark system. In contrast, when the effective reaction size was considered, all three platforms showed almost equal limits of detection and variability. Although dPCR might not always be more appropriate than qPCR for quantification of low copy numbers, dPCR is a suitable method for robust and reproducible quantification of viral DNA, and a promising technology for the higher-order reference measurement method. PMID:26521179

  16. Identification of TCT, a novel knockdown resistance allele mutation and analysis of resistance detection methods in the voltage-gated Na⁺ channel of Culex pipiens pallens from Shandong Province, China.

    PubMed

    Liu, Hong-Mei; Cheng, Peng; Huang, Xiaodan; Dai, Yu-Hua; Wang, Hai-Fang; Liu, Li-Juan; Zhao, Yu-Qiang; Wang, Huai-Wei; Gong, Mao-Qing

    2013-02-01

    The present study aimed to investigate deltamethrin resistance in Culex pipiens pallens (C. pipiens pallens) mosquitoes and its correlation with knockdown resistance (kdr) mutations. In addition, mosquito‑resistance testing methods were analyzed. Using specific primers in polymerase chain reaction (PCR) and allele-specific (AS)-PCR, kdr gene sequences isolated from wild C. pipiens pallens mosquitoes were sequenced. Linear regression analysis was used to determine the correlation between the mutations and deltamethrin resistance. A kdr allelic gene was cloned and sequenced. Analysis of the DNA sequences revealed the presence of two point mutations at the L1014 residue in the IIS6 transmembrane segment of the voltage‑gated sodium channel (VGSC): L1014F, TTA→TTT, replacing a leucine (L) with a phenylalanine (F); L1014S, TTA→TCA, replacing leucine (L) with serine (S). Two alternative kdr-like mutations, L1014F and L1014S, were identified to be positively correlated with the deltamethrin-resistant phenotype. In addition a novel mutation, TCT, was identified in the VGSC of C. pipiens pallens. PCR and AS-PCR yielded consistent results with respect to mosquito resistance. However, the detection rate of PCR was higher than that of AS-PCR. Further studies are required to determine the specific resistance mechanism. PCR and AS-PCR demonstrated suitability for mosquito resistance field tests, however, the former method may be superior to the latter. PMID:23151871

  17. Single-molecule PCR: an artifact-free PCR approach for the analysis of somatic mutations.

    PubMed

    Kraytsberg, Yevgenya; Khrapko, Konstantin

    2005-09-01

    A critical review of the clone-by-clone approach to the analysis of complex spectra of somatic mutations is presented. The study of a priori unknown somatic mutations requires painstaking analysis of complex mixtures of multiple mutant and non-mutant DNA molecules. If mutant fractions are sufficiently high, these mixtures can be dissected by the cloning of individual DNA molecules and scanning of the individual clones for mutations (e.g., by sequencing). Currently, the majority of such cloning is performed using PCR fragments. However, post-PCR cloning may result in various PCR artifacts - PCR errors and jumping PCR - and preferential amplification of certain mutations. This review argues that single-molecule PCR is a simple alternative that promises to evade the disadvantages inherent to post-PCR cloning and enhance mutational analysis in the future. PMID:16149882

  18. Predominance of PCR-ribotypes, 018 (smz) and 369 (trf) of Clostridium difficile in Japan: a potential relationship with other global circulating strains?

    PubMed

    Senoh, Mitsutoshi; Kato, Haru; Fukuda, Tadashi; Niikawa, Akiko; Hori, Yoshiko; Hagiya, Hideharu; Ito, Yoichiro; Miki, Hiroshi; Abe, Yoshifumi; Furuta, Kiyoshi; Takeuchi, Hideki; Tajima, Hirokazu; Tominaga, Harumi; Satomura, Hideyuki; Kato, Hideaki; Morita, Sayuri; Tanada, Ai; Hara, Toshinori; Kawada, Miki; Sato, Yuka; Takahashi, Masahiko; Higuchi, Akiko; Nakajima, Tomoko; Wakamatsu, Yukiko; Toyokawa, Masahiro; Ueda, Akiko; Roberts, Paul; Miyajima, Fabio; Shibayama, Keigo

    2015-10-01

    Global spread and evolutionary links of an epidemic Clostridium difficile strain (PCR-ribotype 027) have been noted in recent decades. However, in Japan, no outbreaks caused by type 027 have been reported to date. A total of 120 C. difficile isolates from patients at 15 hospitals during non-outbreak seasons between 2011 and 2013 as well as 18 and 21 isolates collected from two hospitals in 2010 and 2009, respectively, in outbreak periods in Japan, were examined. Among these 120 isolates, Japan-ribotypes smz and ysmz (subtype variant of smz) were the most predominant (39.2 %) followed by Japan-ribotype trf (15.8 %). Types smz/ysmz and trf were also concurrently predominant at two hospitals in the outbreak settings. Out of the five binary toxin-positive isolates observed, only one was PCR-ribotype 027 and another PCR-ribotype 078. Type smz was later found to correspond to PCR-ribotype 018. High rates of resistance against gatifloxacin, moxifloxacin, erythromycin and clindamycin were observed in the PCR-ribotype 018 isolates. Interestingly, all trf isolates were toxin A-negative, toxin B-positive, but they did not correspond to PCR-ribotype 017, thus being assigned a new ribotype (PCR-ribotype 369). In conclusion, PCR-ribotypes 018 (smz) and 369 (trf) were identified as major circulating strains in both outbreak and non-outbreak settings in Japan. Given their epidemiological relevance, molecular investigations are warranted to clarify potential evolutionary links with related strains found elsewhere, such as PCR-ribotypes 018 and 017 from Europe and North America. PMID:26238868

  19. A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.

    PubMed

    Thierry, Alain R

    2016-01-01

    Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics

  20. Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism

    PubMed Central

    Lito, Piro; Solomon, Martha; Li, Lian-Sheng; Hansen, Rasmus; Rosen, Neal

    2016-01-01

    It is thought that KRAS oncoproteins are constitutively active because their guanosine triphosphatase (GTPase) activity is disabled. Consequently, drugs targeting the inactive or guanosine 5′-diphosphate–bound conformation are not expected to be effective. We describe a mechanism that enables such drugs to inhibit KRASG12C signaling and cancer cell growth. Inhibition requires intact GTPase activity and occurs because drug-bound KRASG12C is insusceptible to nucleotide exchange factors and thus trapped in its inactive state. Indeed, mutants completely lacking GTPase activity and those promoting exchange reduced the potency of the drug. Suppressing nucleotide exchange activity downstream of various tyrosine kinases enhanced KRASG12C inhibition, whereas its potentiation had the opposite effect. These findings reveal that KRASG12C undergoes nucleotide cycling in cancer cells and provide a basis for developing effective therapies to treat KRASG12C-driven cancers. PMID:26841430

  1. Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism.

    PubMed

    Lito, Piro; Solomon, Martha; Li, Lian-Sheng; Hansen, Rasmus; Rosen, Neal

    2016-02-01

    It is thought that KRAS oncoproteins are constitutively active because their guanosine triphosphatase (GTPase) activity is disabled. Consequently, drugs targeting the inactive or guanosine 5'-diphosphate-bound conformation are not expected to be effective. We describe a mechanism that enables such drugs to inhibit KRAS(G12C) signaling and cancer cell growth. Inhibition requires intact GTPase activity and occurs because drug-bound KRAS(G12C) is insusceptible to nucleotide exchange factors and thus trapped in its inactive state. Indeed, mutants completely lacking GTPase activity and those promoting exchange reduced the potency of the drug. Suppressing nucleotide exchange activity downstream of various tyrosine kinases enhanced KRAS(G12C) inhibition, whereas its potentiation had the opposite effect. These findings reveal that KRAS(G12C) undergoes nucleotide cycling in cancer cells and provide a basis for developing effective therapies to treat KRAS(G12C)-driven cancers. PMID:26841430

  2. Allele-Specific Transcription Factor Binding in Pig Calpastatin Promoter Regions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The identification of predictive DNA markers for pork quality would allow U.S. pork producers and breeders to more quickly and efficiently select genetically superior animals for production of consistent, high quality meat. Genome scans have identified QTL for tenderness on pig chromosome 2 which ha...

  3. Site- and allele-specific polycomb dysregulation in T-cell leukaemia.

    PubMed

    Navarro, Jean-Marc; Touzart, Aurore; Pradel, Lydie C; Loosveld, Marie; Koubi, Myriam; Fenouil, Romain; Le Noir, Sandrine; Maqbool, Muhammad Ahmad; Morgado, Ester; Gregoire, Claude; Jaeger, Sebastien; Mamessier, Emilie; Pignon, Charles; Hacein-Bey-Abina, Salima; Malissen, Bernard; Gut, Marta; Gut, Ivo G; Dombret, Hervé; Macintyre, Elizabeth A; Howe, Steven J; Gaspar, H Bobby; Thrasher, Adrian J; Ifrah, Norbert; Payet-Bornet, Dominique; Duprez, Estelle; Andrau, Jean-Christophe; Asnafi, Vahid; Nadel, Bertrand

    2015-01-01

    T-cell acute lymphoblastic leukaemias (T-ALL) are aggressive malignant proliferations characterized by high relapse rates and great genetic heterogeneity. TAL1 is amongst the most frequently deregulated oncogenes. Yet, over half of the TAL1(+) cases lack TAL1 lesions, suggesting unrecognized (epi)genetic deregulation mechanisms. Here we show that TAL1 is normally silenced in the T-cell lineage, and that the polycomb H3K27me3-repressive mark is focally diminished in TAL1(+) T-ALLs. Sequencing reveals that >20% of monoallelic TAL1(+) patients without previously known alterations display microinsertions or RAG1/2-mediated episomal reintegration in a single site 5' to TAL1. Using 'allelic-ChIP' and CrispR assays, we demonstrate that such insertions induce a selective switch from H3K27me3 to H3K27ac at the inserted but not the germline allele. We also show that, despite a considerable mechanistic diversity, the mode of oncogenic TAL1 activation, rather than expression levels, impact on clinical outcome. Altogether, these studies establish site-specific epigenetic desilencing as a mechanism of oncogenic activation. PMID:25615415

  4. Defining a personal, allele-specific, and single-molecule long-read transcriptome.

    PubMed

    Tilgner, Hagen; Grubert, Fabian; Sharon, Donald; Snyder, Michael P

    2014-07-01

    Personal transcriptomes in which all of an individual's genetic variants (e.g., single nucleotide variants) and transcript isoforms (transcription start sites, splice sites, and polyA sites) are defined and quantified for full-length transcripts are expected to be important for understanding individual biology and disease, but have not been described previously. To obtain such transcriptomes, we sequenced the lymphoblastoid transcriptomes of three family members (GM12878 and the parents GM12891 and GM12892) by using a Pacific Biosciences long-read approach complemented with Illumina 101-bp sequencing and made the following observations. First, we found that reads representing all splice sites of a transcript are evident for most sufficiently expressed genes ≤3 kb and often for genes longer than that. Second, we added and quantified previously unidentified splicing isoforms to an existing annotation, thus creating the first personalized annotation to our knowledge. Third, we determined SNVs in a de novo manner and connected them to RNA haplotypes, including HLA haplotypes, thereby assigning single full-length RNA molecules to their transcribed allele, and demonstrated Mendelian inheritance of RNA molecules. Fourth, we show how RNA molecules can be linked to personal variants on a one-by-one basis, which allows us to assess differential allelic expression (DAE) and differential allelic isoforms (DAI) from the phased full-length isoform reads. The DAI method is largely independent of the distance between exon and SNV--in contrast to fragmentation-based methods. Overall, in addition to improving eukaryotic transcriptome annotation, these results describe, to our knowledge, the first large-scale and full-length personal transcriptome. PMID:24961374

  5. Allele-specific distribution of RNA polymerase II on female X chromosomes.

    PubMed

    Kucera, Katerina S; Reddy, Timothy E; Pauli, Florencia; Gertz, Jason; Logan, Jenae E; Myers, Richard M; Willard, Huntington F

    2011-10-15

    While the distribution of RNA polymerase II (PolII) in a variety of complex genomes is correlated with gene expression, the presence of PolII at a gene does not necessarily indicate active expression. Various patterns of PolII binding have been described genome wide; however, whether or not PolII binds at transcriptionally inactive sites remains uncertain. The two X chromosomes in female cells in mammals present an opportunity to examine each of the two alleles of a given locus in both active and inactive states, depending on which X chromosome is silenced by X chromosome inactivation. Here, we investigated PolII occupancy and expression of the associated genes across the active (Xa) and inactive (Xi) X chromosomes in human female cells to elucidate the relationship of gene expression and PolII binding. We find that, while PolII in the pseudoautosomal region occupies both chromosomes at similar levels, it is significantly biased toward the Xa throughout the rest of the chromosome. The general paucity of PolII on the Xi notwithstanding, detectable (albeit significantly reduced) binding can be observed, especially on the evolutionarily younger short arm of the X. PolII levels at genes that escape inactivation correlate with the levels of their expression; however, additional PolII sites can be found at apparently silenced regions, suggesting the possibility of a subset of genes on the Xi that are poised for expression. Consistent with this hypothesis, we show that a high proportion of genes associated with PolII-accessible sites, while silenced in GM12878, are expressed in other female cell lines. PMID:21791549

  6. SNP Detection in mRNA in Living Cells Using Allele Specific FRET Probes

    PubMed Central

    Dahan, Liya; Huang, Lingyan; Kedmi, Ranit; Behlke, Mark A.; Peer, Dan

    2013-01-01

    Live mRNA detection allows real time monitoring of specific transcripts and genetic alterations. The main challenge of live genetic detection is overcoming the high background generated by unbound probes and reaching high level of specificity with minimal off target effects. The use of Fluorescence Resonance Energy Transfer (FRET) probes allows differentiation between bound and unbound probes thus decreasing background. Probe specificity can be optimized by adjusting the length and through use of chemical modifications that alter binding affinity. Herein, we report the use of two oligonucleotide FRET probe system to detect a single nucleotide polymorphism (SNP) in murine Hras mRNA, which is associated with malignant transformations. The FRET oligonucleotides were modified with phosphorothioate (PS) bonds, 2′OMe RNA and LNA residues to enhance nuclease stability and improve SNP discrimination. Our results show that a point mutation in Hras can be detected in endogenous RNA of living cells. As determined by an Acceptor Photobleaching method, FRET levels were higher in cells transfected with perfect match FRET probes whereas a single mismatch showed decreased FRET signal. This approach promotes in vivo molecular imaging methods and could further be applied in cancer diagnosis and theranostic strategies. PMID:24039756

  7. Transgene Detection by Digital Droplet PCR

    PubMed Central

    Moser, Dirk A.; Braga, Luca; Raso, Andrea; Zacchigna, Serena; Giacca, Mauro; Simon, Perikles

    2014-01-01

    Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term ‘gene doping’ in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future. PMID:25375130

  8. Selecting optimal oligonucleotide primers for multiplex PCR.

    PubMed

    Nicodème, P; Steyaert, J M

    1997-01-01

    We investigate the problem of designing efficient multiplex PCR for medical applications. We show that the problem is NP-complete by transformation to the Multiple Choice Matching problem and give an efficient approximation algorithm. We developed this algorithm in a computer program that predicts which genomic regions may be simultaneously amplified by PCR. Practical use of the software shows that the method can treat 250 non-polymorphic loci with less than 5 simultaneous experiments. PMID:9322038

  9. Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols.

    PubMed

    Giannattasio, S; Bobba, A; Jurgelevicius, V; Vacca, R A; Lattanzio, P; Merafina, R S; Utkus, A; Kucinskas, V; Marra, E

    2006-01-01

    Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes. PMID:17020467

  10. A naked-eye colorimetric "PCR developer"

    NASA Astrophysics Data System (ADS)

    Valentini, Paola; Pompa, Pier Paolo

    2016-04-01

    Despite several advances in molecular biology and diagnostics, Polymerase Chain Reaction (PCR) is currently the gold standard for nucleic acids amplification and detection, due to its versatility, low-cost and universality, with estimated <10 billion reactions per year and a worldwide market of several billion dollars/year. Nevertheless, PCR still relies on the laborious, time-consuming, and multi-step gel electrophoresis-based detection, which includes gel casting, electrophoretic run, gel staining, and gel visualization. In this work, we propose a "PCR developer", namely a universal one-step, one-tube method, based on controlled aggregation of gold nanoparticles (AuNPs), to detect PCR products by naked eye in few minutes, with no need for any instrumentation. We demonstrated the specificity and sensitivity of the PCR developer on different model targets, suitable for a qualitative detection in real-world diagnostics (i.e., gene rearrangements, genetically modified organisms, and pathogens). The PCR developer proved to be highly specific and ultra-sensitive, discriminating down to few copies of HIV viral DNA, diluted in an excess of interfering human genomic DNA, which is a clinically relevant viral load. Hence, it could be a valuable tool for both academic research and clinical applications.

  11. Graft-versus-leukemia activity may overcome therapeutic resistance of chronic lymphocytic leukemia with unmutated immunoglobulin variable heavy-chain gene status: implications of minimal residual disease measurement with quantitative PCR.

    PubMed

    Ritgen, Matthias; Stilgenbauer, Stephan; von Neuhoff, Nils; Humpe, Andreas; Brüggemann, Monika; Pott, Christiane; Raff, Thorsten; Kröber, Alexander; Bunjes, Donald; Schlenk, Richard; Schmitz, Norbert; Döhner, Hartmut; Kneba, Michael; Dreger, Peter

    2004-10-15

    The aim of this study was to investigate if graft-versus-leukemia (GVL) activity conferred by allogeneic stem cell transplantation (allo-SCT) is effective in chronic lymphocytic leukemia (CLL) with unmutated V(H) gene status. The kinetics of residual disease (MRD) were measured by quantitative allele-specific immunoglobulin heavy chain (IgH) polymerase chain reaction (PCR) in 9 patients after nonmyeloablative allo-SCT for unmutated CLL. Despite an only modest decrease in the early posttransplantation phase, MRD became undetectable in 7 of 9 patients (78%) from day +100 onwards subsequent to chronic graft-versus-host disease or donor lymphocyte infusions. With a median follow-up of 25 months (range, 14-37 months), these 7 patients remain in continuous clinical and molecular remission. In contrast, PCR negativity was achieved in only 6 of 26 control patients (23%) after autologous SCT for unmutated CLL and it was not durable. Taken together, this study shows for the first time that GVL-mediated immunotherapy might be effective in CLL with unmutated V(H). PMID:15205268

  12. A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR.

    PubMed

    Gao, Song; He, Dan; Li, Guangquan; Zhang, Yanhua; Lv, Huiying; Wang, Li

    2016-09-15

    Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample. PMID:27393656

  13. A web server for performing electronic PCR

    PubMed Central

    Rotmistrovsky, Kirill; Jang, Wonhee; Schuler, Gregory D.

    2004-01-01

    ‘Electronic PCR’ (e-PCR) refers to a computational procedure that is used to search DNA sequences for sequence tagged sites (STSs), each of which is defined by a pair of primer sequences and an expected PCR product size. To gain speed, our implementation extracts short ‘words’ from the 3′ end of each primer and stores them in a sorted hash table that can be accessed efficiently during the search. One recent improvement is the use of overlapping discontinuous words to allow matches to be found despite the presence of a mismatch. Moreover, it is possible to allow gaps in the alignment between the primer and the sequence. The effect of these changes is to improve sensitivity without significantly affecting specificity. The new software provides a search mode using a query STS against a sequence database to augment the previously available mode using a query sequence against an STS database. Finally, e-PCR may now be used through a web service, with search results linked to other web resources such as the UniSTS database and the MapViewer genome browser. The e-PCR web server may be found at www.ncbi.nlm.nih.gov/sutils/e-pcr. PMID:15215361

  14. Chemically modified primers for improved multiplex PCR

    PubMed Central

    Shum, Jonathan; Paul, Natasha

    2009-01-01

    Multiplexed PCR, the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR set-ups. These complications include a greater probability for non-specific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in such applications as pathogen detection, RNA quantification, mutation analysis and now, next generation DNA sequencing. Herein, we investigate the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses reveal a decrease in off-target amplification and subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex set-ups revealed a greater limit of detection and more uniform amplification of each target as compared to unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue in improving multiplex PCR performance. PMID:19258004

  15. PCR+ In Diesel Fuels and Emissions Research

    SciTech Connect

    McAdams, H.T.

    2002-04-15

    In past work for the U.S. Department of Energy (DOE) and Oak Ridge National Laboratory (ORNL), PCR+ was developed as an alternative methodology for building statistical models. PCR+ is an extension of Principal Components Regression (PCR), in which the eigenvectors resulting from Principal Components Analysis (PCA) are used as predictor variables in regression analysis. The work was motivated by the observation that most heavy-duty diesel (HDD) engine research was conducted with test fuels that had been ''concocted'' in the laboratory to vary selected fuel properties in isolation from each other. This approach departs markedly from the real world, where the reformulation of diesel fuels for almost any purpose leads to changes in a number of interrelated properties. In this work, we present new information regarding the problems encountered in the conventional approach to model-building and how the PCR+ method can be used to improve research on the relationship between fuel characteristics and engine emissions. We also discuss how PCR+ can be applied to a variety of other research problems related to diesel fuels.

  16. Diagnostic PCR of dermatophytes--an overview.

    PubMed

    Gräser, Yvonne; Czaika, Viktor; Ohst, Torsten

    2012-10-01

    The prevalence of onychomycosis is increasing steadily, sevenfold alone in the US within the last twenty years. An important aspect in this development is the demographic development of the human population of the industrial countries like Germany. A fast and accurate laboratory diagnosis is essential for successful treatment because 50% of the cases are misdiagnosed when relying on the clinical appearance only. The current diagnosis of dermatophytosis, based on direct microscopy and culture of the clinical specimen, is problematic given the lacking specificity of the former and the length of time needed for the latter. Molecular techniques can help to solve these problems. In recent years, a number of in-house PCR assays have been developed to identify dermatophytes directly from clinical specimens. Based on the "Mikrobiologisch-infektiologischen Qualitätsstandards (MIQ) für Nukleinsäure-Amplifikationstechniken" and the MIQE guideline (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) 11 studies are reviewed which were published between 2007 and 2010. The present article evaluates the quality of the PCR assays regarding false positive and false negative results due to contamination, PCR format, statistical analysis, and diagnostic performance of the studies. It shows that we are only at the beginning of providing high quality PCR diagnosis of dermatophytes. PMID:23013298

  17. Real-time PCR array chip with capillary-driven sample loading and reactor sealing for point-of-care applications.

    PubMed

    Ramalingam, Naveen; Liu, Hao-Bing; Dai, Chang-Chun; Jiang, Yu; Wang, Hui; Wang, Qinghui; M Hui, Kam; Gong, Hai-Qing

    2009-10-01

    A major challenge for the lab-on-a-chip (LOC) community is to develop point-of-care diagnostic chips that do not use instruments. Such instruments include pumping or liquid handling devices for distribution of patient's nucleic-acid test sample among an array of reactors and microvalves or mechanical parts to seal these reactors. In this paper, we report the development of a primer pair pre-loaded PCR array chip, in which the loading of the PCR mixture into an array of reactors and subsequent sealing of the reactors were realized by a novel capillary-based microfluidics with a manual two-step pipetting operations. The chip is capable of performing simultaneous (parallel) analyses of multiple gene targets and its performance was tested by amplifying twelve different gene targets against cDNA template from human hepatocellular carcinoma using SYBR Green I fluorescent dye. The versatility and reproducibility of the PCR-array chip are demonstrated by real-time PCR amplification of the BNI-1 fragment of SARS cDNA cloned in a plasmid vector. The reactor-to-reactor diffusion of the pre-loaded primer pairs in the chip is investigated to eliminate the possibility of primer cross-contamination. Key technical issues such as PCR mixture loss in gas-permeable PDMS chip layer and bubble generation due to different PDMS-glass bonding methods are investigated. PMID:19421862

  18. Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

    EPA Science Inventory

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

  19. Utility of PCR in diagnosing pulmonary tuberculosis.

    PubMed Central

    Bennedsen, J; Thomsen, V O; Pfyffer, G E; Funke, G; Feldmann, K; Beneke, A; Jenkins, P A; Hegginbothom, M; Fahr, A; Hengstler, M; Cleator, G; Klapper, P; Wilkins, E G

    1996-01-01

    At present, the rapid diagnosis of pulmonary tuberculosis rests with microscopy. However, this technique is insensitive and many cases of pulmonary tuberculosis cannot be initially confirmed. Nucleic acid amplification techniques are extremely sensitive, but when they are applied to tuberculosis diagnosis, they have given variable results. Investigators at six centers in Europe compared a standardized PCR system (Amplicor; Roche) against conventional culture methods. Defined clinical information was collected. Discrepant samples were retested, and inhibition assays and backup amplification with a separate primer pair were performed. Mycobacterium tuberculosis complex organisms were recovered from 654 (9.1%) of 7,194 samples and 293 (7.8%) of 3,738 patients. Four hundred fifty-two of the M. tuberculosis isolates from 204 patients were smear positive and culture positive. Among the culture-positive specimens, PCR had a sensitivity of 91.4% for smear-positive specimens and 60.9% for smear-negative specimens, with a specificity of 96.1%. Analysis of 254 PCR-positive, culture-negative specimens with discrepant results revealed that 130 were from patients with recently diagnosed tuberculosis and 94 represented a presumed laboratory error. Similar analysis of 118 PCR-negative, culture-positive specimens demonstrated that 27 discrepancies were due to presumed uneven aliquot distribution and 11 were due to presumed laboratory error; PCR inhibitors were detected in 8 specimens. Amplicor enables laboratories with little previous experience with nucleic acid amplification to perform PCR. Disease in more than 60% of the patients with tuberculosis with smear-negative, culture-positive specimens can be diagnosed at the time of admission, and potentially all patients with smear-positive specimens can immediately be confirmed as being infected with M. tuberculosis, leading to improved clinical management. PMID:8735089

  20. PCR microfluidic devices for DNA amplification.

    PubMed

    Zhang, Chunsun; Xu, Jinliang; Ma, Wenli; Zheng, Wenling

    2006-01-01

    The miniaturization of biological and chemical analytical devices by micro-electro-mechanical-systems (MEMS) technology has posed a vital influence on such fields as medical diagnostics, microbial detection and other bio-analysis. Among many miniaturized analytical devices, the polymerase chain reaction (PCR) microchip/microdevices are studied extensively, and thus great progress has been made on aspects of on-chip micromachining (fabrication, bonding and sealing), choice of substrate materials, surface chemistry and architecture of reaction vessel, handling of necessary sample fluid, controlling of three or two-step temperature thermocycling, detection of amplified nucleic acid products, integration with other analytical functional units such as sample preparation, capillary electrophoresis (CE), DNA microarray hybridization, etc. However, little has been done on the review of above-mentioned facets of the PCR microchips/microdevices including the two formats of flow-through and stationary chamber in spite of several earlier reviews [Zorbas, H. Miniature continuous-flow polymerase chain reaction: a breakthrough? Angew Chem Int Ed 1999; 38 (8):1055-1058; Krishnan, M., Namasivayam, V., Lin, R., Pal, R., Burns, M.A. Microfabricated reaction and separation systems. Curr Opin Biotechnol 2001; 12:92-98; Schneegabeta, I., Köhler, J.M. Flow-through polymerase chain reactions in chip themocyclers. Rev Mol Biotechnol 2001; 82:101-121; deMello, A.J. DNA amplification: does 'small' really mean 'efficient'? Lab Chip 2001; 1: 24N-29N; Mariella, Jr. R. MEMS for bio-assays. Biomed Microdevices 2002; 4 (2):77-87; deMello AJ. Microfluidics: DNA amplification moves on. Nature 2003; 422:28-29; Kricka, L.J., Wilding, P. Microchip PCR. Anal BioAnal Chem 2003; 377:820-825]. In this review, we survey the advances of the above aspects among the PCR microfluidic devices in detail. Finally, we also illuminate the potential and practical applications of PCR microfluidics to some fields such

  1. Real-time PCR: Advanced technologies and applications

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This book brings together contributions from 20 experts in the field of PCR, providing a broad perspective of the applications of quantitative real-time PCR (qPCR). The editors state in the preface that the aim is to provide detailed insight into underlying principles and methods of qPCR to provide ...

  2. PCR AS A DIAGNOSTIC TOOL FOR BRUCELLOSIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Numerous PCR-based assays have been developed for the identification of Brucella to improve diagnostic capabilities. Collectively, the repertoire of assays addresses several aspects of the diagnostic process. For some purposes, the simple identification of Brucella is adequate (e.g. diagnosis of ...

  3. The Power of Real-Time PCR

    ERIC Educational Resources Information Center

    Valasek, Mark A.; Repa, Joyce J.

    2005-01-01

    In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this technology is the rapid and accurate assessment of changes in gene…

  4. Handheld real-time PCR device.

    PubMed

    Ahrberg, Christian D; Ilic, Bojan Robert; Manz, Andreas; Neužil, Pavel

    2016-02-01

    Here we report one of the smallest real-time polymerase chain reaction (PCR) systems to date with an approximate size of 100 mm × 60 mm × 33 mm. The system is an autonomous unit requiring an external 12 V power supply. Four simultaneous reactions are performed in the form of virtual reaction chambers (VRCs) where a ≈200 nL sample is covered with mineral oil and placed on a glass cover slip. Fast, 40 cycle amplification of an amplicon from the H7N9 gene was used to demonstrate the PCR performance. The standard curve slope was -3.02 ± 0.16 cycles at threshold per decade (mean ± standard deviation) corresponding to an amplification efficiency of 0.91 ± 0.05 per cycle (mean ± standard deviation). The PCR device was capable of detecting a single deoxyribonucleic acid (DNA) copy. These results further suggest that our handheld PCR device may have broad, technologically-relevant applications extending to rapid detection of infectious diseases in small clinics. PMID:26753557

  5. Real-time PCR (qPCR) primer design using free online software.

    PubMed

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. PMID:21445907

  6. Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases.

    PubMed

    Mock, Ulrike; Hauber, Ilona; Fehse, Boris

    2016-03-01

    Genome editing using designer nucleases such as transcription activator-like effector nucleases (TALENs) or clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 nucleases is an emerging technology in basic and applied research. Whereas the application of editing tools, namely CRISPR-Cas9, has recently become very straightforward, quantification of resulting gene knockout rates still remains a bottleneck. This is particularly true if the product of a targeted gene is not easily detectable. To address this problem, we devised a novel gene-editing frequency digital PCR (GEF-dPCR) technique. GEF-dPCR exploits two differently labeled probes that are placed within one amplicon at the gene-editing target site to simultaneously detect wild-type and nonhomologous end-joining (NHEJ)-affected alleles. Taking advantage of the principle of dPCR, this enables concurrent quantification of edited and wild-type alleles in a given sample. We propose that our method is optimal for the monitoring of gene-edited cells in vivo, e.g., in clinical settings. Here we describe preparation, design of primers and probes, and setup and analysis of GEF-dPCR. The setup of GEF-dPCR requires up to 2 weeks (depending on the starting point); once the dPCR has been established, the protocol for sample analysis takes <1 d. PMID:26914317

  7. A comparison of the effects of PCR inhibition in quantitative PCR and forensic STR analysis.

    PubMed

    Funes-Huacca, Maribel E; Opel, Kerry; Thompson, Robyn; McCord, Bruce R

    2011-04-01

    In this paper we compare the effects of three representative PCR inhibitors using quantitative PCR (qPCR) and multiplex STR amplification in order to determine the effect of inhibitor concentration on allele dropout and to develop better ways to interpret forensic DNA data. We have used humic acid, collagen and calcium phosphate at different concentrations to evaluate the profiles of alleles inhibited in these amplifications. These data were correlated with previously obtained results from quantitative PCR including melt curve effects, efficiency changes and cycle threshold (Ct) values. Overall, the data show that there are two competing processes that result from PCR inhibition. The first process is a general loss of larger alleles. This appears to occur with all inhibitors. The second process is more sequence specific and occurs when the inhibitor binds DNA, altering the cycle threshold and the melt curve. This sequence-specific inhibition results in patterns of allele loss that occur in addition to the overall loss of larger alleles. The data demonstrate the applicability of utilizing real-time PCR results to predict the presence of certain types of PCR inhibition in STR analysis. PMID:21462225

  8. Identification of bacterial plant pathogens using multilocus PCR and electrospray ionization-mass spectrometry (PCR/ESI-MS)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS, previously known as “TIGER”) utilizes PCR with broad range primers to amplify products from wide array of organisms within a taxonomic group, followed by analysis of PCR amplicons using mass spectrometry. Computer analysis of precise masses ...

  9. Rapid Simultaneous Detection of Enterovirus and Parechovirus RNAs in Clinical Samples by One-Step Real-Time Reverse Transcription-PCR Assay ▿

    PubMed Central

    Bennett, Susan; Harvala, Heli; Witteveldt, Jeroen; McWilliam Leitch, E. Carol; McLeish, Nigel; Templeton, Kate; Gunson, Rory; Carman, William F.; Simmonds, Peter

    2011-01-01

    Enteroviruses (EVs) are recognized as the major etiological agent in meningitis in children and young adults. The use of molecular techniques, such as PCR, has substantially improved the sensitivity of enterovirus detection compared to that of virus culture methods. PCR-based methods also can detect a much wider range of EV variants, including those within species A, as well as human parechoviruses (HPeVs) that often grow poorly in vitro and which previously have been underdiagnosed by traditional methods. To exploit these developments, we developed a real-time one-step reverse transcription-PCR (RT-PCR) for the rapid and sensitive detection of EV and HPeV in clinical specimens. Two commercially available RT-PCR kits were used (method I, Platinum one-step kit; method II, Express qPCR one-step kit) with primers and probes targeting the EV and HPeV 5′-untranslated regions (5′UTR). Amplification dynamics (threshold cycle [CT]values and efficiencies) of absolutely quantified full-length RNA transcripts representative of EV species A to D and HPeV were similar, demonstrating the effectiveness of both assays across the range of currently described human EV and HPeV variants. Probit analysis of multiple endpoint replicates demonstrated comparable sensitivities of the assays for EV and HPeV (method I, approximately 10 copies per reaction for both targets; method II, 20 copies per reaction). CT values were highly reproducible on repeat testing of positive controls within assays and between assay runs. Considering the sample turnaround time of less than 3 h, the multiplexed one-step RT-PCR method provides rapid diagnostic testing for EV and HPeV in cases of suspected central nervous system infections in a clinically relevant time frame. PMID:21593263

  10. Rapid PCR thermocycling using microscale thermal convection.

    PubMed

    Muddu, Radha; Hassan, Yassin A; Ugaz, Victor M

    2011-01-01

    Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed(1). Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format. Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations(2-9). Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument. One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time(10,11). Here we describe results of our efforts to probe the full 3-D velocity and

  11. Clinical Utility of Droplet Digital PCR for Human Cytomegalovirus

    PubMed Central

    Sedlak, Ruth Hall; Cook, Linda; Cheng, Anqi; Magaret, Amalia

    2014-01-01

    Human cytomegalovirus (CMV) has historically been the major infectious cause of morbidity and mortality among patients receiving hematopoietic cell or organ transplant. Standard care in a transplant setting involves frequent monitoring of CMV viral load over weeks to months to determine when antiviral treatment may be required. Quantitative PCR (qPCR) is the standard molecular diagnostic method for monitoring. Recently, digital PCR (dPCR) has shown promise in viral diagnostics, although current dPCR systems have lower throughput than qPCR systems. Here, we compare qPCR and droplet digital PCR (ddPCR) for CMV detection in patient plasma samples. Droplet digital PCR exhibits increased precision over qPCR at viral loads of ≥4 log10 with equivalent sensitivity. However, retrospective analysis of longitudinal samples from transplant patients with CMV viral loads near therapeutic thresholds did not provide evidence that the improved precision of ddPCR would be of clinical benefit. Given the throughput advantages of current qPCR systems, a widespread switch to dPCR for CMV monitoring would appear premature. PMID:24871214

  12. The effects of metal ion PCR inhibitors on results obtained with the Quantifiler(®) Human DNA Quantification Kit.

    PubMed

    Combs, Laura Gaydosh; Warren, Joseph E; Huynh, Vivian; Castaneda, Joanna; Golden, Teresa D; Roby, Rhonda K

    2015-11-01

    Forensic DNA samples may include the presence of PCR inhibitors, even after extraction and purification. Studies have demonstrated that metal ions, co-purified at specific concentrations, inhibit DNA amplifications. Metal ions are endogenous to sample types, such as bone, and can be introduced from environmental sources. In order to examine the effect of metal ions as PCR inhibitors during quantitative real-time PCR, 2800 M DNA was treated with 0.0025-18.750 mM concentrations of aluminum, calcium, copper, iron, nickel, and lead. DNA samples, both untreated and metal-treated, were quantified using the Quantifiler(®) Human DNA Quantification Kit. Quantification cycle (Cq) values for the Quantifiler(®) Human DNA and internal PCR control (IPC) assays were measured and the estimated concentrations of human DNA were obtained. Comparisons were conducted between metal-treated and control DNA samples to determine the accuracy of the quantification estimates and to test the efficacy of the IPC inhibition detection. This kit is most resistant to the presence of calcium as compared to all metals tested; the maximum concentration tested does not affect the amplification of the IPC or quantification of the sample. This kit is most sensitive to the presence of aluminum; concentrations greater than 0.0750 mM negatively affected the quantification, although the IPC assay accurately assessed the presence of PCR inhibition. The Quantifiler(®) Human DNA Quantification Kit accurately quantifies human DNA in the presence of 0.5000 mM copper, iron, nickel, and lead; however, the IPC does not indicate the presence of PCR inhibition at this concentration of these metals. Unexpectedly, estimates of DNA quantity in samples treated with 18.750 mM copper yielded values in excess of the actual concentration of DNA in the samples; fluorescence spectroscopy experiments indicated this increase was not a direct interaction between the copper metal and 6-FAM dye used to label the probe that

  13. The Next-Generation PCR-Based Quantification Method for Ambient Waters: Digital PCR.

    PubMed

    Cao, Yiping; Griffith, John F; Weisberg, Stephen B

    2016-01-01

    Real-time quantitative PCR (qPCR) is increasingly being used for ambient water monitoring, but development of digital polymerase chain reaction (digital PCR) has the potential to further advance the use of molecular techniques in such applications. Digital PCR refines qPCR by partitioning the sample into thousands to millions of miniature reactions that are examined individually for binary endpoint results, with DNA density calculated from the fraction of positives using Poisson statistics. This direct quantification removes the need for standard curves, eliminating the labor and materials associated with creating and running standards with each batch, and removing biases associated with standard variability and mismatching amplification efficiency between standards and samples. Confining reactions and binary endpoint measurements to small partitions also leads to other performance advantages, including reduced susceptibility to inhibition, increased repeatability and reproducibility, and increased capacity to measure multiple targets in one analysis. As such, digital PCR is well suited for ambient water monitoring applications and is particularly advantageous as molecular methods move toward autonomous field application. PMID:27460373

  14. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri.

    PubMed

    Zhao, Yun; Xia, Qingyan; Yin, Youping; Wang, Zhongkang

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

  15. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri

    PubMed Central

    Yin, Youping; Wang, Zhongkang

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

  16. PCR identification of Mycobacterium bovis BCG.

    PubMed Central

    Talbot, E A; Williams, D L; Frothingham, R

    1997-01-01

    The attenuated bacillus Calmette-Guérin (BCG) vaccine strain is derived from a virulent strain of Mycobacterium bovis. BCG is difficult to differentiate from other strains of M. bovis and other members of the M. tuberculosis complex by conventional methods. Recently, a genomic region designated RD1 was found to be present in all virulent M. bovis and M. tuberculosis strains tested but deleted from all BCG strains tested. With this information, a multiplex PCR method was developed to detect the RD1 deletion. A large collection of BCG and other M. tuberculosis complex strains from diverse host and geographic origins was tested. RD1 was deleted in 23 of 23 BCG strains. RD1 was present in 129 of 129 other M. tuberculosis complex strains. This multiplex PCR method can be used as a tool for the rapid and specific identification of BCG. PMID:9041390

  17. Replaceable Microfluidic Cartridges for a PCR Biosensor

    NASA Technical Reports Server (NTRS)

    Francis, Kevin; Sullivan, Ron

    2005-01-01

    The figure depicts a replaceable microfluidic cartridge that is a component of a miniature biosensor that detects target deoxyribonucleic acid (DNA) sequences. The biosensor utilizes (1) polymerase chain reactions (PCRs) to multiply the amount of DNA to be detected, (2) fluorogenic polynucleotide probe chemicals for labeling the target DNA sequences, and (3) a high-sensitivity epifluorescence-detection optoelectronic subsystem. Microfluidics is a relatively new field of device development in which one applies techniques for fabricating microelectromechanical systems (MEMS) to miniature systems for containing and/or moving fluids. Typically, microfluidic devices are microfabricated, variously, from silicon or polymers. The development of microfluidic devices for applications that involve PCR and fluorescence-based detection of PCR products poses special challenges

  18. Aspergillus Collagen-Like Genes (acl): Identification, Sequence Polymorphism, and Assessment for PCR-Based Pathogen Detection

    PubMed Central

    Tuntevski, Kiril; Durney, Brandon C.; Snyder, Anna K.; LaSala, P. Rocco; Nayak, Ajay P.; Green, Brett J.; Beezhold, Donald H.; Rio, Rita V. M.; Holland, Lisa A.

    2013-01-01

    The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects. PMID:24123732

  19. Digital PCR analysis of circulating nucleic acids.

    PubMed

    Hudecova, Irena

    2015-10-01

    Detection of plasma circulating nucleic acids (CNAs) requires the use of extremely sensitive and precise methods. The commonly used quantitative real-time polymerase chain reaction (PCR) poses certain technical limitations in relation to the precise measurement of CNAs whereas the costs of massively parallel sequencing are still relatively high. Digital PCR (dPCR) now represents an affordable and powerful single molecule counting strategy to detect minute amounts of genetic material with performance surpassing many quantitative methods. Microfluidic (chip) and emulsion (droplet)-based technologies have already been integrated into platforms offering hundreds to millions of nanoliter- or even picoliter-scale reaction partitions. The compelling observations reported in the field of cancer research, prenatal testing, transplantation medicine and virology support translation of this technology into routine use. Extremely sensitive plasma detection of rare mutations originating from tumor or placental cells among a large background of homologous sequences facilitates unraveling of the early stages of cancer or the detection of fetal mutations. Digital measurement of quantitative changes in plasma CNAs associated with cancer or graft rejection provides valuable information on the monitoring of disease burden or the recipient's immune response and subsequent therapy treatment. Furthermore, careful quantitative assessment of the viral load offers great value for effective monitoring of antiviral therapy for immunosuppressed or transplant patients. The present review describes the inherent features of dPCR that make it exceptionally robust in precise and sensitive quantification of CNAs. Moreover, I provide an insight into the types of potential clinical applications that have been developed by researchers to date. PMID:25828047

  20. Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

    SciTech Connect

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2010-08-27

    Research highlights: {yields} Incubating PCR products at a high temperature causes smears in gel electrophoresis. {yields} Smears interfere with the interpretation of methylation analysis using COBRA. {yields} Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. {yields} The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 {sup o}C or 65 {sup o}C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

  1. Clostridium difficile PCR Ribotypes in Calves, Canada

    PubMed Central

    Stämpfli, Henry R.; Duffield, Todd; Peregrine, Andrew S.; Trotz-Williams, Lise A.; Arroyo, Luis G.; Brazier, Jon S.; Weese, J. Scott

    2006-01-01

    We investigated Clostridium difficile in calves and the similarity between bovine and human C. difficile PCR ribotypes by conducting a case-control study of calves from 102 dairy farms in Canada. Fecal samples from 144 calves with diarrhea and 134 control calves were cultured for C. difficile and tested with an ELISA for C. difficile toxins A and B. C. difficile was isolated from 31 of 278 calves: 11 (7.6%) of 144 with diarrhea and 20 (14.9%) of 134 controls (p = 0.009). Toxins were detected in calf feces from 58 (56.8%) of 102 farms, 57 (39.6%) of 144 calves with diarrhea, and 28 (20.9%) of 134 controls (p = 0.0002). PCR ribotyping of 31 isolates showed 8 distinct patterns; 7 have been identified in humans, 2 of which have been associated with outbreaks of severe disease (PCR types 017 and 027). C. difficile may be associated with calf diarrhea, and cattle may be reservoirs of C. difficile for humans. PMID:17283624

  2. Quantitative PCR marker genes for endometrial adenocarcinoma.

    PubMed

    Kölbl, Alexandra C; Victor, Lisa-Marie; Birk, Amelie E; Jeschke, Udo; Andergassen, Ulrich

    2016-09-01

    Endometrial adenocarcinoma is a common malignancy in women worldwide, with formation of remote metastasis occurring following oncological treatment. Circulating tumor cells (CTCs) are regarded to be the origin of haematogenous metastasis formation. The present study aimed to identify suitable marker genes using a quantitative polymerase chain reaction (qPCR) approach to detect CTCs from blood samples of patients with endometrial carcinoma. Therefore, RNA was isolated from endometrial adenocarcinoma cell lines and from healthy endometrial tissue and reverse transcribed to cDNA, which was then used in qPCR on a number of marker genes. Cytokeratin 19 and claudin 4 were identified as suitable marker genes for CTCs in endometrial adenocarcinoma, due to their high expression in the majority of the cell lines investigated. The expression values of the genes examined varied widely between the different cell lines, which is similar to the variation in the patient samples. Therefore, the necessity for a set of genes for CTC detection and not one single marker gene is demonstrated. qPCR is a fast, cost‑efficient and easy to perform technique, which may be used in the detection of CTCs. Investigation of the occurrence of CTCs in cancer patients would aid in the prevention of metastasis and thereby refine treatment. PMID:27431566

  3. TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR

    PubMed Central

    Zhang, Qian; Wang, Jing; Deng, Fang; Yan, Zhengjian; Xia, Yinglin; Wang, Zhongliang; Ye, Jixing; Deng, Youlin; Zhang, Zhonglin; Qiao, Min; Li, Ruifang; Denduluri, Sahitya K.; Wei, Qiang; Zhao, Lianggong; Lu, Shun; Wang, Xin; Tang, Shengli; Liu, Hao; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan; Jiang, Li

    2015-01-01

    The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification of gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is the most commonly-used platform due to its inexpensive nature and robust chemistry, quantifying the expression of genes with low abundance or RNA samples extracted from highly restricted or limited sources can be challenging because the detection sensitivity of SYBR Green-based qPCR is limited. Here, we develop a novel and effective touchdown qPCR (TqPCR) protocol by incorporating a 4-cycle touchdown stage prior to the quantification amplification stage. Using the same cDNA templates, we find that TqPCR can reduce the average Cq values for Gapdh, Rps13, and Hprt1 reference genes by 4.45, 5.47, and 4.94 cycles, respectively, when compared with conventional qPCR; the overall average Cq value reduction for the three reference genes together is 4.95. We further find that TqPCR can improve PCR amplification efficiency and thus increase detection sensitivity. When the quantification of Wnt3A-induced target gene expression in mesenchymal stem cells is analyzed, we find that, while both conventional qPCR and TqPCR can detect the up-regulation of the relatively abundant target Axin2, only TqPCR can detect the up-regulation of the lowly-expressed targets Oct4 and Gbx2. Finally, we demonstrate that the MRQ2 and MRQ3 primer pairs derived from mouse reference gene Tbp can be used to validate the RNA/cDNA integrity of qPCR samples. Taken together, our results strongly suggest that TqPCR may increase detection sensitivity and PCR amplification efficiency. Overall, TqPCR should be advantageous over conventional qPCR in expression quantification, especially when the transcripts of interest are lowly expressed, and/or the availability of total RNA is highly restricted or limited. PMID:26172450

  4. Viral diagnostics in the era of digital PCR

    PubMed Central

    Sedlak, Ruth Hall; Jerome, Keith R.

    2012-01-01

    Unlike quantitative PCR (qPCR), digital PCR (dPCR) achieves sensitive and accurate absolute quantitation of a DNA sample without the need for a standard curve. A single PCR reaction is divided into many separate reactions that each have a positive or negative signal. By applying Poisson statistics, the number of DNA molecules in the original sample is directly calculated from the number of positive and negative reactions. The recent availability of multiple commercial dPCR platforms has led to increased interest in clinical diagnostic applications, such as low viral load detection and low abundance mutant detection, where dPCR could be superior to traditional qPCR.Here we review current literature that demonstrates dPCR’s potential utility in viral diagnostics, particularly through absolute quantification of target DNA sequences and rare mutant allele detection. PMID:23182074

  5. Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR).

    PubMed

    Floren, C; Wiedemann, I; Brenig, B; Schütz, E; Beck, J

    2015-04-15

    Species fraud and product mislabelling in processed food, albeit not being a direct health issue, often results in consumer distrust. Therefore methods for quantification of undeclared species are needed. Targeting mitochondrial DNA, e.g. CYTB gene, for species quantification is unsuitable, due to a fivefold inter-tissue variation in mtDNA content per cell resulting in either an under- (-70%) or overestimation (+160%) of species DNA contents. Here, we describe a reliable two-step droplet digital PCR (ddPCR) assay targeting the nuclear F2 gene for precise quantification of cattle, horse, and pig in processed meat products. The ddPCR assay is advantageous over qPCR showing a limit of quantification (LOQ) and detection (LOD) in different meat products of 0.01% and 0.001%, respectively. The specificity was verified in 14 different species. Hence, determining F2 in food by ddPCR can be recommended for quality assurance and control in production systems. PMID:25466124

  6. Lab-on-a-chip PCR: real time PCR in miniaturized format for HLA diagnostics

    NASA Astrophysics Data System (ADS)

    Gaertner, Claudia; Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Sewart, René; Frank, Rainer; Willems, Andreas

    2014-05-01

    In case of transplantation or the identification of special metabolic diseases like coeliac disease, HLA typing has to be done fast and reliably with easy-to-handle devices by using limited amount of sample. Against this background a lab-on-a-chip device was realized enabling a fast HLA typing via miniaturized Real-time PCR. Hereby, two main process steps were combined, namely the extraction of DNA from whole blood and the amplification of the target DNA by Real-time PCR giving rise-to a semi-quantitative analysis. For the implementation of both processes on chip, a sample preparation and a real-time module were used. Sample preparation was carried out by using magnetic beads that were stored directly on chip as dry powder, together with all lysis reagents. After purification of the DNA by applying a special buffer regime, the sample DNA was transferred into the PCR module for amplification and detection. Coping with a massively increased surface-to-volume ratio, which results in a higher amount of unspecific binding on the chip surface, special additives needed to be integrated to compensate for this effect. Finally the overall procedure showed a sensitivity comparable to standard Real-time PCR but reduced the duration of analysis to significantly less than one hour. The presented work demonstrates that the combination of lab-on-a-chip PCR with direct optical read-out in a real-time fashion is an extremely promising tool for molecular diagnostics.

  7. Multiplex PCR serogrouping of Listeria monocytogenes isolated in Japan.

    PubMed

    Shimojima, Yukako; Ida, Miki; Nishino, Yukari; Ishitsuka, Rie; Kuroda, Sumiyo; Hirai, Akihiko; Sadamasu, Kenji; Nakama, Akiko; Kai, Akemi

    2016-04-01

    PCR serogrouping methods were used to examine strains of L. monocytogenes isolated in Japan. Among 187 strains, 99.5% were classified into 4 PCR serogroups corresponding to conventional serotypes. Only one isolate had a new PCR profile, which may be a variant of serogroup IVb. PMID:26537550

  8. Multiplex PCR serogrouping of Listeria monocytogenes isolated in Japan

    PubMed Central

    SHIMOJIMA, Yukako; IDA, Miki; NISHINO, Yukari; ISHITSUKA, Rie; KURODA, Sumiyo; HIRAI, Akihiko; SADAMASU, Kenji; NAKAMA, Akiko; KAI, Akemi

    2015-01-01

    PCR serogrouping methods were used to examine strains of L. monocytogenes isolated in Japan. Among 187 strains, 99.5% were classified into 4 PCR serogroups corresponding to conventional serotypes. Only one isolate had a new PCR profile, which may be a variant of serogroup IVb. PMID:26537550

  9. Determining Fungi rRNA Copy Number by PCR

    EPA Science Inventory

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within ...

  10. Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression.

    PubMed

    Lewis, April M; Rice, Kelly C

    2016-01-01

    Quantitative real-time polymerase chain reaction (qPCR) is a sensitive tool that can be used to quantify and compare the amount of specific RNA transcripts between different biological samples. This chapter describes the use of a "two-step" qPCR method to calculate the relative fold change of expression of genes of interest in S. aureus. Using this work-flow, cDNA is synthesized from RNA templates (previously checked for the absence of significant genomic DNA contamination) using a cocktail of random primers and reverse-transcriptase enzyme. The cDNA pools generated can then be assessed for expression of specific genes of interest using SYBR Green-based qPCR and quantification of relative fold-change expression. PMID:25646613

  11. Coupling Spore Traps and Quantitative PCR Assays for Detection of the Downy Mildew Pathogens of Spinach (Peronospora effusa) and Beet (P. schachtii)

    PubMed Central

    Klosterman, Steven J.; Anchieta, Amy; McRoberts, Neil; Koike, Steven T.; Subbarao, Krishna V.; Voglmayr, Hermann; Choi, Young-Joon; Thines, Marco; Martin, Frank N.

    2016-01-01

    Downy mildew of spinach (Spinacia oleracea), caused by Peronospora effusa, is a production constraint on production worldwide, including in California, where the majority of U.S. spinach is grown. The aim of this study was to develop a real-time quantitative polymerase chain reaction (qPCR) assay for detection of airborne inoculum of P. effusa in California. Among oomycete ribosomal DNA (rDNA) sequences examined for assay development, the highest nucleotide sequence identity was observed between rDNA sequences of P. effusa and P. schachtii, the cause of downy mildew on sugar beet and Swiss chard in the leaf beet group (Beta vulgaris subsp. vulgaris). Single-nucleotide polymorphisms were detected between P. effusa and P. schachtii in the 18S rDNA regions for design of P. effusa- and P. schachtii-specific TaqMan probes and reverse primers. An allele-specific probe and primer amplification method was applied to determine the frequency of both P. effusa and P. schachtii rDNA target sequences in pooled DNA samples, enabling quantification of rDNA of P. effusa from impaction spore trap samples collected from spinach production fields. The rDNA copy numbers of P. effusa were, on average, ≈3,300-fold higher from trap samples collected near an infected field compared with those levels recorded at a site without a nearby spinach field. In combination with disease-conducive weather forecasting, application of the assays may be helpful to time fungicide applications for disease management. PMID:24964150

  12. 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.

  13. Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

    PubMed Central

    O'Sullivan, Matthew V. N.; Zhou, Fei; Sintchenko, Vitali; Kong, Fanrong; Gilbert, Gwendolyn L.

    2011-01-01

    Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific. Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms. The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane. PMID:21847083

  14. DNA fingerprinting of medically important microorganisms by use of PCR.

    PubMed Central

    van Belkum, A

    1994-01-01

    Selected segments of any DNA molecule can be amplified exponentially by PCR. This technique provides a powerful tool to detect and identify minimal numbers of microorganisms. PCR is applicable both in diagnosis and in epidemiology. By amplification of hypervariable DNA domains, differences can be detected even among closely related strains. PCR fingerprinting is a valuable tool for medical microbiologists, epidemiologists, and microbial taxonomists. The current state of PCR-mediated genotyping is reviewed, and a comparison with conventional molecular typing methods is included. Because of its speed and versatility, PCR fingerprinting will play an important role in microbial genetics, epidemiology, and systematics. Images PMID:8055466

  15. Rapid identification of Brucella isolates to the species level by real time PCR based single nucleotide polymorphism (SNP) analysis

    PubMed Central

    Gopaul, Krishna K; Koylass, Mark S; Smith, Catherine J; Whatmore, Adrian M

    2008-01-01

    Background Brucellosis, caused by members of the genus Brucella, remains one of the world's major zoonotic diseases. Six species have classically been recognised within the family Brucella largely based on a combination of classical microbiology and host specificity, although more recently additional isolations of novel Brucella have been reported from various marine mammals and voles. Classical identification to species level is based on a biotyping approach that is lengthy, requires extensive and hazardous culturing and can be difficult to interpret. Here we describe a simple and rapid approach to identification of Brucella isolates to the species level based on real-time PCR analysis of species-specific single nucleotide polymorphisms (SNPs) that were identified following a robust and extensive phylogenetic analysis of the genus. Results Seven pairs of short sequence Minor Groove Binding (MGB) probes were designed corresponding to SNPs shown to possess an allele specific for each of the six classical Brucella spp and the marine mammal Brucella. Assays were optimised to identical reaction parameters in order to give a multiple outcome assay that can differentiate all the classical species and Brucella isolated from marine mammals. The scope of the assay was confirmed by testing of over 300 isolates of Brucella, all of which typed as predicted when compared to other phenotypic and genotypic approaches. The assay is sensitive being capable of detecting and differentiating down to 15 genome equivalents. We further describe the design and testing of assays based on three additional SNPs located within the 16S rRNA gene that ensure positive discrimination of Brucella from close phylogenetic relatives on the same platform. Conclusion The multiple-outcome assay described represents a new tool for the rapid, simple and unambiguous characterisation of Brucella to the species level. Furthermore, being based on a robust phylogenetic framework, the assay provides a platform

  16. Denoising PCR-amplified metagenome data

    PubMed Central

    2012-01-01

    Background PCR amplification and high-throughput sequencing theoretically enable the characterization of the finest-scale diversity in natural microbial and viral populations, but each of these methods introduces random errors that are difficult to distinguish from genuine biological diversity. Several approaches have been proposed to denoise these data but lack either speed or accuracy. Results We introduce a new denoising algorithm that we call DADA (Divisive Amplicon Denoising Algorithm). Without training data, DADA infers both the sample genotypes and error parameters that produced a metagenome data set. We demonstrate performance on control data sequenced on Roche’s 454 platform, and compare the results to the most accurate denoising software currently available, AmpliconNoise. Conclusions DADA is more accurate and over an order of magnitude faster than AmpliconNoise. It eliminates the need for training data to establish error parameters, fully utilizes sequence-abundance information, and enables inclusion of context-dependent PCR error rates. It should be readily extensible to other sequencing platforms such as Illumina. PMID:23113967

  17. In silico PCR primer designing and validation.

    PubMed

    Kumar, Anil; Chordia, Nikita

    2015-01-01

    Polymerase chain reaction (PCR) is an enzymatic reaction whose efficiency and sensitivity largely depend on the efficiency of the primers that are used for the amplification of a concerned gene/DNA fragment. Selective amplification of nucleic acid molecules initially present in minute quantities provides a powerful tool for analyzing nucleic acids. In silico method helps in designing primers. There are various programs available for PCR primer design. Here we described designing of primers using web-based tools like "Primer3" and "Web Primer". For designing the primer, DNA template sequence is required that can be taken from any of the available sequence databases, e.g., RefSeq database. The in silico validation can be carried out using BLAST tool and Gene Runner software, which check their efficiency and specificity. Thereafter, the primers designed in silico can be validated in the wet lab. After that, these validated primers can be synthesized for use in the amplification of concerned gene/DNA fragment. PMID:25697657

  18. An evaluation of direct PCR amplification

    PubMed Central

    Hall, Daniel E.; Roy, Reena

    2014-01-01

    Aim To generate complete DNA profiles from blood and saliva samples deposited on FTA® and non-FTA® paper substrates following a direct amplification protocol. Methods Saliva samples from living donors and blood samples from deceased individuals were deposited on ten different FTA® and non-FTA® substrates. These ten paper substrates containing body fluids were kept at room temperature for varying lengths of time ranging from one day to approximately one year. For all assays in this research, 1.2 mm punches were collected from each substrate containing one type of body fluid and amplified with reagents provided in the nine commercial polymerase chain reaction (PCR) amplification kits. The substrates were not subjected to purification reagent or extraction buffer prior to amplification. Results Success rates were calculated for all nine amplification kits and all ten substrates based on their ability to yield complete DNA profiles following a direct amplification protocol. Six out of the nine amplification kits, and four out of the ten paper substrates had the highest success rates overall. Conclusion The data show that it is possible to generate complete DNA profiles following a direct amplification protocol using both standard (non-direct) and direct PCR amplification kits. The generation of complete DNA profiles appears to depend more on the success of the amplification kit rather than the than the FTA®- or non-FTA®-based substrates. PMID:25559837

  19. Accurate quantification of supercoiled DNA by digital PCR.

    PubMed

    Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

    2016-01-01

    Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry. PMID:27063649

  20. Accurate quantification of supercoiled DNA by digital PCR

    PubMed Central

    Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

    2016-01-01

    Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry. PMID:27063649

  1. Digital Droplet PCR: CNV Analysis and Other Applications.

    PubMed

    Mazaika, Erica; Homsy, Jason

    2014-01-01

    Digital droplet PCR (ddPCR) is an assay that combines state-of-the-art microfluidics technology with TaqMan-based PCR to achieve precise target DNA quantification at high levels of sensitivity and specificity. Because quantification is achieved without the need for standard assays in an easy to interpret, unambiguous digital readout, ddPCR is far simpler, faster, and less error prone than real-time qPCR. The basic protocol can be modified with minor adjustments to suit a wide range of applications, such as CNV analysis, rare variant detection, SNP genotyping, and transcript quantification. This unit describes the ddPCR workflow in detail for the Bio-Rad QX100 system, but the theory and data interpretation are generalizable to any ddPCR system. PMID:25042719

  2. Rapid micro-PCR system for hepatitis C virus amplification

    NASA Astrophysics Data System (ADS)

    Lin, Yu-Cheng; Huang, Ming-Yuan; Young, Kung-Chia; Chang, Ting-Tsung; Wu, Ching-Yi

    2000-08-01

    A rapid micro-polymerase chain reaction ((mu) -PCR) system was integrated to amplify the complementary DNA (cDNA) molecules of hepatitis C virus (HCV). This system consists of a rapid thermal cycling system and a (mu) PCR chip fabricated by MEMS fabrication techniques. This rapid (mu) PCR system is verified by using serum samples from patients with chronic hepatitis C. The HCV amplicon of the rapid (mu) PCR system was analyzed by slab gel electrophoresis with separation of DNA marker in parallel. The (mu) PCR chip was fabricated on silicon wafer and Pyrex glass using photolithography, wet etching, and anodic bonding methods. Using silicon material to fabricate the raction well improves the temperature uniformity of sample and helps to reach the desired temperature faster. The rapid close loop thermal cycling system comprises power supplies, a thermal generator, a computer control PID controller, and a data acquisition subsystem. The thermoelectric (T.E.) cooler is used to work as the thermal generator and a heat sink by controlling the polarity of supplied power. The (mu) PCR system was verified with traditional PCR equipment by loading the same PCR mixture with HCV cDNA and running the same cycle numbers, then comparing both HCV amplicon slab gel electrophoresis. The HCV amplicon from the (mu) PCR system shows a DNA fragment with an expected size of 145 base pairs. The background is lower with the (mu) PCR system than that with the tradional PCR equipment. Comparing the traditional PCR equipment which spends 5.5 hours for 30 cycles to gain the detectable amount of HCV amplicon in slab gel separation, this (mu) PCR system takes 30 minutes to finish the 30 thermal cycles. This work has demonstrated that this rapid (mu) PCR system can provide rapid heat generation and dissipation, improved temperature uniformity in DNA amplification.

  3. PCR and real-time PCR assays to detect fungi of Alternaria alternata species.

    PubMed

    Kordalewska, Milena; Brillowska-Dąbrowska, Anna; Jagielski, Tomasz; Dworecka-Kaszak, Bożena

    2015-01-01

    Fungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria infections may be indistinguishable from other fungal diseases. Therefore, a diagnostic result is often delayed or even not achieved at all. In this paper we present easy to perform and interpret PCR and real-time PCR assays enabling detection of A. alternata species. On the basis of alignment of β-tubulin gene sequences, A. alternata-specific primers were designed. DNA from fungal isolates, extracted in a two-step procedure, were used in PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The assays specificity was confirmed, since positive results were obtained for all A. alternata isolates, and no positive results were obtained neither for other molds, dermatophytes, yeast-like fungi, nor human DNA. The assays developed here enable fast and unambiguous identification of A. alternata pathogens. PMID:26610309

  4. Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR)

    PubMed Central

    Felder, Eva; Mossbrugger, Ilona; Lange, Mirko; Wölfel, Roman

    2012-01-01

    Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix. PMID:23105972

  5. A quadruplex PCR (qxPCR) assay for adulteration in dairy products.

    PubMed

    Agrimonti, Caterina; Pirondini, Andrea; Marmiroli, Marta; Marmiroli, Nelson

    2015-11-15

    This study describes the development of a quadruplex quantitative Real Time PCR (qxPCR) based on SYBR®GreenER chemistry, for rapid identification of DNA of cow, goat, sheep and buffalo in dairy products, and for quantification of cow DNA in these products. The platform was applied to: (i) mixes of milks at fixed percentages; (ii) cheeses prepared with the same mixes; (iii) commercial dairy products. The methodology enabled the detection of DNA from cow in mixes of milk and cheeses with a limit of detection (LOD) of 0.1%. When applied to commercial dairy products the qxPCR gave results comparable with each single-plex Real Time PCR. A good correlation (R(2)>0.9) between peaks' area of derivative of melting curves of amplicons and percentages of cow milk in milk mixes and cheeses, allows for an estimation of cow DNA in a dynamic range varying from 0.1-5% to 1-25%. PMID:25976998

  6. Sensitivity of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood

    PubMed Central

    da Costa Lima, Manoel Sebastião; Zorzenon, Denielly Christina Rodrigues; Dorval, Maria Elizabeth Cavalheiros; Pontes, Elenir Rose Jardim Cury; Oshiro, Elisa Teruya; Cunha, Rodrigo; Andreotti, Renato; Matos, Maria de Fatima Cepa

    2013-01-01

    Objective To evaluate the effectiveness of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood samples. Methods DNA extraction was performed using Promega Wizard® Genomic kits. PCR employing RV1/RV2 primers yielded 145-bp amplicons. Real-time PCR was performed with the same primers and SYBR Green ROX Plus mix. These techniques were used to analyze 100 peripheral blood samples from patients with clinical signs of the disease. Results The sensitivity and specificity levels were 91,3%% and 29,6%, respectively, for real-time PCR and 97.78% and 61.82%, respectively, for PCR. Conclusions Real-time PCR proved to be a satisfactory method for the diagnosis of human visceral leishmaniasis.

  7. Identification of Actinobacillus actinomycetemcomitans in subgingival plaque by PCR.

    PubMed Central

    Flemmig, T F; Rüdiger, S; Hofmann, U; Schmidt, H; Plaschke, B; Strätz, A; Klaiber, B; Karch, H

    1995-01-01

    The purpose of this study was to assess the sensitivity and specificity of the PCR in detecting Actinobacillus actinomycetemcomitans. The PCR's detection capability was compared with those of three other methods: culture-enhanced PCR (CE-PCR), colony hybridization (CH), and conventional culture with presumptive biochemical identification. A 285-bp stretch of the leukotoxin gene lktA of A. actinomycetemcomitans was amplified by PCR with primers TT-15 and TT-16. For CH, the PCR product was labeled with digoxigenin and used as a hybridization probe. Nucleotide sequence analysis of the PCR product of A. actinomycetemcomitans 1D4 and 1664 and three clinical isolates revealed complete homology among the tested strains, with only one base substitution (at position 1344) in comparison with the published sequence. With artificially infected subgingival plaque, the detection limit of PCR for A. actinomycetemcomitans was 10(3) CFU/ml of plaque suspension. Culturing subgingival plaque on tryptic soy-serum-bacitracin-vancomycin agar prior to PCR (CE-PCR) improved the limit of detection to 10(2) CFU/ml. Analysis of subgingival plaque samples from 35 patients with periodontal disease and 10 periodontally healthy subjects revealed that CE-PCR and CH had the highest overall rate of A. actinomycetemcomitans detection (both 58%), followed by PCR and culture (both 42%). With CH as the "gold standard", the sensitivities of CE-PCR, PCR, and culture were 88, 65, and 58%, respectively; the specificities were 84, 89, and 79%, respectively. The CE-PCR provided acceptable positive and negative predictive values (> or = 70%) when the prevalence of A. actinomycetemcomitans varied between 30 and 70%. PCR alone provided comparable predictive values over a narrower range of prevalence rates (30 to 50%), while culture did not afford acceptable predictive values at any prevalence rate. PCR and CE-PCR were found to be superior to culture with presumptive biochemical identification and should be the

  8. STS-102 MPLM Leonardo moves into PCR

    NASA Technical Reports Server (NTRS)

    2001-01-01

    KENNEDY SPACE CENTER, Fla. -- In the payload changeout room on the Rotating Service Structure, Launch Pad 39B, workers move the Multi-Purpose Logistics Module Leonardo out of the payload canister. From the PCR Leonardo then will be transferred into Space Shuttle Discovery'''s payload bay. One of Italy'''s major contributions to the International Space Station program, Leonardo is a reusable logistics carrier. It is the primary delivery system used to resupply and return Station cargo requiring a pressurized environment. Leonardo is the primary payload on mission STS-102 and will deliver up to 10 tons of laboratory racks filled with equipment, experiments and supplies for outfitting the newly installed U.S. Laboratory Destiny. STS-102 is scheduled to launch March 8 at 6:45 a.m. EST.

  9. Reference genes in real-time PCR.

    PubMed

    Kozera, Bartłomiej; Rapacz, Marcin

    2013-11-01

    This paper aims to discuss various aspects of the use of reference genes in qPCR technique used in the thousands of present studies. Most frequently, these are housekeeping genes and they must meet several criteria so that they can lay claim to the name. Lots of papers report that in different conditions, for different organisms and even tissues the basic assumption—the constant level of the expression is not maintained for many genes that seem to be perfect candidates. Moreover, their transcription can not be affected by experimental factors. Sounds simple and clear but a great number of designed protocols and lack of consistency among them brings confusion on how to perform experiment properly. Since during selection of the most stable normalizing gene we can not use any reference gene, different ways and algorithms for their selection were developed. Such methods, including examples of best normalizing genes in some specific cases and possible mistakes are presented based on available sources. Numerous examples of reference genes applications, which are usually in too few numbers in relevant articles not allowing to make a solid fundament for a reader, will be shown along with instructive compilations to make an evidence for presented statements and an arrangement of future qPCR experiments. To include all the pitfalls and problems associated with the normalization methods there is no way not to begin from sample preparation and its storage going through candidate gene selection, primer design and statistical analysis. This is important because numerous short reviews available cover the topic only in lesser extent at the same time giving the reader false conviction of complete topic recognition. PMID:24078518

  10. Diagnostic utility of droplet digital PCR for HIV reservoir quantification.

    PubMed

    Trypsteen, Wim; Kiselinova, Maja; Vandekerckhove, Linos; De Spiegelaere, Ward

    2016-01-01

    Quantitative real-time PCR (qPCR) is implemented in many molecular laboratories worldwide for the quantification of viral nucleic acids. However, over the last two decades, there has been renewed interest in the concept of digital PCR (dPCR) as this platform offers direct quantification without the need for standard curves, a simplified workflow and the possibility to extend the current detection limit. These benefits are of great interest in terms of the quantification of low viral levels in HIV reservoir research because changes in the dynamics of residual HIV reservoirs will be important to monitor HIV cure efforts. Here, we have implemented a systematic literature screening and text mining approach to map the use of droplet dPCR (ddPCR) in the context of HIV quantification. In addition, several technical aspects of ddPCR were compared with qPCR: accuracy, sensitivity, precision and reproducibility, to determine its diagnostic utility. We have observed that ddPCR was used in different body compartments in multiple HIV-1 and HIV-2 assays, with the majority of reported assays focusing on HIV-1 DNA-based applications (i.e. total HIV DNA). Furthermore, ddPCR showed a higher accuracy, precision and reproducibility, but similar sensitivity when compared to qPCR due to reported false positive droplets in the negative template controls with a need for standardised data analysis (i.e. threshold determination). In the context of a low level of detection and HIV reservoir diagnostics, ddPCR can offer a valid alternative to qPCR-based assays but before this platform can be clinically accredited, some remaining issues need to be resolved. PMID:27482456

  11. IDH1 mutation detection by droplet digital PCR in glioma.

    PubMed

    Wang, Jing; Zhao, Yi-ying; Li, Jian-feng; Guo, Cheng-cheng; Chen, Fu-rong; Su, Hong-kai; Zhao, Hua-fu; Long, Ya-kang; Shao, Jian-yong; To, Shing shun Tony; Chen, Zhong-ping

    2015-11-24

    Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation. PMID:26485760

  12. IDH1 mutation detection by droplet digital PCR in glioma

    PubMed Central

    Wang, Jing; Zhao, Yi-ying; Li, Jian-feng; Guo, Cheng-cheng; Chen, Fu-rong; Su, Hong-kai; Zhao, Hua-fu; Long, Ya-kang; Shao, Jian-yong; Tony To, Shing-shun; Chen, Zhong-ping

    2015-01-01

    Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation. PMID:26485760

  13. DNA rehybridization during PCR: the 'Cot effect' and its consequences.

    PubMed

    Mathieu-Daudé, F; Welsh, J; Vogt, T; McClelland, M

    1996-06-01

    The rate of amplification of abundant PCR products generally declines faster than that of less abundant products in the same tube in the later cycles of PCR. As a consequence, differences in product abundance diminish as the number of PCR cycles increases. Rehybridization of PCR products which may interfere with primer binding or extension can explain this significant feature in late cycles. Rehybridization occurs with a half-time dependent on the reciprocal of the DNA concentration. Thus, if multiple PCR products are amplified in the same tube, reannealing occurs faster for the more abundant PCR products. In RT-PCR using an internal control, this results in a systematic bias against the more abundant of the two PCR products. In RNA fingerprinting by arbitrarily primed PCR (or differentially display of cDNAs), very large or absolute differences in the expression of a transcript between samples are preserved but smaller real differences may be gradually erased as the PCR reaction proceeds. Thus, this 'Cot effect' may systematically cause an underestimate of the true difference in starting template concentrations. However, differences in starting template concentrations will be better preserved in the less abundant PCR products. Furthermore, the slow down in amplification of abundant products will allow these rarer products to become more visible in the fingerprint which may, in turn, allow rarer cDNAs to be sampled more efficiently. In some applications, where the object is to stochiometrically amplify a mixture of nucleic acids, the bias against abundant PCR products can be partly overcome by limiting the number of PCR cycles and, thus, the concentration of the products. In other cases, abundance normalization at later cycles may be useful, such as in the production of normalized libraries. PMID:8668539

  14. Rapid separation and concentration of food-borne pathogens in food samples prior to quantification by viable-cell counting and real-time PCR.

    PubMed

    Fukushima, Hiroshi; Katsube, Kazunori; Hata, Yukiko; Kishi, Ryoko; Fujiwara, Satomi

    2007-01-01

    Buoyant density gradient centrifugation has been used to separate bacteria from complex food matrices, as well as to remove compounds that inhibit rapid detection methods, such as PCR, and to prevent false-positive results due to DNA originating from dead cells. Applying a principle of buoyant density gradient centrifugation, we developed a method for rapid separation and concentration following filtration and low- and high-speed centrifugation, as well as flotation and sedimentation buoyant density centrifugation, for 12 food-borne pathogens (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Vibrio cholerae O139, Vibrio parahaemolyticus O3K6, Vibrio vulnificus, Providencia alcalifaciens, Aeromonas hydrophila, Bacillus cereus, Staphylococcus aureus, and Clostridium perfringens) in 13 different food homogenates. This method can be used prior to real-time quantitative PCR (RTi-qPCR) and viable-cell counting. Using this combined method, the target organisms in the food samples theoretically could be concentrated 250-fold and detected at cell concentrations as low as 10(1) to 10(3) CFU/g using the RTi-qPCR assay, and amounts as small as 10(0) to 10(1) CFU/g could be isolated using plate counting. The combined separation and concentration methods and RTi-qPCR confirmed within 3 h the presence of 10(1) to 10(2) CFU/g of Salmonella and C. jejuni directly in naturally contaminated chicken and the presence of S. aureus directly in remaining food items in a poisoning outbreak. These results illustrated the feasibility of using these assays for rapid inspection of bacterial food contamination during a real-world outbreak. PMID:17056684

  15. Simultaneous identification and DNA barcoding of six Eimeria species infecting turkeys using PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus.

    PubMed

    Hafeez, Mian A; Shivaramaiah, Srichaitanya; Dorsey, Kristi Moore; Ogedengbe, Mosun E; El-Sherry, Shiem; Whale, Julia; Cobean, Julie; Barta, John R

    2015-05-01

    Species-specific PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus were generated that allow for the specific identification of the most common Eimeria species infecting turkeys (i.e., Eimeria adenoeides, Eimeria meleagrimitis, Eimeria gallopavonis, Eimeria meleagridis, Eimeria dispersa, and Eimeria innocua). PCR reaction chemistries were optimized with respect to divalent cation (MgCl2) and dNTP concentrations, as well as PCR cycling conditions (particularly anneal temperature for primers). Genomic DNA samples from single oocyst-derived lines of six Eimeria species were tested to establish specificity and sensitivity of these newly designed primer pairs. A mixed 60-ng total DNA sample containing 10 ng of each of the six Eimeria species was used as DNA template to demonstrate specific amplification of the correct product using each of the species-specific primer pairs. Ten nanograms of each of the five non-target Eimeria species was pooled to provide a non-target, control DNA sample suitable to test the specificity of each primer pair. The amplifications of the COI region with species-specific primer pairs from pooled samples yielded products of expected sizes (209 to 1,012 bp) and no amplification of non-target Eimeria sp. DNA was detected using the non-target, control DNA samples. These primer pairs specific for Eimeria spp. of turkeys did not amplify any of the seven Eimeria species infecting chickens. The newly developed PCR primers can be used as a diagnostic tool capable of specifically identifying six turkey Eimeria species; additionally, sequencing of the PCR amplification products yields sequence-based genotyping data suitable for identification and molecular phylogenetics. PMID:25678350

  16. Real-time cdPCR opens a window into events occurring in the first few PCR amplification cycles.

    PubMed

    Duewer, David L; Kline, Margaret C; Romsos, Erica L

    2015-12-01

    Polymerase chain reaction (PCR) end-point limiting dilution techniques, collectively termed "digital PCR (dPCR)", have been proposed as providing a potentially primary method for DNA quantification. We are evaluating several commercially available dPCR systems for use in certifying mass concentration in human genomic DNA reference materials. To better understand observed anomalies among results from chamber- and droplet-dPCR (cdPCR and ddPCR) systems, we have developed a graphical tool for evaluating and documenting the performance of PCR assays in real-time cdPCR systems: the ogive plot, the cumulative distribution of crossing threshold values. The ogive structure appears to embed information about early amplification events. We have successfully simulated ogives observed with different assays and reaction conditions using a four-stage amplification model parameterized by the probability of creating an intact 1) first generation "long" amplicon of indeterminate length from an original DNA target, 2) second generation defined-length amplicon from a long amplicon, and 3) defined-length amplicon from another defined-length amplicon. We are using insights from this model to optimize dPCR assay design and reaction conditions and to help validate assays proposed for use in value-assigning DNA reference materials. PMID:26438478

  17. A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

    PubMed Central

    Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T

    2014-01-01

    Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells. PMID:25437051

  18. Optimized MOL-PCR for Characterization of Microbial Pathogens.

    PubMed

    Wuyts, Véronique; Roosens, Nancy H C; Bertrand, Sophie; Marchal, Kathleen; De Keersmaecker, Sigrid C J

    2016-01-01

    Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL-PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL-PCR assay for subtyping of Salmonella Typhimurium. PMID:26742655

  19. Patho-Genes.org: a website dedicated to gene sequences of potential bioterror bacteria and PCR primers used to amplify them.

    PubMed

    Gardès, Julien; Bachar, Dipankar; Croce, Olivier; Christen, Richard

    2012-09-01

    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

  20. Variation in copy number of the 28S rDNA of Aspergillus fumigatus measured by droplet digital PCR and analog quantitative real-time PCR.

    PubMed

    Alanio, Alexandre; Sturny-Leclère, Aude; Benabou, Marion; Guigue, Nicolas; Bretagne, Stéphane

    2016-08-01

    Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis. PMID:27316653

  1. Loop-linker PCR: an advanced PCR technique for genome walking.

    PubMed

    Trinh, Quoclinh; Shi, Hui; Xu, Wentao; Hao, Junran; Luo, Yunbo; Huang, Kunlun

    2012-10-01

    In this article, we developed a novel PCR method, termed loop-linker PCR, to isolate flanking sequences in transgenic crops. The novelty of this approach is its use of a stem-loop structure to design a loop-linker adapter. The adapter is designed to form a nick site when ligated with restricted DNA. This modification not only can prevent the self-ligation of adapters but also promotes the elongation of the 3' end of the loop-linker adapter to generate a stem-loop structure in the ligation products. Moreover, the suppressive effect of the stem-loop structure decreases nonspecific amplification and increases the success rate of the approach; all extension products will suppress exponential amplification except from the ligation product that contains the specific primer binding site. Using this method, 442, 1830, 107, and 512 bp left border flanking sequences were obtained from the transgenic maizes LY038, DAS-59122-7, Event 3272, and the transgenic soybean MON89788, respectively. The experimental results demonstrated that loop-linker PCR is an efficient, reliable, and cost-effective method for identifying flanking sequences in transgenic crops and could be applied for other genome walking applications. PMID:23008115

  2. A simple, universal, efficient PCR-based gene synthesis method: sequential OE-PCR gene synthesis.

    PubMed

    Zhang, Pingping; Ding, Yingying; Liao, Wenting; Chen, Qiuli; Zhang, Huaqun; Qi, Peipei; He, Ting; Wang, Jinhong; Deng, Songhua; Pan, Tianyue; Ren, Hao; Pan, Wei

    2013-07-25

    Herein we present a simple, universal, efficient gene synthesis method based on sequential overlap extension polymerase chain reactions (OE-PCRs). This method involves four key steps: (i) the design of paired complementary 54-mer oligonucleotides with 18 bp overlaps, (ii) the utilisation of sequential OE-PCR to synthesise full-length genes, (iii) the cloning and sequencing of four positive T-clones of the synthesised genes and (iv) the resynthesis of target genes by OE-PCR with correct templates. Mispriming and secondary structure were found to be the principal obstacles preventing successful gene synthesis and were easily identified and solved in this method. Compensating for the disadvantages of being laborious and time-consuming, this method has many attractive advantages, such as the ability to guarantee successful gene synthesis in most cases and good allowance for Taq polymerase, oligonucleotides, PCR conditions and a high error rate. Thus, this method provides an alternative tool for individual gene synthesis without strict needs of the high-specialised experience. PMID:23597923

  3. Evaluation of PCR based coprodiagnosis of human opisthorchiasis.

    PubMed

    Stensvold, C R; Saijuntha, W; Sithithaworn, P; Wongratanacheewin, S; Strandgaard, H; Ornbjerg, N; Johansen, M V

    2006-01-01

    In this study, a recently developed PCR test for the detection of Opisthorchis viverrini in human faecal samples was evaluated using two parasitological methods as references. During a survey of foodborne trematodes (FBT) in the Vientiane Province, Lao PDR, 85 samples were collected and evaluated for FBT eggs by the Kato Katz (KK) technique, the formalin ethyl acetate concentration technique (FECT) and a PCR analysis for the distinction between O. viverrini and other FBT. The two parasitological methods did not differ in the ability of detecting FBT eggs, and a single KK reading was characterized by a sensitivity of 85% when compared to two FECT readings. The PCR tested positive only in cases where eggs had been demonstrated by parasitological examination. However, the PCR tested negative in some samples with very high egg counts. Demonstrating a PCR sensitivity of approximately 50% in samples with faecal egg counts>1000, the previously reported PCR sensitivity based on in vitro studies was not supported. It is believed that technical problems rather than diagnostic reference related issues were responsible for the relatively low PCR performance. Further studies should aim at optimizing DNA extraction and amplification, and future PCR evaluation should include specificity control such as the scanning electron microscopy of eggs in test samples or the expulsion of adult trematodes from PCR tested individuals. PMID:16253202

  4. Detection of virulence genes of Clostridium difficile by multiplex PCR.

    PubMed

    Antikainen, Jenni; Pasanen, Tanja; Mero, Sointu; Tarkka, Eveliina; Kirveskari, Juha; Kotila, Saara; Mentula, Silja; Könönen, Eija; Virolainen-Julkunen, Anni-Riitta; Vaara, Martti; Tissari, Päivi

    2009-08-01

    Clostridium difficile strains belonging to the PCR ribotype 027, pulse-field gel electrophoresis (PFGE) type NAP1, toxinotype III and restriction endonuclease analysis group BI harbouring mutations in the tcdC gene and possessing binary toxin components A and B have been described to cause epidemics with increased morbidity and mortality. In the present study we developed a conventional multiplex PCR designed to detect selected virulence associated markers of the hypervirulent C. difficile PCR ribotype 027. The multiplex PCR assay detected the major toxins A and B, binary toxin components A and B as well as a possible deletion in the tcdC gene: a characteristic pattern of amplification products for the PCR ribotype 027 strains was detected. This rather simple method was specific for the screening of this hypervirulent C. difficile strain. The correlation between the multiplex PCR and PCR ribotyping methods was excellent. The sensitivity and specificity were 100% in our epidemiological situation. In conclusion, this multiplex PCR was found useful in the preliminary screening for the hypervirulent C. difficile PCR ribotype 027. PMID:19664132

  5. Detection and quantification of chimerism by droplet digital PCR.

    PubMed

    George, David; Czech, Juliann; John, Bobby; Yu, Min; Jennings, Lawrence J

    2013-01-01

    Accurate quantification of chimerism and microchimerism is proving to be increasingly valuable for hematopoietic cell transplantation as well as non-transplant conditions. However, methods that are available to quantify low-level chimerism lack accuracy. Therefore, we developed and validated a method for quantifying chimerism based on digital PCR technology. We demonstrate accurate quantification that far exceeds what is possible with analog qPCR down to 0.01% with the potential to go even lower. Also, this method is inherently more informative than qPCR. We expect the advantages of digital PCR will make it the preferred method for chimerism analysis. PMID:23974275

  6. Antigenic typing of canine parvovirus using differential PCR.

    PubMed

    Kaur, Gurpreet; Chandra, Mudit; Dwivedi, P N; Sharma, N S

    2014-12-01

    Canine parvovirus (CPV) is an enteric pathogen causing hemorrhagic enteritis in pups of 3-6 months of age and is mainly transmitted via feco-oral route. In the present study, a total of 85 animals rectal swabs suspected of CPV were tested using a PCR, nested PCR and a newly designed differential PCR. Using PCR 7 (8.23 %) animals were positive whereas 39 (45.88 %) were positive by using nested PCR and 40 (47.05 %) were positive for either one or more than one antigenic types of CPV using differential PCR. Using differential PCR it was found that CPV-2a and CPV-2b were the most prevailing antigenic types. Also it was found that dogs that were vaccinated too yielded positive CPV indicating a possible presence of additional CPV antigenic types. Thus, the primers used in differential PCR can be used in a single PCR reaction to detect various antigenic types of CPV. PMID:25674626

  7. Buoyancy-Driven Polymerase Chain Reaction (PCR) Devices

    SciTech Connect

    Ness, K D; Wheeler, E K; Benett, W; Stratton, P; Christian, A; Chen, A; Ortega, J; Weisgraber, T H; Goodson, K E

    2004-09-28

    Polymerase chain reaction (PCR) facilitates DNA detection by significantly increasing the concentration of specific DNA segments. A new class of PCR instruments uses a buoyancy-driven re-circulating flow to thermally cycle the DNA sample and benefits from reduced cycle times, low sample volumes, a miniaturized format, and low power consumption. This paper analyzes a specific buoyancy PCR device in a micro-channel ''race-track'' geometry to determine key parameters about PCR cycle times and other figures of merit as functions of device dimensions. The 1-D model balances the buoyancy driving force with frictional losses. A hydrostatic pressure imbalance concept is used between the left and right sides of the fluid loop to calculate the buoyancy driving force. Velocity and temperature distributions within the channels are determined from two-dimensional analysis of the channel section, with developing region effects included empirically through scaled values of the local Nusselt number. Good agreement between four independent verification steps validate the 1-D simulation approach: (1) analytical expressions for the thermal entrance length are compared against, (2) comparison with a full 3-D finite element simulation, (3) comparison with an experimental flow field characterization, and (4) calculation of the minimum PCR runtime required to get a positive PCR signal from the buoyancy-driven PCR device. The 1-D approach closely models an actual buoyancy-driven PCR device and can further be used as a rapid design tool to simulate buoyancy PCR flows and perform detailed design optimizations studies.

  8. Highly precise measurement of HIV DNA by droplet digital PCR.

    PubMed

    Strain, Matthew C; Lada, Steven M; Luong, Tiffany; Rought, Steffney E; Gianella, Sara; Terry, Valeri H; Spina, Celsa A; Woelk, Christopher H; Richman, Douglas D

    2013-01-01

    Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR (long terminal repeat) circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it. PMID:23573183

  9. Highly Precise Measurement of HIV DNA by Droplet Digital PCR

    PubMed Central

    Strain, Matthew C.; Lada, Steven M.; Luong, Tiffany; Rought, Steffney E.; Gianella, Sara; Terry, Valeri H.; Spina, Celsa A.; Woelk, Christopher H.; Richman, Douglas D.

    2013-01-01

    Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR (long terminal repeat) circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it. PMID:23573183

  10. Global RT-PCR and RT-qPCR Analysis of the mRNA Expression of the Human PTPome.

    PubMed

    Nunes-Xavier, Caroline E; Pulido, Rafael

    2016-01-01

    Comprehensive comparative gene expression analysis of the tyrosine phosphatase superfamily members (PTPome) under cell- or tissue-specific growth conditions may help to define their individual and specific role in physiology and disease. Semi-quantitative and quantitative PCR are commonly used methods to analyze and measure gene expression. Here, we describe technical aspects of PTPome mRNA expression analysis by semi-quantitative RT-PCR and quantitative RT-PCR (RT-qPCR). We provide a protocol for each method consisting in reverse transcription followed by PCR using a global platform of specific PTP primers. The chapter includes aspects from primer validation to the setup of the PTPome RT-qPCR platform. Examples are given of PTP-profiling gene expression analysis using a human breast cancer cell line upon long-term or short-term treatment with cell signaling-activation agents. PMID:27514798

  11. Quantification of Bacterial Transcripts during Infection Using Competitive Reverse Transcription-PCR (RT-PCR) and LightCycler RT-PCR

    PubMed Central

    Goerke, Christiane; Bayer, Manfred G.; Wolz, Christiane

    2001-01-01

    Bacteria have evolved sophisticated regulatory circuits to modulate their gene expression in response to disparate environments. In order to monitor bacterial gene expression and regulation in the host, methods for direct transcript analysis from clinical specimens are needed. For most bacterial infections, amplification of the mRNAs of interest is necessary due to the low numbers of cells present and the low levels of specific transcripts. Here we compare two methods of quantitative reverse transcription-PCR (RT-PCR)—competitive RT-PCR using a one-tube system followed by standard gel analysis and the real-time detection of PCR product formation by fluorescence resonance energy transfer technology using the LightCycler unit. We isolated Staphylococcus aureus RNA directly from clinical specimens obtained from cystic fibrosis patients with chronic S. aureus lung infection and from an animal model of foreign-body infection with no further cultivation of the bacteria. Competitive RT-PCR and LightCycler RT-PCR were tested for their ability to quantify the transcription of a constitutively expressed gyrase gene (gyr) and a highly regulated α-toxin gene (hla) of S. aureus. Reproducible results were obtained with both methods. A sensitivity of 104 (gyr) and 103 (hla) copies, respectively, was reached, which was sufficient for the quantification of transcripts during bacterial infection. Overall, the competitive RT-PCR is a robust technique which does not need special RNA purification. On the negative side, it is labor intensive and time consuming, thus limiting the numbers of samples which can be analyzed at a given time. LightCycler RT-PCR is very susceptible to even traces of inhibitors, but it allows high-throughput processing of samples. PMID:11238208

  12. ANIMAL DNA IN PCR REAGENTS PLAGUES ANCIENT DNA RESEARCH

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high-cycle PCR amplification targ...

  13. REAL-TIME PCR ASSAY DEVELOPMENT FOR MULTIPLE MAIZE PATHOGENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This talk presents updates on the development of real-time PCR assays for two seedborne pathogens of maize, Pantoea (Erwinia) stewartii, the causal agent of Stewart's bacterial wilt, and Stenocarpella (Diplodia) maydis, the causal agent of Diplodia ear rot. We developed primers and a real-time PCR p...

  14. Interlaboratory Comparison of Quantitative PCR Test Results for Dehalococcoides

    EPA Science Inventory

    Quantitative PCR (qPCR) techniques have been widely used to measure Dehalococcoides (Dhc) DNA in the groundwater at field sites for several years. Interpretation of these data may be complicated when different laboratories using alternate methods conduct the analysis. An...

  15. DNA probes and PCR for diagnosis of parasitic infections.

    PubMed Central

    Weiss, J B

    1995-01-01

    DNA probe and PCR-based assays to identify and detect parasites are technically complex; however, they have high sensitivity, directly detect parasites independent of the immunocompetence or previous clinical history of the patient, and can distinguish between organisms that are morphologically similar. Diagnosis of parasites is often based on direct detection by microscopy, which is insensitive and laborious and can lack specificity. Most PCR-based assays were more sensitive than DNA probe assays. The development of PCR-based diagnostic assays requires multiple steps following the initial selection of oligonucleotide primers and reporter probe. Generally, the ability to detect the DNA of one parasite was attained by PCR; however, advances in the preparation of samples for PCR (extraction of DNA while removing PCR inhibitors) will be required to achieve that sensitivity with human specimens. Preliminary PCR systems have been developed for many different parasites, yet few have been evaluated with a large number of clinical specimens and/or under field conditions. Those evaluations are essential for determination of clinical and field utility and performance and of the most appropriate application of the assay. Several situations in which PCR-based diagnosis will result in epidemiologic, medical, or public health advances have been identified. PMID:7704890

  16. Quantitative PCR Method for Diagnosis of Citrus Bacterial Canker†

    PubMed Central

    Cubero, J.; Graham, J. H.; Gottwald, T. R.

    2001-01-01

    For diagnosis of citrus bacterial canker by PCR, an internal standard is employed to ensure the quality of the DNA extraction and that proper requisites exist for the amplification reaction. The ratio of PCR products from the internal standard and bacterial target is used to estimate the initial bacterial concentration in citrus tissues with lesions. PMID:11375206

  17. Predicting Salmonella enterica serotypes by repetitive sequence-based PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Repetitive extragenic palindromic sequence-based PCR (rep-PCR) utilizing a semi-automated system, was evaluated as a method to determine Salmonella serotypes. A group of 216 Salmonella isolates belonging to 13 frequently isolated serotypes and one rarer serotype from poultry were used to create a D...

  18. Urine sample used for congenital toxoplasmosis diagnosis by PCR.

    PubMed Central

    Fuentes, I; Rodriguez, M; Domingo, C J; del Castillo, F; Juncosa, T; Alvar, J

    1996-01-01

    The diagnosis of toxoplasmosis in congenitally infected infants can be difficult; serology is unreliable, and diagnosis must be based on the combination of symptomatology and direct demonstration of the parasite. Four infants suspected of having Toxoplasma gondii infection were studied by serological analysis, tissue culture, and PCR determination. T. gondii was isolated from the urine of one patient. The parasite was detected by PCR in the blood and cerebrospinal fluid of three infants and in the urine in all patients. Because nested PCR proved to be a sensitive, relatively rapid, and specific method and because it can be applied to a variety of different clinical samples, PCR can be a valuable technique for the identification of T. gondii infections in children. The present study indicates that PCR examination of urine, a fluid never before used for diagnosis in this age group, may be valuable in diagnosing cases of congenital toxoplasmosis. PMID:8880481

  19. Engineered DNA polymerase improves PCR results for plastid DNA1

    PubMed Central

    Schori, Melanie; Appel, Maryke; Kitko, AlexaRae; Showalter, Allan M.

    2013-01-01

    • Premise of the study: Secondary metabolites often inhibit PCR and sequencing reactions in extractions from plant material, especially from silica-dried and herbarium material. A DNA polymerase that is tolerant to inhibitors improves PCR results. • Methods and Results: A novel DNA amplification system, including a DNA polymerase engineered via directed evolution for improved tolerance to common plant-derived PCR inhibitors, was evaluated and PCR parameters optimized for three species. An additional 31 species were then tested with the engineered enzyme and optimized protocol, as well as with regular Taq polymerase. • Conclusions: PCR products and high-quality sequence data were obtained for 96% of samples for rbcL and 79% for matK, compared to 29% and 21% with regular Taq polymerase. PMID:25202519

  20. Investigation of vesicular rashes for HSV and VZV by PCR.

    PubMed

    Beards, G; Graham, C; Pillay, D

    1998-03-01

    Vesicular fluid from rashes of 132 patients was tested by a multiplex PCR shown to be specific for herpes simplex virus (HSV) type 1 and 2, and varicella zoster virus (VZV) genomic DNA. The results were compared with those obtained by examination by electron microscopy and virus isolation by cell culture. The PCR did not differentiate between HSV 1 and 2. By PCR, 64 HSV infections and 53 VZV infections were identified, with presumed 100% sensitivity and specificity. Fifteen specimens tested negative by PCR, electron microscopy, and virus isolation for herpes viruses. The sensitivities of virus isolation and electron microscopy for detection of herpes simplex virus were 56% and 80%. For varicella zoster virus, the sensitivities of virus isolation and electron microscopy were 47% and 60%. These data illustrate the advantage of rapid PCR diagnosis of herpes simplex virus and varicella zoster virus in vesicle fluids. PMID:9515761

  1. A thermally baffled device for highly stabilized convective PCR.

    PubMed

    Chang, Hsiao-Fen Grace; Tsai, Yun-Long; Tsai, Chuan-Fu; Lin, Ching-Ko; Lee, Pei-Yu; Teng, Ping-Hua; Su, Chen; Jeng, Chien-Chung

    2012-05-01

    Rayleigh-Bénard convective PCR is a simple and effective design for amplification of DNA. Convective PCR is, however, extremely sensitive to environmental temperature fluctuations, especially when using small- diameter test tubes. Therefore, this method is inherently unstable with limited applications. Here, we present a convective PCR device that has been modified by adding thermal baffles. With this thermally baffled device the influence from fluctuations in environmental temperature were significantly reduced, even in a wind tunnel (1 m/s). The thermally baffled PCR instrument described here has the potential to be used as a low-cost, point-of-care device for PCR-based molecular diagnostics in the field. PMID:22241586

  2. Epidemiological typing of Stenotrophomonas (Xanthomonas) maltophilia by PCR.

    PubMed Central

    Chatelut, M; Dournes, J L; Chabanon, G; Marty, N

    1995-01-01

    We used two PCR methods for epidemiological typing of Stenotrophomonas (Xanthomonas) maltophilia with either arbitrary primers (random amplified polymorphic DNA) or enterobacterial repetitive intergenic consensus sequences as primers (ERIC-PCR). The analysis was performed with 38 isolates of S. maltophilia, comprising 9 nosocomial isolates from a burn unit, 20 other clinical isolates epidemiologically unrelated, and 9 isolates from one cystic fibrosis patient. Both methods indicated that all of the nosocomial episodes were independent. In contrast, the nine isolates from the cystic fibrosis patient were assigned to very closely related profiles, especially by ERIC-PCR. We conclude that random amplified polymorphic DNA and ERIC-PCR have comparable reproducible and discriminatory powers for epidemiological typing of S. maltophilia, but ERIC-PCR profiles can be more easily evaluated. PMID:7790459

  3. Multiplex PCR for rapid diagnosis of tuberculous meningitis.

    PubMed

    Kusum, Sharma; Aman, Sharma; Pallab, Ray; Kumar, Sharma Shiv; Manish, Modi; Sudesh, Prabhakar; Subhash, Varma; Meera, Sharma

    2011-10-01

    Rapid and specific diagnosis of tubercular meningitis is of paramount importance to decrease morbidity and mortality. The aim of the study was to evaluate multiplex PCR using protein b, MPB 64, and IS6110 primers directed against M. tuberculosis complex for the diagnosis of tuberculous meningitis (TBM). Multiplex PCR was performed on 18 TBM confirmed cases (culture was positive), 92 clinically suspected TBM cases and 100 non-TBM (control group) patients. Multiplex PCR had a sensitivity of 94.4% for confirmed cases and specificity of 100% for confirmed TBM cases. In 92 clinically diagnosed but unconfirmed TBM cases, multiplex PCR was positive in 84.78% cases. The overall sensitivity of microscopy, culture and multiplex cases were 1.81, 16.73, and 86.63% and specificity was 100, 100, and 100% respectively. Multiplex PCR using protein b, MPB 64, and IS6110 primers has a high sensitivity and specificity in diagnosis of tubercular meningitis. PMID:21455603

  4. Use of Repetitive Element Palindromic-PCR (rep-PCR) for the Epidemiologic Discrimination of Food-Borne Pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of defined primers for polymerase chain reactions (PCR) amplicifcations of interspersed repetitive DNA elements present at distinct locations in prokaryotic genomes is referred to as Repetitive Element Palindromic Sequences Based-Polymerase Chain Reactions, rep-PCR. The initial discovery of...

  5. How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments.

    PubMed

    Svec, David; Tichopad, Ales; Novosadova, Vendula; Pfaffl, Michael W; Kubista, Mikael

    2015-03-01

    We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1-16 qPCR replicates per concentration and we tested 2-10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3-4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range. PMID:27077029

  6. Comparison of droplet digital PCR and seminested real-time PCR for quantification of cell-associated HIV-1 RNA.

    PubMed

    Kiselinova, Maja; Pasternak, Alexander O; De Spiegelaere, Ward; Vogelaers, Dirk; Berkhout, Ben; Vandekerckhove, Linos

    2014-01-01

    Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2 = 0.97, msRNA: R2 = 0.92) and patient-derived samples (usRNA: R2 = 0.51, msRNA: R2 = 0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used

  7. Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA

    PubMed Central

    De Spiegelaere, Ward; Vogelaers, Dirk; Berkhout, Ben; Vandekerckhove, Linos

    2014-01-01

    Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2 = 0.97, msRNA: R2 = 0.92) and patient-derived samples (usRNA: R2 = 0.51, msRNA: R2 = 0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used

  8. Detection of beet yellows virus by RT-PCR and immunocapture RT-PCR in Tetragonia expansa and Beta vulgaris.

    PubMed

    Kundu, K; Rysánek, P

    2004-01-01

    Two sensitive methods, RT-PCR with phenol-extracted RNA or Triton X-100-released RNA and immunocapture RT-PCR (IR-RT-PCR) were used for the detection of Beet yellows virus (BYV) in young and old leaves of Tetragonia expansa and sugar beet (Beta vulgaris) and in sugar beet roots. Four oligonucleotide primer pairs proved suitable for the detection of BYV. The release of BYV RNA with Triton X-100 was shown to be a very effective and easy as compared to isolation of total RNA by phenol extraction with the same or higher sensitivity of subsequent PCR. Using the Triton X-100 release of RNA and IC-RT-PCR the sensitivity of detection was so high that pg amounts of BYV RNA occurring in dilutions up to 10(-6) of saps from young Tetragonia and sugar beet leaves could be detected. PMID:15595212

  9. Field effect sensors for PCR applications

    NASA Astrophysics Data System (ADS)

    Taing, Meng-Houit; Sweatman, Denis R.

    2004-03-01

    The use of field effect sensors for biological and chemical sensing is widely employed due to its ability to make detections based on charge and surface potential. Because proteins and DNA almost always carry a charge [1], silicon can be used to micro fabricate such a sensor. The EIS structure (Electrolyte on Insulator on Silicon) provides a novel, label-free and simple to fabricate way to make a field effect DNA detection sensor. The sensor responds to fluctuating capacitance caused by a depletion layer thickness change at the surface of the silicon substrate through DNA adsorption onto the dielectric oxide/PLL (Poly-L-Lysine) surface. As DNA molecules diffuse to the sensor surface, they are bound to their complimentary capture probes deposited on the surface. The negative charge exhibited by the DNA forces negative charge carriers in the substrate to move away from the surface. This causes an n-type depletion layer substrate to thicken and a p-type to thin. The depletion layer thickness can be measured by its capacitance using an LCR meter. This experiment is conducted using the ConVolt (constant voltage) approach. Nucleic acids are amplified by an on chip PCR (Polymerase Chain Reaction) system and then fed into the sensor. The low ionic solution strength will ensure that counter-ions do not affect the sensor measurements. The sensor surface contains capture probes that bind to the pathogen. The types of pathogens we"ll be detecting include salmonella, campylobacter and E.Coli DNA. They are held onto the sensor surface by the positively charged Poly-L-Lysine layer. The electrolyte is biased through a pseudo-reference electrode. Pseudo reference electrodes are usually made from metals such as Platinum or Silver. The problem associated with "floating" biasing electrodes is they cannot provide stable biasing potentials [2]. They drift due to surface charging effects and trapped charges on the surface. To eliminate this, a differential system consisting of 2 sensors

  10. 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

  11. Sequence variation at the rice blast resistance gene Pi-km locus: Implications for the development of allele specific markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The recently cloned blast resistance (R) gene Pi-km protects rice crops against specific races of the fungal pathogen Magnaporthe oryzae in a gene-for-gene manner. The use of blast R genes remains the most cost-effective method for an integrated disease management strategy. To facilitate rice breed...

  12. Transcriptome and Allele Specificity Associated with a 3BL Locus for Fusarium Crown Rot Resistance in Bread Wheat

    PubMed Central

    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

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

    PubMed

    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

  14. 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

  15. 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

  16. PCR in laboratory diagnosis of human Borrelia burgdorferi infections.

    PubMed Central

    Schmidt, B L

    1997-01-01

    The laboratory diagnosis of Lyme borreliosis, the most prevalent vector-borne disease in the United States and endemic in parts of Europe and Asia, is currently based on serology with known limitations. Direct demonstration of Borrelia burgdorferi by culture may require weeks, while enzyme-linked immunosorbent assays for antigen detection often lack sensitivity. The development of the PCR has offered a new dimension in the diagnosis. Capable of amplifying minute amounts of DNA into billions of copies in just a few hours, PCR facilitates the sensitive and specific detection of DNA or RNA of pathogenic organisms. This review is restricted to applications of PCR methods in the diagnosis of human B. burgdorferi infections. In the first section, methodological aspects, e.g., sample preparation, target selection, primers and PCR methods, and detection and control of inhibition and contamination, are highlighted. In the second part, emphasis is placed on diagnostic aspects, where PCR results in patients with dermatological, neurological, joint, and ocular manifestations of the disease are discussed. Here, special attention is given to monitoring treatment efficacy by PCR tests. Last, specific guidelines on how to interpret PCR results, together with the advantages and limitations of these new techniques, are presented. PMID:8993863

  17. A comparison of four methods for PCR inhibitor removal.

    PubMed

    Hu, Qingqing; Liu, Yuxuan; Yi, Shaohua; Huang, Daixin

    2015-05-01

    Biological samples collected from the crime scenes often contain some compounds that can inhibit the polymerase chain reaction (PCR). The removal of PCR inhibitors from the extracts prior to the PCR amplification is vital for successful forensic DNA typing. This paper aimed to evaluate the ability of four different methods (PowerClean® DNA Clean-Up kit, DNA IQ™ System, Phenol-Chloroform extraction and Chelex®-100 methods) to remove eight commonly encountered PCR inhibitors including: melanin, humic acid, collagen, bile salt, hematin, calcium ions, indigo and urea. Each of these PCR inhibitors was effectively removed by the PowerClean® DNA Clean-Up kit and DNA IQ™ System as demonstrated by generating more complete short tandem repeat (STR) profiles from the cleaned up inhibitor samples than from the raw inhibitor samples. The Phenol-Chloroform extraction and Chelex®-100 methods, however, could only remove some of eight PCR inhibitors. Our results demonstrated that the PowerClean® DNA Clean-Up kit and DNA IQ™ System were very effective for the removal of known PCR inhibitors that are routinely found in DNA extracts from forensic samples. PMID:25553520

  18. Interaction of quantitative PCR components with polymeric surfaces.

    PubMed

    Gonzalez, Asensio; Grimes, Ronan; Walsh, Edmond J; Dalton, Tara; Davies, Mark

    2007-04-01

    This study investigated the effect of exposing a polymerase chain reaction (PCR) mixture to capillary tubing of different materials and lengths, at different contact times and flow rates and the adsorption of major reaction components into the tubing wall. Using 0.5 mm ID tubing, lengths of 40 cm and residence times up to 45 min, none of the tested polymeric materials was found to affect subsequent PCR amplification. However, after exposure of the mixture to tubing lengths of 3 m or reduction of sample volume, PCR inhibition occurred, increasing with the volume to length ratio. Different flow velocities did not affect PCR yield. When the adsorption of individual PCR components was studied, significant DNA adsorption and even more significant adsorption of the fluorescent dye Sybr Green I was found. The results indicate that PCR inhibition in polymeric tubing results from adsorption of reaction components to wall surfaces, increasing substantially with tubing length or sample volume reduction, but not with contact time or flow velocities typical in dynamic PCR amplification. The data also highlight that chemical compatibility of polymeric capillaries with DNA dyes should be carefully considered for the design of quantitative microfluidic devices. PMID:17180709

  19. PCR assays for detection of Baylisascaris procyonis eggs and larvae.

    PubMed

    Dangoudoubiyam, Sriveny; Vemulapalli, Ramesh; Kazacos, Kevin R

    2009-06-01

    The objective of this study was to develop polymerase chain reaction (PCR) assays for detection of Baylisascaris procyonis eggs and larvae in fecal, environmental, and tissue samples. We have optimized conventional and real-time PCR assays for B. procyonis using the mitochondrial cytochrome oxidase 2 gene as the target for amplification. The lower limit of detection of the parasite genomic DNA was 10 pg in the conventional PCR and 100 fg in the real-time PCR. In both PCR assays, specific amplification of a 146 bp product was achieved with DNA extracted from a single in vitro hatched B. procyonis larva and also from canine fecal samples spiked with as few as 20 unembryonated B. procyonis eggs per gram of feces. The PCR assays were successfully used for detection of B. procyonis eggs and larvae in fecal, environmental, and tissue samples. No DNA amplification was seen when the genomic DNA of related ascarids (including B. transfuga) and a hookworm was used as template in the PCR; however, amplification was seen with the very closely related B. columnaris. PMID:19090651

  20. Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species.

    PubMed

    Doi, Hideyuki; Takahara, Teruhiko; Minamoto, Toshifumi; Matsuhashi, Saeko; Uchii, Kimiko; Yamanaka, Hiroki

    2015-05-01

    Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors. PMID:25850372

  1. Sensitive detection of sample interference in environmental qPCR.

    PubMed

    Green, Hyatt C; Field, Katharine G

    2012-06-15

    Sample interference in environmental applications of quantitative PCR (qPCR) can prevent accurate estimations of molecular markers in the environment. We developed a spike-and-recovery approach using a mutant strain of Escherichia coli that contains a chromosomal insertion of a mutant GFP gene. The method was tested in water samples by separately reducing extraction efficiency or adding humic acids and ethanol, compounds that often contaminate environmental DNA extracts, and analyzing qPCR amplification of the spiked E. coli control and human fecal Bacteroides markers (HF183 and HF134). This approach, coupled with previously developed kinetic outlier detection (KOD) methods, allowed sensitive detection of PCR inhibition at much lower inhibitor concentrations than alternative approaches using Cq values or amplification efficiencies. Although HF183 was more sensitive to the effects of qPCR inhibitors than the E. coli control assay, KOD methods correctly identified inhibition of both control and HF183 assays in samples containing as little as 0.1 ng humic acids per reaction or 5% ethanol. Because sigmoidal modeling methods allow distinction of qPCR inhibition from poor DNA recovery, we were able to simultaneously identify qPCR-inhibited reactions and estimate recovery of nucleic acids in environmental samples using a single control assay. Since qPCR is currently used to estimate important water quality parameters that have serious economic and human health outcomes, these results are timely. While we demonstrate the methods in the context of water quality regulation, they will be useful in all areas of environmental research that use qPCR. PMID:22560896

  2. Predicting Gene Structures from Multiple RT-PCR Tests

    NASA Astrophysics Data System (ADS)

    Kováč, Jakub; Vinař, Tomáš; Brejová, Broňa

    It has been demonstrated that the use of additional information such as ESTs and protein homology can significantly improve accuracy of gene prediction. However, many sources of external information are still being omitted from consideration. Here, we investigate the use of product lengths from RT-PCR experiments in gene finding. We present hardness results and practical algorithms for several variants of the problem and apply our methods to a real RT-PCR data set in the Drosophila genome. We conclude that the use of RT-PCR data can improve the sensitivity of gene prediction and locate novel splicing variants.

  3. Efficient PCR amplification by an unnatural base pair system.

    PubMed

    Kimoto, Michiko; Kawai, Rie; Mitsui, Tsuneo; Yokoyama, Shigeyuki; Hirao, Ichiro

    2008-01-01

    Expansion of the genetic alphabet by an unnatural base pair system enables the site-specific incorporation of extra functional components into nucleic acids and proteins. In this system, PCR amplification of DNA templates containing unnatural base pairs is essential for modern biotechnology. We present a new unnatural base pair system, in which DNA duplexes containing the unnatural base pairs can be efficiently amplified by PCR. The system also provides a method for the site-specific incorporation of functional components into amplified DNA fragments by PCR, using unnatural base substrates linked with functional groups of interest. PMID:18776457

  4. PCR amplification on microarrays of gel immobilized oligonucleotides

    DOEpatents

    Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

    2003-11-04

    The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

  5. Preparation of DNA-containing extract for PCR amplification

    DOEpatents

    Dunbar, John M.; Kuske, Cheryl R.

    2006-07-11

    Environmental samples typically include impurities that interfere with PCR amplification and DNA quantitation. Samples of soil, river water, and aerosol were taken from the environment and added to an aqueous buffer (with or without detergent). Cells from the sample are lysed, releasing their DNA into the buffer. After removing insoluble cell components, the remaining soluble DNA-containing extract is treated with N-phenacylthiazolium bromide, which causes rapid precipitation of impurities. Centrifugation provides a supernatant that can be used or diluted for PCR amplification of DNA, or further purified. The method may provide a DNA-containing extract sufficiently pure for PCR amplification within 5–10 minutes.

  6. Analysis of mutations using PCR and denaturing gradient gel electrophoresis

    SciTech Connect

    Cariello, N.F.; Swenberg, J.A. Duke Univ., Durham, NC ); DeBellis, A.; Skopek, T.R. )

    1991-01-01

    Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on primary sequence. Under the appropriate conditions, all base pair (bp) substitutions, frameshifts, and deletions less than about 10 bp can be resolved from the wild type sequence using DGGE. Polymerase chain reaction (PCR) permits facile amplification of a given region of the genome. The authors have combined PCR and DGGE to: (1) localize mutations in the X-linked human androgen receptor gene; (2) analyze thousands of thioguanine-resistant mutants simultaneously; (3) examine the fidelity of several DNA polymerases used in PCR.

  7. Identification and quantification of ice nucleation active microorganisms by digital droplet PCR (ddPCR)

    NASA Astrophysics Data System (ADS)

    Linden, Martin; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine

    2015-04-01

    Several bioaerosol types, including bacteria, fungi, pollen and lichen, have been identified as sources of biological ice nucleators (IN) which induce ice formation already at temperatures as high as -10 °C or above. Accordingly, they potentially contribute widely to environmental ice nucleation in the atmosphere and are of great interest in the study of natural heterogenous ice nucleation processes. Ice nucleation active microorganisms have been found and studied among bacteria (Proteobacteria) and fungi (phyla Basidiomycota and Ascomycota). The mechanisms enabling the microorganisms to ice nucleation are subject to ongoing research. While it has been demonstrated that whole cells can act as ice nucleators in the case of bacteria due to the presence of specific membrane proteins, cell-free ice nucleation active particles seem to be responsible for this phenomenon in fungi and lichen. The identification and quantification of these ice nucleation active microorganisms and their IN in atmospheric samples is crucial to understand their contribution to the pool of atmospheric IN. This is not a trivial task since the respective microorganisms are often prevalent in lowest concentrations and a variety of states, be it viable cells, spores or cell debris from dead cells. Molecular biology provides tools to identify and quantify ice nucleation active microorganisms independent of their state by detecting genetic markers specific for the organism of interest. Those methods are not without their drawbacks in terms of sample material concentration required or reliable standardization. Digital Droplet Polymerase Chain Reaction (ddPCR) was chosen for our demands as a more elegant, quick and specific method in the investigation of ice nucleation active microorganisms in atmospheric samples. The advantages of ddPCR lie in the simultaneous detection and quantification of genetic markers and their original copy numbers in a sample. This is facilitated by the fractionation of the

  8. Sensitive Simultaneous Detection of Seven Sexually Transmitted Agents in Semen by Multiplex-PCR and of HPV by Single PCR

    PubMed Central

    de Abreu, André Luelsdorf Pimenta; Irie, Mary Mayumi Taguti; Esquiçati, Isis Baroni; Malagutti, Natália; Vasconcellos, Vinícius Rodrigo Bulla; Discacciati, Michele Garcia; Bonini, Marcelo Gialluisi; Maria-Engler, Silvya Stuchi; Consolaro, Marcia Edilaine Lopes

    2014-01-01

    Sexually transmitted diseases (STDs) may impair sperm parameters and functions thereby promoting male infertility. To date limited molecular studies were conducted to evaluate the frequency and type of such infections in semen Thus, we aimed at conceiving and validating a multiplex PCR (M-PCR) assay for the simultaneous detection of the following STD pathogens in semen: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Herpes virus simplex (HSV) −1 and −2, and Treponema pallidum; We also investigated the potential usefulness of this M-PCR assay in screening programs for semen pathogens. In addition, we aimed: to detect human Papillomavirus (HPV) and genotypes by single PCR (sPCR) in the same semen samples; to determine the prevalence of the seven STDs, HPV and co-infections; to assess the possibility that these infections affect semen parameters and thus fertility. The overall validation parameters of M-PCR were extremely high including agreement (99.2%), sensitivity (100.00%), specificity (99.70%), positive (96.40%) and negative predictive values (100.00%) and accuracy (99.80%). The prevalence of STDs was very high (55.3%). Furthermore, associations were observed between STDs and changes in semen parameters, highlighting the importance of STD detection in semen. Thus, this M-PCR assay has great potential for application in semen screening programs for pathogens in infertility and STD clinics and in sperm banks. PMID:24921247

  9. Oligoribonucleotide (ORN) Interference-PCR (ORNi-PCR): A Simple Method for Suppressing PCR Amplification of Specific DNA Sequences Using ORNs

    PubMed Central

    Tanigawa, Naoki; Fujita, Toshitsugu; Fujii, Hodaka

    2014-01-01

    Polymerase chain reaction (PCR) amplification of multiple templates using common primers is used in a wide variety of molecular biological techniques. However, abundant templates sometimes obscure the amplification of minor species containing the same primer sequences. To overcome this challenge, we used oligoribonucleotides (ORNs) to inhibit amplification of undesired template sequences without affecting amplification of control sequences lacking complementarity to the ORNs. ORNs were effective at very low concentrations, with IC50 values for ORN-mediated suppression on the order of 10 nM. DNA polymerases that retain 3′–5′ exonuclease activity, such as KOD and Pfu polymerases, but not those that retain 5′–3′ exonuclease activity, such as Taq polymerase, could be used for ORN-mediated suppression. ORN interference-PCR (ORNi-PCR) technology should be a useful tool for both molecular biology research and clinical diagnosis. PMID:25405983

  10. A highly specific q-RT-PCR assay to address the relevance of the JAK2WT and JAK2V617F expression levels and control genes in Ph-negative myeloproliferative neoplasms.

    PubMed

    Fantasia, Francesca; Di Capua, Emma Nora; Cenfra, Natalia; Pessina, Gloria; Mecarocci, Sergio; Rago, Angela; Cotroneo, Ettore; Busanello, Anna; Equitani, Francesco; Lo-Coco, Francesco; Nervi, Clara; Cimino, Giuseppe

    2014-04-01

    In Ph- myeloproliferative neoplasms, the quantification of the JAK2V617F transcripts may provide some advantages over the DNA allele burden determination. We developed a q-RT-PCR to assess the JAK2WT and JAK2V617F mRNA expression in 105 cases (23 donors, 13 secondary polycythemia, 22 polycythemia vera (PV), 38 essential thrombocythemia (ET), and 9 primary myelofibrosis (PMF)). Compared with the standard allele-specific oligonucleotide (ASO)-PCR technique, our assay showed a 100 % concordance rate detecting the JAK2V617F mutation in 22/22 PV (100 %), 29/38 (76.3 %) ET, and 5/9 (55.5 %) PMF cases, respectively. The sensitivity of the assay was 0.01 %. Comparing DNA and RNA samples, we found that the JAK2V617F mutational ratios were significantly higher at the RNA level both in PV (p = 0.005) and ET (p = 0.001) samples. In PV patients, JAK2WT expression levels positively correlated with the platelets (PLTs) (p = 0.003) whereas a trend to negative correlation was observed with the Hb levels (p = 0.051). JAK2V617F-positive cases showed the lowest JAK2WT and ABL1 mRNA expression levels. In all the samples, the expression pattern of beta-glucoronidase (GUSB) was more homogeneous than that of ABL1 or β2 microglobulin (B2M). Using GUSB as normalizator gene, a significant increase of the JAK2V617F mRNA levels was seen in two ET patients at time of progression to PV. In conclusion, the proposed q-RT-PCR is a sensitive and accurate method to quantify the JAK2 mutational status that can also show clinical correlations suggesting the impact of the residual amount of the JAK2WT allele on the Ph- MPN disease phenotype. Our observations also preclude the use of ABL1 as a housekeeping gene for these neoplasms. PMID:24173087

  11. Introducing basic molecular biology to Turkish rural and urban primary school children via hands-on PCR and gel electrophoresis activities.

    PubMed

    Selli, Cigdem; Yıldırım, Gokce; Kaymak, Aysegul; Karacicek, Bilge; Ogut, Deniz; Gungor, Turkan; Erem, Erdem; Ege, Mehmet; Bümen, Nilay; Tosun, Metiner

    2014-01-01

    This study includes the results of a 2-day education project titled "Molecular Biology Laboratory Summer School, MoBiLYO." The project was held at a University Research Center by scientists from Department of Pharmacology and graduate students. The project was composed of introductory lectures, model construction, DNA isolation, polymerase chain reaction (PCR), and gel electrophoresis. The participants were 13-year-old eighth-graders attending primary schools affiliated with Ministry of National Education in urban and rural areas of Izmir, Turkey. The purpose of this study was to introduce basic molecular biology concepts through individually performed experiments such as PCR and gel electrophoresis integrated with creative drama. The students were assessed at the beginning and the end of each project day via mini-tests, experimental and presentation skills evaluation forms. Data showed that students' knowledge about DNA structure and basic molecular biology techniques significantly increased. On the basis of experimental and presentational skills, there was no significant difference between kids from urban and rural schools or between public and boarding public schools, whereas the average score of girls was significantly higher than that of boys. In conclusion, individually performed experiments integrated with creative drama significantly increased students' perception of complex experimental procedures on basic molecular biology concepts. Data suggests that integration of these concepts into the science and technology curriculum of Turkish primary education may support the recruitment of future scientists who can handle rapidly developing genomic techniques that will affect our everyday life. PMID:24474053

  12. The clinical potential of Enhanced-ice-COLD-PCR.

    PubMed

    Tost, Jörg

    2016-01-01

    Enhanced-ice-COLD-PCR (E-ice-COLD-PCR) is a novel assay format that allows for the efficient enrichment and sensitive detection of all mutations in a region of interest using a chemically modified blocking oligonucleotide, which impedes the amplification of wild-type sequences. The assay is compatible with DNA extracted from tissue and cell-free circulating DNA. The main features of E-ice-COLD-PCR are the simplicity of the setup and the optimization of the assay, the use of standard laboratory equipment and the very short time to results (~4 h including DNA extraction, enrichment and sequence-based identification of mutations). E-ice-COLD-PCR is therefore a highly promising technology for a number of basic research as well as clinical applications including detection of clinically relevant mutated subclones and monitoring of treatment response or disease recurrence. PMID:26589575

  13. Quantitative PCR for Genetic Markers of Human Fecal Pollution

    EPA Science Inventory

    Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantificationapproach. We report the development of quantitative PCR assays for quantification of two recently described human-...

  14. Quantitative PCR for genetic markers of human fecal pollution

    EPA Science Inventory

    Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for enumeration of two recently described hum...

  15. DNA Microarray-Based PCR Ribotyping of Clostridium difficile

    PubMed Central

    Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian

    2014-01-01

    This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray. PMID:25411174

  16. MPprimer: a program for reliable multiplex PCR primer design

    PubMed Central

    2010-01-01

    Background Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility. Results A program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs) for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2× to 5× plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy), which has 79 exons, for 20×, 20×, 20×, 14×, and 5× plex PCR reactions in five tubes to detect underlying exon deletions. Conclusions MPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays. PMID:20298595

  17. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces

    PubMed Central

    Verhaegen, Bavo; De Reu, Koen; De Zutter, Lieven; Verstraete, Karen; Heyndrickx, Marc; Van Coillie, Els

    2016-01-01

    Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan® Environmental Master Mix 2.0; UMM: TaqMan® Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material. PMID:27213452

  18. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces.

    PubMed

    Verhaegen, Bavo; De Reu, Koen; De Zutter, Lieven; Verstraete, Karen; Heyndrickx, Marc; Van Coillie, Els

    2016-01-01

    Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan(®) Environmental Master Mix 2.0; UMM: TaqMan(®) Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material. PMID:27213452

  19. Quantification of HEV RNA by Droplet Digital PCR.

    PubMed

    Nicot, Florence; Cazabat, Michelle; Lhomme, Sébastien; Marion, Olivier; Sauné, Karine; Chiabrando, Julie; Dubois, Martine; Kamar, Nassim; Abravanel, Florence; Izopet, Jacques

    2016-01-01

    The sensitivity of real-time PCR for hepatitis E virus (HEV) RNA quantification differs greatly among techniques. Standardized tools that measure the real quantity of virus are needed. We assessed the performance of a reverse transcription droplet digital PCR (RT-ddPCR) assay that gives absolute quantities of HEV RNA. Analytical and clinical validation was done on HEV genotypes 1, 3 and 4, and was based on open reading frame (ORF)3 amplification. The within-run and between-run reproducibilities were very good, the analytical sensitivity was 80 HEV RNA international units (IU)/mL and linearities of HEV genotype 1, 3 and 4 were very similar. Clinical validation based on 45 samples of genotype 1, 3 or 4 gave results that correlated well with a validated reverse transcription quantitative PCR (RT-qPCR) assay (Spearman rs = 0.89, p < 0.0001). The RT-ddPCR assay is a sensitive method and could be a promising tool for standardizing HEV RNA quantification in various sample types. PMID:27548205

  20. Partially overlapping primer-based PCR for genome walking.

    PubMed

    Li, Haixing; Ding, Dongqin; Cao, Yusheng; Yu, Bo; Guo, Liang; Liu, Xiaohua

    2015-01-01

    Current genome walking methods are cumbersome to perform and can result in non-specific products. Here, we demonstrate the use of partially overlapping primer-based PCR (POP-PCR), a direct genome walking technique for the isolation of unknown flanking regions. This method exploits the partially overlapping characteristic at the 3' ends of a set of POP primers (walking primers), which guarantees that the POP primer only anneals to the POP site of the preceding PCR product at relatively low temperatures. POP primer adaptation priming at the genomic DNA/POP site occurs only once due to one low-/reduced-stringency cycle in each nested PCR, resulting in the synthesis of a pool of single-stranded DNA molecules. Of this pool, the target single-stranded DNA is replicated to the double-stranded form bound by the specific primer and the POP primer in the subsequent high-stringency cycle due to the presence of the specific primer-binding site. The non-target single stranded DNA does not become double stranded due to the absence of a binding site for any of the primers. Therefore, the POP-PCR enriches target DNA while suppressing non-target products. We successfully used POP-PCR to retrieve flanking regions bordering the gadA locus in Lactobacillus brevis NCL912, malQ in Pichia pastoris GS115, the human aldolase A gene, and hyg in rice. PMID:25811779

  1. Improved PCR Amplification of Broad Spectrum GC DNA Templates

    PubMed Central

    Guido, Nicholas; Starostina, Elena; Leake, Devin; Saaem, Ishtiaq

    2016-01-01

    Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10–90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content. PMID:27271574

  2. Rapid diagnosis of goose viral infections by multiplex PCR.

    PubMed

    Chen, Zongyan; Li, Chuanfeng; Li, Guoxin; Yu, Hai; Jiang, Yifeng; Yan, Liping; Meng, Chunchun; Zhou, Yanjun; Tong, Guangzhi; Liu, Guangqing

    2013-08-01

    Goose parvovirus (GPV), newcastle disease virus (NDV), goose herpesvirus (GHV) and goose adenovirus (GAV) are considered collectively to be four of the most important and widespread viruses of geese. Because all of these viruses cause similar pathological changes, histological differentiation among these viruses is difficult. A reliable, specific and sensitive multiplex PCR (mPCR) assay was developed for the combined detection of GPV, NDV, GHV and GAV in clinical samples of geese. Using the mPCR technique, single infections with GPV (28/76; 36.8%), NDV (9/76; 11.8%), GHV (3/76; 3.9%) and GAV (12/76; 15.8%) were identified in the samples; co-infections with GAV and either GPV or NDV (31.6%; 24/76) were also identified with this approach. The results for all of the samples tested were the same in both the uPCR and mPCR systems. The mPCR approach is considered to be useful for routine molecular diagnosis and epidemiological applications in geese. PMID:23518397

  3. Quantification of HEV RNA by Droplet Digital PCR

    PubMed Central

    Nicot, Florence; Cazabat, Michelle; Lhomme, Sébastien; Marion, Olivier; Sauné, Karine; Chiabrando, Julie; Dubois, Martine; Kamar, Nassim; Abravanel, Florence; Izopet, Jacques

    2016-01-01

    The sensitivity of real-time PCR for hepatitis E virus (HEV) RNA quantification differs greatly among techniques. Standardized tools that measure the real quantity of virus are needed. We assessed the performance of a reverse transcription droplet digital PCR (RT-ddPCR) assay that gives absolute quantities of HEV RNA. Analytical and clinical validation was done on HEV genotypes 1, 3 and 4, and was based on open reading frame (ORF)3 amplification. The within-run and between-run reproducibilities were very good, the analytical sensitivity was 80 HEV RNA international units (IU)/mL and linearities of HEV genotype 1, 3 and 4 were very similar. Clinical validation based on 45 samples of genotype 1, 3 or 4 gave results that correlated well with a validated reverse transcription quantitative PCR (RT-qPCR) assay (Spearman rs = 0.89, p < 0.0001). The RT-ddPCR assay is a sensitive method and could be a promising tool for standardizing HEV RNA quantification in various sample types. PMID:27548205

  4. Detection of Treponema pallidum in the vitreous by PCR

    PubMed Central

    Müller, M; Ewert, I; Hansmann, F; Tiemann, C; Hagedorn, H J; Solbach, W; Roider, J; Nölle, B; Laqua, H; Hoerauf, H

    2007-01-01

    Background Ocular involvement of syphilis still poses a clinical challenge due to the chameleonic behaviour of the disease. As the serodiagnosis has significant limitations, the direct detection of Treponema pallidum (TP) in the vitreous represents a desirable diagnostic tool. Methods Real‐time polymerase chain reaction (PCR) for the detection of TP was applied in diagnostic vitrectomies of two patients with acute chorioretinitis. Qualitative verification of TP by real‐time PCR and melting point analysis according to a modified protocol was ruled out. Patients underwent complete ophthalmological examination with fundus photographs, fluorescein angiography, serological examination, antibiotic treatment and follow‐up. Results In two cases of acute chorioretinitis of unknown origin, real‐time PCR of vitreous specimens of both patients provided evidence of TP and was 100% specific. Initial diagnosis of presumed viral retinitis was ruled out by PCR of vitreous specimen. Patients were treated with systemic antibiotics and showed prompt improvement in visual function and resolution of fundus lesions. Conclusions With real‐time PCR, detection of TP in the vitreous was possible and delivered a sensitive, quick and inexpensive answer to a disease rather difficult to assess. In cases of acute chorioretinitis, the use of PCR‐based assays of vitreous specimens in the diagnostic evaluation of patients is advisable. Although syphilitic chorioretinitis is a rare disease, PCR should include search for TP, as diagnostic dilemmas prolong definitive treatment in a sight‐threatening disease. PMID:17108014

  5. Real-time PCR for sexing Schistosoma mansoni cercariae.

    PubMed

    Chevalier, Frédéric D; Le Clec'h, Winka; Alves de Mattos, Ana Carolina; LoVerde, Philip T; Anderson, Timothy J C

    2016-01-01

    The gender of cercarial larvae can only be determined using molecular methods. End point PCR methods that amplify repetitive markers on the W chromosome of the female (ZW) parasites have been developed, but sometimes results are ambiguous or incorrect. To more effectively distinguish sexes, and to determine why end point PCR can be incorrect, we quantified the W6 repeat sequence and a specific Z chromosome gene using real-time PCR. The ratio between copy number of W6 and a Z chromosome marker unambiguously identifies gender: females have higher ratios (421-4371) than males (0-21). However, some males have low numbers of W6 elements in their genome, and qPCR demonstrated significantly higher W6/Z marker ratios for male genotypes giving ambiguous end point PCR results compared with males giving clear end point results. The quantitative PCR sexing method developed will be particularly useful where reliable sexing of cercariae is critical, for example when staging genetic crosses. PMID:27021570

  6. Simulation of between Repeat Variability in Real Time PCR Reactions

    PubMed Central

    Lievens, Antoon; Van Aelst, Stefan; Van den Bulcke, Marc; Goetghebeur, Els

    2012-01-01

    While many decisions rely on real time quantitative PCR (qPCR) analysis few attempts have hitherto been made to quantify bounds of precision accounting for the various sources of variation involved in the measurement process. Besides influences of more obvious factors such as camera noise and pipetting variation, changing efficiencies within and between reactions affect PCR results to a degree which is not fully recognized. Here, we develop a statistical framework that models measurement error and other sources of variation as they contribute to fluorescence observations during the amplification process and to derived parameter estimates. Evaluation of reproducibility is then based on simulations capable of generating realistic variation patterns. To this end, we start from a relatively simple statistical model for the evolution of efficiency in a single PCR reaction and introduce additional error components, one at a time, to arrive at stochastic data generation capable of simulating the variation patterns witnessed in repeated reactions (technical repeats). Most of the variation in values was adequately captured by the statistical model in terms of foreseen components. To recreate the dispersion of the repeats' plateau levels while keeping the other aspects of the PCR curves within realistic bounds, additional sources of reagent consumption (side reactions) enter into the model. Once an adequate data generating model is available, simulations can serve to evaluate various aspects of PCR under the assumptions of the model and beyond. PMID:23189123

  7. Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

    PubMed

    Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

    2016-12-15

    Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number. PMID:27451195

  8. Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR.

    PubMed

    Villalobos, Michael J; Betti, Christopher J; Vaughan, Andrew T M

    2005-01-01

    Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks introduced into any identifiable genomic location. PMID:15502230

  9. Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species

    PubMed Central

    Wang, Xiaoyu; Seo, Dong Joo; Lee, Min Hwa

    2014-01-01

    This study aimed to develop a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Arcobacter species. Specific primers targeting the 23S ribosomal RNA gene were used to detect Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. The specificity of the LAMP primer set was assessed using DNA samples from a panel of Arcobacter and Campylobacter species, and the sensitivity was determined using serial dilutions of Arcobacter species cultures. LAMP showed a 10- to 1,000-fold-higher sensitivity than multiplex PCR, with a detection limit of 2 to 20 CFU per reaction in vitro. Whereas multiplex PCR showed cross-reactivity with Campylobacter species, the LAMP method developed in this study was more sensitive and reliable than conventional PCR or multiplex PCR for the detection of Arcobacter species. PMID:24478488

  10. Comparing Rapid and Specific Detection of Brucella in Clinical Samples by PCR-ELISA and Multiplex-PCR Method

    PubMed Central

    Mohammad Hasani, Sharareh; Mirnejad, Reza; Amani, Jafar; Vafadar, Mohamad javad

    2016-01-01

    Background: Rapid diagnosis and differentiation of Brucella is of high importance due to the side effects of antibiotics for the treatment of brucellosis. This study aimed to identify and compare PCR-ELISA as a more accurate diagnositc test with other common molecular and serological tests. Methods: In this experimental and sectional study, during March 2014 to Sep 2015, 52 blood samples of suspected patients with clinical symptoms of brucellosis were evaluated in medical centers all over Iran with serum titers higher than 1:80. Using two pairs of specific primers of Brucella abortus, B. melitensis and DIG-dUTP, Fragment IS711 (The common gene fragment in B. melitensis and B. abortus) was amplified. DIG-ELISA was performed using specific probes of these 2 species of Brucella and patterns were subsequently analyzed, then positive responses were compared by detecting gel electrophoresis. Results: PCR-ELISA method detected all 28 samples from 52 positive samples. Its sensitivity was 6.0 pg concentration of genomic DNA of Brucella. In gel electrophoresis method, 22 samples of all positive samples were detected. PCR-ELISA was more efficient than PCR and bacterial culture method at P-value <0.05. Conclusion: PCR-ELISA molecular method is more sensitive than other molecular methods, lack of mutagenic color and also a semi-quantitative ability. This method is more effective and more accurate compared to PCR, serology and culture of bacteria. PCR-ELISA does not have false responses. The limitation of this method is detection of bacteria in the genus compared to Multiplex PCR and Gel Electrophoresis. PMID:27499776

  11. DNA Barcode-Based PCR-RFLP and Diagnostic PCR for Authentication of Jinqian Baihua She (Bungarus Parvus)

    PubMed Central

    Li, Xiaolei; Zeng, Weiping; Liao, Jing; Liang, Zhenbiao; Huang, Shuhua

    2015-01-01

    We established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and diagnostic PCR based on cytochrome C oxidase subunit I (COI) barcodes of Bungarus multicinctus, genuine Jinqian Baihua She (JBS), and adulterant snake species. The PCR-RFLP system utilizes the specific restriction sites of SpeI and BstEII in the COI sequence of B. multicinctus to allow its cleavage into 3 fragments (120 bp, 230 bp, and 340 bp); the COI sequences of the adulterants do not contain these restriction sites and therefore remained intact after digestion with SpeI and BstEII (except for that of Zaocys dhumnades, which could be cleaved into a 120 bp and a 570 bp fragment). For diagnostic PCR, a pair of species-specific primers (COI37 and COI337) was designed to amplify a specific 300 bp amplicon from the genomic DNA of B. multicinctus; no such amplicons were found in other allied species. We tested the two methods using 11 commercial JBS samples, and the results demonstrated that barcode-based PCR-RFLP and diagnostic PCR both allowed effective and accurate authentication of JBS. PMID:26078770

  12. DNA Barcode-Based PCR-RFLP and Diagnostic PCR for Authentication of Jinqian Baihua She (Bungarus Parvus).

    PubMed

    Li, Xiaolei; Zeng, Weiping; Liao, Jing; Liang, Zhenbiao; Huang, Shuhua; Chao, Zhi

    2015-01-01

    We established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and diagnostic PCR based on cytochrome C oxidase subunit I (COI) barcodes of Bungarus multicinctus, genuine Jinqian Baihua She (JBS), and adulterant snake species. The PCR-RFLP system utilizes the specific restriction sites of SpeI and BstEII in the COI sequence of B. multicinctus to allow its cleavage into 3 fragments (120 bp, 230 bp, and 340 bp); the COI sequences of the adulterants do not contain these restriction sites and therefore remained intact after digestion with SpeI and BstEII (except for that of Zaocys dhumnades, which could be cleaved into a 120 bp and a 570 bp fragment). For diagnostic PCR, a pair of species-specific primers (COI37 and COI337) was designed to amplify a specific 300 bp amplicon from the genomic DNA of B. multicinctus; no such amplicons were found in other allied species. We tested the two methods using 11 commercial JBS samples, and the results demonstrated that barcode-based PCR-RFLP and diagnostic PCR both allowed effective and accurate authentication of JBS. PMID:26078770

  13. Determining Fungi rRNA Copy Number by PCR

    PubMed Central

    Black, Jonathan; Dean, Timothy; Byfield, Grace; Foarde, Karin; Menetrez, Marc

    2013-01-01

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within a standard qPCR reaction. The first control developed was the internal standard control gene, benA. This gene encodes for β-tubulin and was selected based on its single-copy nature. The second control developed was the standard control plasmid, which contained a fragment of the ribosomal RNA (rRNA) gene and produced a specific PCR product. The results confirm the multicopy nature of the rRNA region in several filamentous fungi and show that we can quantify fungi of unknown genome size over a range of spore extractions by inclusion of these two standard controls. Advances in qPCR have led to extremely sensitive and quantitative methods for single-copy genes; however, it has not been well established that the rRNA can be used to quantitate fungal contamination. We report on the use of qPCR, combined with two controls, to identify and quantify indoor fungal contaminants with a greater degree of confidence than has been achieved previously. Advances in indoor environmental health have demonstrated that contamination of the built environment by the filamentous fungi has adverse impacts on the health of building occupants. This study meets the need for more accurate and reliable methods for fungal identification and quantitation in the indoor environment. PMID:23543828

  14. Inverse PCR for subtyping of Acinetobacter baumannii carrying ISAba1.

    PubMed

    Kim, Shukho; Park, Yun-Ju; Kim, Jungmin

    2016-05-01

    Acinetobacter baumannii has been prevalent in nosocomial infections, often causing outbreaks in intensive care units. ISAba1 is an insertion sequence that has been identified only in A. baumannii and its copy number varies among strains. It has been reported that ISAba1 provides a promoter for bla OXA-51-like, bla OXA-23-like, and bla ampC, which are associated with the resistance of A. baumannii to carbapenems and cephalosporins. The main purpose of this study was to develop a novel inverse PCR method capable of typing A. baumannii strains. The method involves three major steps: cutting of genomic DNA with a restriction enzyme, ligation, and PCR. In the first step, bacterial genomic DNA was digested with DpnI. In the second step, the digested genomic DNAs were ligated to form intramolecular circular DNAs. In the last step, the ligated circular DNAs were amplified by PCR with primers specific for ISAba1 and the amplified PCR products were electrophoresed. Twenty-two clinical isolates of A. baumannii were used for the evaluation of the inverse PCR (iPCR) typing method. Dendrogram analysis revealed two major clusters, similar to pulsed-field gel electrophoresis (PFGE) results. Three ISAba1-associated genes - bla ampC, bla OXA-66-like, and csuD - were amplified and detected in the clinical isolates. This novel iPCR typing method is comparable to PFGE in its ability to discriminate A. baumannii strains, and is a promising molecular epidemiological tool for investigating A. baumannii carrying ISAba1. PMID:27095456

  15. Droplet-based micro oscillating-flow PCR chip

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Li, Zhi-Xin; Luo, Rong; Lü, Shu-Hai; Xu, Ai-Dong; Yang, Yong-Jun

    2005-08-01

    Polymerase chain reactions (PCR), thermally activated chemical reactions which are widely used for nucleic acid amplification, have recently received much attention in microelectromechanical systems and micro total analysis systems because a wide variety of DNA/RNA molecules can be enriched by PCR for further analyses. In the present work, a droplet-based micro oscillating-flow PCR chip was designed and fabricated by the silicon microfabrication technique. Three different temperature zones, which were stable at denaturation, extension and annealing temperatures and isolated from each other by a thin-wall linkage, were integrated with a single, simple and straight microchannel to form the chip's basic functional structure. The PCR mixture was injected into the chip as a single droplet and flowed through the three temperature zones in the main microchannel in an oscillating manner to achieve the temperature maintenance and transitions. The chip's thermal performance was theoretically analyzed and numerically simulated. The results indicated that the time needed for the temperature of the droplet to change to the target value is less than 1 s, and the root mean square error of temperature is less than 0.2 °C. A droplet of 1 µl PCR mixture with standard HPV (Human Papilloma Virus)-DNA sample inside was amplified by the present chip and the results were analyzed by slab gel electrophoresis with separation of DNA markers in parallel. The electrophoresis results demonstrated that the micro oscillating-flow PCR chip successfully amplified the HPV-DNA, with a processing time of about 15 min which is significantly reduced compared to that for the conventional PCR instrument.

  16. The methylation status of plant genomic DNA influences PCR efficiency.

    PubMed

    Kiselev, K V; Dubrovina, A S; Tyunin, A P

    2015-03-01

    During the polymerase chain reaction (PCR), which is a versatile and widely used method, certain DNA sequences are rapidly amplified through thermocycling. Although there are numerous protocols of PCR optimization for different applications, little is known about the effect of DNA modifications, such as DNA methylation, on PCR efficiency. Recent studies show that cytosine methylation alters DNA mechanical properties and suggest that DNA methylation may directly or indirectly influence the effectiveness of DNA amplification during PCR. In the present study, using plant DNA, we found that highly methylated plant DNA genomic regions were amplified with lower efficiencies compared to that for the regions methylated at a lower level. The correlation was observed when amplifying stilbene synthase (STS1, STS10) genes of Vitis amurensis, the Actin2 gene of Arabidopsis thaliana, the internal transcribed spacer (AtITS), and tRNAPro of A. thaliana. The level of DNA methylation within the analyzed DNA regions has been analyzed with bisulfite sequencing. The obtained data show that efficient PCRs of highly methylated plant DNA regions can be hampered. Proteinase K treatment of the plant DNA prior to PCR and using HotTaq DNA polymerase improved amplification of the highly methylated plant DNA regions. We suggest that increased DNA denaturation temperatures of the highly methylated DNA and contamination with DNA-binding proteins contribute to the hampered PCR amplification of highly methylated DNA. The data show that it is necessary to use current DNA purification protocols and commercial kits with caution to ensure appropriate PCR product yield and prevent bias toward unmethylated DNA amplification in PCRs. PMID:25506767

  17. Early diagnosis of Lassa fever by reverse transcription-PCR.

    PubMed Central

    Demby, A H; Chamberlain, J; Brown, D W; Clegg, C S

    1994-01-01

    We developed a method based on a coupled reverse transcription-PCR (RT-PCR) for the detection of Lassa virus using primers specific for regions of the S RNA segment which are well conserved between isolates from Sierra Leone, Liberia, and Nigeria. The specificity of the assay was confirmed by Southern blotting with a chemiluminescent probe. The assay was able to detect 1 to 10 copies of a plasmid or an RNA transcript containing the target sequence. There was complete concordance between RT-PCR and virus culture for the detection of Lassa virus in a set of 29 positive and 32 negative serum samples obtained on admission to the hospital from patients suspected of having Lassa fever in Sierra Leone. Specificity was confirmed by the failure of amplification of specific products from serum samples collected from 129 healthy blood donors in Sierra Leone or from tissue culture supernatants from cells infected with related arenaviruses (Mopeia, lymphocytic choriomeningitis, Tacaribe, and Pichinde viruses). Sequential serum samples from 29 hospitalized patients confirmed to have Lassa fever were tested by RT-PCR and for Lassa virus-specific antibodies by indirect immunofluorescence (IF). RT-PCR detected virus RNA in 79% of the patients at the time of admission, comparing favorably with IF, which detected antibodies in only 21% of the patients. Lassa virus RNA was detected by RT-PCR in all 29 patients by the third day of admission, whereas antibody was detectable by IF in only 52% of the patients. These results point to an important role for RT-PCR in the management of suspected cases of Lassa fever. Images PMID:7883875

  18. Anti-PcrV antibody strategies against virulent Pseudomonas aeruginosa.

    PubMed

    Sawa, Teiji; Ito, Emi; Nguyen, Vinh Huu; Haight, Matthew

    2014-01-01

    Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes fatal acute lung infections in critically ill individuals. Its pathogenesis is associated with bacterial virulence conferred by the type III secretion system (TTSS), through which P. aeruginosa causes necrosis of the lung epithelium and disseminates into the circulation, resulting in bacteremia, sepsis, and mortality. TTSS allows P. aeruginosa to directly translocate cytotoxins into eukaryotic cells, inducing cell death. The P. aeruginosa V-antigen PcrV, a homolog of the Yersinia V-antigen LcrV, is an indispensable contributor to TTS toxin translocation. Vaccination against PcrV ensures the survival of challenged mice and decreases lung inflammation and injury. Both the rabbit polyclonal anti-PcrV antibody and the murine monoclonal anti-PcrV antibody, mAb166, inhibit TTS toxin translocation. mAb166 IgG was cloned, and a molecular engineered humanized anti-PcrV IgG antigen-binding fragment, KB001, was developed for clinical use. KB001 is currently undergoing Phase-II clinical trials for ventilator-associated pneumonia in France and chronic pneumonia in cystic fibrosis in USA. In these studies, KB001 has demonstrated its safety, a favorable pharmacokinetic profile, and promising potential as a nonantibiotic strategy to reduce airway inflammation and damage in P. aeruginosa pneumonia. PMID:25483637

  19. Reproducibility problems with the AMPLICOR PCR Chlamydia trachomatis test.

    PubMed Central

    Peterson, E M; Darrow, V; Blanding, J; Aarnaes, S; de la Maza, L M

    1997-01-01

    In an attempt to use an expanded "gold standard" in an evaluation of an antigen detection test for Chlamydia trachomatis, the AMPLICOR (Roche Diagnostics Systems, Inc., Branchburg, N.J.) PCR Chlamydia trachomatis test and culture were used with 591 sets of cervical specimens. Of the 591 specimens assayed, 35 were retested due to either an equivocal result by the PCR (19 samples) or a discrepancy between the results of culture, PCR, and the antigen detection method. During the repeat testing of the samples with equivocal and discrepant results, all but one interpretation change was due to the PCR result. In addition, upon repeat testing the PCR assay value measured in optical density units varied widely for 13 of these specimens. These 13 specimens were then tested in triplicate by the manufacturer with primers to the chlamydia plasmid and in duplicate with primers to the major outer membrane protein. Only 3 of the 13 specimens gave the same interpretation with these five replicates. In summary, reproducibility problems with the AMPLICOR test should be considered before it is incorporated as part of routine testing or used as an expanded gold standard for chlamydia testing. PMID:9157161

  20. Multiplex PCR: Optimization and Application in Diagnostic Virology

    PubMed Central

    Elnifro, Elfath M.; Ashshi, Ahmed M.; Cooper, Robert J.; Klapper, Paul E.

    2000-01-01

    PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance. PMID:11023957

  1. Performance of Nanoliter-Sized Droplet-based Microfluidic PCR

    PubMed Central

    Wang, Fang; Burns, Mark A.

    2010-01-01

    A microfluidic device was used to characterize PCR in aqueous-in-oil droplets for potential point-of-care applications. Droplets with a volume range of 5–250nL can be formed on-chip reproducibly, and PCR in the droplets shows amplification efficiencies comparable to benchtop reactions with no evaporation loss. A higher polymerase concentration is required in the reaction droplet while the optimal Magnesium ion concentration is the same for both on-chip and benchtop systems. The optimal hold time is 9 and 30 seconds for denaturation and annealing/extension in thermal cycling, respectively. With the optimized cycling parameters, the total reaction time is reduced to half of that required for benchtop PCR. For the droplets containing the same quantity of template DNA, the PCR yield is approximately the same with either fixed droplet size or fixed template DNA concentration. The droplet-based PCR can be monitored in real time with FRET probes, and provide amplification with a cycle threshold of ~10 cycles earlier than the benchtop instruments. PMID:19479169

  2. [Pnemocystis jiroveci pneumonia: Comparison between conventional PCR and staining techniques].

    PubMed

    Kaouech, E; Kallel, K; Anane, S; Belhadj, S; Abdellatif, S; Mnif, K; Ben Othmane, T; Ben Lakhal, S; Kilani, B; Ben Châabane, T; Chaker, E

    2009-07-01

    Diagnosis of pneumocystis pneumonia is usually based on clinical features and X-rays photography and confirmed in the laboratory by visualisation of Pneumocystis organisms in stained preparations of respiratory specimens using several techniques (Gomori-Grocott, May-Grünwald Giemsa, bleu de toluidine O). Actually, PCR has considerably increased sensitivity of detection of Pneumocystis. The aim of this study is to compare conventional PCR results to those of staining techniques (Gomori-Grocott, May-Grünwald Giemsa) in addition to the X-ray and clinical findings in order to evaluate the contribution of each method. Sixty-four respiratory specimens were collected from 54 immuno-compromised patients with clinical symptoms of pulmonary infection. We diagnosed pneumocystis pneumonia in 16 patients according to staining techniques and/or typical clinical and radiological findings and/or response to treatment. Of the 15 patients, 14 were positive by PCR and only five were positive by direct examination, yielding a sensitivity and specificity of 93.3 and 87.1% for PCR and 33.3 and 100% for staining techniques. Conventional PCR provides a sensitive and objective method for the detection Pneumocystis jiroveci from less invasive sample. PMID:19038508

  3. Digital PCR using micropatterned superporous absorbent array chips.

    PubMed

    Wang, Yazhen; Southard, Kristopher M; Zeng, Yong

    2016-06-21

    Digital PCR (dPCR) is an emerging technology for genetic analysis and clinical diagnostics. To facilitate the widespread application of dPCR, here we developed a new micropatterned superporous absorbent array chip (μSAAC) which consists of an array of microwells packed with highly porous agarose microbeads. The packed beads construct a hierarchically porous microgel which confers superior water adsorption capacity to enable spontaneous filling of PDMS microwells for fluid compartmentalization without the need of sophisticated microfluidic equipment and operation expertise. Using large λ-DNA as the model template, we validated the μSAAC for stochastic partitioning and quantitative digital detection of DNA molecules. Furthermore, as a proof-of-concept, we conducted dPCR detection and single-molecule sequencing of a mutation prevalent in blood cancer, the chromosomal translocation t(14;18), demonstrating the feasibility of the μSAAC for analysis of disease-associated mutations. These experiments were carried out using the standard molecular biology techniques and instruments. Because of its low cost, ease of fabrication, and equipment-free liquid partitioning, the μSAAC is readily adaptable to general lab settings, which could significantly facilitate the widespread application of dPCR technology in basic research and clinical practice. PMID:27010726

  4. A QUANTITATIVE MODEL OF ERROR ACCUMULATION DURING PCR AMPLIFICATION

    PubMed Central

    Pienaar, E; Theron, M; Nelson, M; Viljoen, HJ

    2006-01-01

    The amplification of target DNA by the polymerase chain reaction (PCR) produces copies which may contain errors. Two sources of errors are associated with the PCR process: (1) editing errors that occur during DNA polymerase-catalyzed enzymatic copying and (2) errors due to DNA thermal damage. In this study a quantitative model of error frequencies is proposed and the role of reaction conditions is investigated. The errors which are ascribed to the polymerase depend on the efficiency of its editing function as well as the reaction conditions; specifically the temperature and the dNTP pool composition. Thermally induced errors stem mostly from three sources: A+G depurination, oxidative damage of guanine to 8-oxoG and cytosine deamination to uracil. The post-PCR modifications of sequences are primarily due to exposure of nucleic acids to elevated temperatures, especially if the DNA is in a single-stranded form. The proposed quantitative model predicts the accumulation of errors over the course of a PCR cycle. Thermal damage contributes significantly to the total errors; therefore consideration must be given to thermal management of the PCR process. PMID:16412692

  5. Optimizing methods for PCR-based analysis of predation

    PubMed Central

    Sint, Daniela; Raso, Lorna; Kaufmann, Rüdiger; Traugott, Michael

    2011-01-01

    Molecular methods have become an important tool for studying feeding interactions under natural conditions. Despite their growing importance, many methodological aspects have not yet been evaluated but need to be considered to fully exploit the potential of this approach. Using feeding experiments with high alpine carabid beetles and lycosid spiders, we investigated how PCR annealing temperature affects prey DNA detection success and how post-PCR visualization methods differ in their sensitivity. Moreover, the replicability of prey DNA detection among individual PCR assays was tested using beetles and spiders that had digested their prey for extended times postfeeding. By screening all predators for three differently sized prey DNA fragments (range 116–612 bp), we found that only in the longest PCR product, a marked decrease in prey detection success occurred. Lowering maximum annealing temperatures by 4 °C resulted in significantly increased prey DNA detection rates in both predator taxa. Among the three post-PCR visualization methods, an eightfold difference in sensitivity was observed. Repeated screening of predators increased the total number of samples scoring positive, although the proportion of samples testing positive did not vary significantly between different PCRs. The present findings demonstrate that assay sensitivity, in combination with other methodological factors, plays a crucial role to obtain robust trophic interaction data. Future work employing molecular prey detection should thus consider and minimize the methodologically induced variation that would also allow for better cross-study comparisons. PMID:21507208

  6. Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

    PubMed Central

    Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

    2016-01-01

    Synopsis A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laboratory developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. We will focus on two FDA cleared commercial syndromic multiplex tests: Luminex xTAG GPP and Biofire FimArray GI test. These multiplex tests can detect and identify multiple enteric pathogens in one test and provide results within hours. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens. The high negative predictive value of these multiplex tests has led to the suggestion that they be used as screening tools especially in outbreaks. Although the clinical utility and benefit of multiplex PCR test are to be further investigated, implementing these multiplex PCR tests in gastroenterology diagnostic algorithm has the potential to improve diagnosis of infectious gastroenteritis. PMID:26004652

  7. Distinguishing human and possum faeces using PCR markers.

    PubMed

    Devane, M; Robson, B; Nourozi, F; Wood, D; Gilpin, B J

    2013-09-01

    Specificity testing of two published polymerase chain reaction (PCR) markers for the detection of human faecal pollution, revealed 100% false-positive rates to brush-tailed possum faeces (n = 10), but low false-positive rates against other potential pollution sources. Cross-reaction with possums could be a problem with other human-specific markers; therefore, a possum PCR marker was developed for use in conjunction with human PCR markers. The possum PCR marker was based on Bacteroidales 16S ribosomal ribonucleic acid sequences, and was tested on 233 individual faecal samples from 11 other animal species. Sensitivity of the possum marker in possum faeces (n = 36) was high at 83.3%. Cross-reactivity of the possum marker was limited to black swan (7/20 samples), human (2/48 samples) and rabbit (1/10) faecal samples, all at marker concentrations at least four orders of magnitude lower than possum faeces. The possum marker was not detected in human sewage or the faeces of other animal species. Specificity of the possum PCR marker, therefore, was high at 95.7%. To exclude the possibility that only possum pollution is being detected, additional testing by other faecal source tracking methods is required where the water sample is positive for both human and possum markers. PMID:23981869

  8. Application of PCR and real-time PCR for monitoring cyanobacteria, Microcystis spp. and Cylindrospermopsis raciborskii in Macau freshwater reservoir

    NASA Astrophysics Data System (ADS)

    Zhang, Weiying; Lou, Inchio; Ung, Wai Kin; Kong, Yijun; Mok, Kai Meng

    2014-06-01

    Freshwater algal blooms have become a growing concern world-wide. They are caused by a high level of cyanobacteria, predominantly Microcystis spp. and Cylindrospermopsis raciborskii, which can produce microcystin and cylindrospermopsin, respectively. Longtime exposure to these cyanotoxins may affect public health, thus reliable detection, quantification, and enumeration of these harmful algae species has become a priority in water quality management. Traditional manual enumeration of algal bloom cells primarily involves microscopic identification which limited by inaccuracy and time-consumption.With the development of molecular techniques and an increasing number of microbial sequences available in the Genbank database, the use of molecular methods can be used for more rapid, reliable, and accurate detection and quantification. In this study, multiplex polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) techniques were developed and applied for monitoring cyanobacteria Microcystis spp. and C. raciborskii in the Macau Storage Reservoir (MSR). The results showed that the techniques were successful for identifying and quantifying the species in pure cultures and mixed cultures, and proved to be a potential application for water sampling in MSR. When the target species were above 1 million cells/L, similar cell numbers estimated by microscopic enumeration and qPCR were obtained. Further quantification in water samples indicated that the ratio of the estimated number of cell by microscopy and qPCR was 0.4-12.9 for cyanobacteria and 0.2-3.9 for C. raciborskii. However, Microcystis spp. was not observed by manual enumeration, while it was detected at low levels by qPCR, suggesting that qPCR is more sensitive and accurate. Thus the molecular approaches provide an additional reliable monitoring option to traditional microscopic enumeration for the ecosystems monitoring program.

  9. An improved, PCR-based strategy for the detection of Trypanosoma cruzi in human blood samples.

    PubMed

    Ribeiro-dos-Santos, G; Nishiya, A S; Sabino, E C; Chamone, D F; Saez-Alquézar, A

    1999-10-01

    Attempts were made to improve the PCR-based detection of Trypanosoma cruzi in blood samples, primarily for screening blood donors. Samples were obtained from candidate donors who were reactive in one or two of three serological tests for Chagas disease (and therefore considered 'indeterminate') or in all three tests (3+). Each sample was then examined using three different, PCR-based techniques: 'PCR-I' (in which the target DNA is a nuclear repetitive sequence); 'PCR-II' [amplifying a conserved region of the T. cruzi kinetoplast DNA (kDNA)]; and 'PCR-III' (a new strategy in which the target kDNA is amplified by 'nested' PCR). Among the samples from 3+ individuals, PCR-I, PCR-II and PCR-III amplified two (3.8%) out of 52, four (4.5%) out of 88, and 27 (25.7%) out of 105 samples tested, respectively. Seven, 69 and 70 samples from 'indeterminate' subjects were tested by PCR-I, PCR-II and PCR-III, respectively; there was not a single positive result by PCR-I or PCR-II, but three (4.3%) of the samples tested by PCR-III were positive. In a reconstruction experiment, in conditions in which PCR-I and PCR-II could not detect 10,000 parasites/ml, PCR-III was able to detect one parasite/ml. Although all three PCR-based strategies examined had rather poor sensitivities, PCR-III was far more sensitive than PCR-I or PCR-II. PMID:10715696

  10. Using PCR to Target Misconceptions about Gene Expression †

    PubMed Central

    Wright, Leslie K.; Newman, Dina L.

    2013-01-01

    We present a PCR-based laboratory exercise that can be used with first- or second-year biology students to help overcome common misconceptions about gene expression. Biology students typically do not have a clear understanding of the difference between genes (DNA) and gene expression (mRNA/protein) and often believe that genes exist in an organism or cell only when they are expressed. This laboratory exercise allows students to carry out a PCR-based experiment designed to challenge their misunderstanding of the difference between genes and gene expression. Students first transform E. coli with an inducible GFP gene containing plasmid and observe induced and un-induced colonies. The following exercise creates cognitive dissonance when actual PCR results contradict their initial (incorrect) predictions of the presence of the GFP gene in transformed cells. Field testing of this laboratory exercise resulted in learning gains on both knowledge and application questions on concepts related to genes and gene expression. PMID:23858358

  11. Identification of neotropical felid faeces using RCP-PCR.

    PubMed

    Roques, S; Adrados, B; Chavez, C; Keller, C; Magnusson, W E; Palomares, F; Godoy, J A

    2011-01-01

    Faeces similarity among sympatric felid species has generally hampered their use in distributional, demographic and dietary studies. Here, we present a new and simple approach based on a set of species-specific primers, for the unambiguous identification of faeces from sympatric neotropical felids (i.e. puma, jaguar, jaguarundi and ocelot/ margay). This method, referred to as rapid classificatory protocol-PCR (RCP-PCR), consists of a single-tube multiplex PCR yielding species-specific banding patterns on agarose gel. The method was optimized with samples of known origin (14 blood and 15 fresh faeces) and validated in faecal samples of unknown origin (n = 138), for some of which (n = 40) we also obtained species identification based on mtDNA sequencing. This approach proved reliable and provides high identification success rates from faeces. Its simplicity and cost effectiveness should facilitate its application for routine surveys of presence and abundance of these species. PMID:21429118

  12. Development of a PCR Assay for Rapid Detection of Enterococci

    PubMed Central

    Ke, Danbing; Picard, François J.; Martineau, Francis; Ménard, Christian; Roy, Paul H.; Ouellette, Marc; Bergeron, Michel G.

    1999-01-01

    Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the tuf gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (Enterococcus avium, E. faecalis, E. faecium, and E. gallinarum). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR

  13. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    NASA Astrophysics Data System (ADS)

    Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

    2008-10-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  14. Detection and Quantification of Wallemia sebi in Aerosols by Real-Time PCR, Conventional PCR, and Cultivation

    PubMed Central

    Zeng, Qing-Yin; Westermark, Sven-Olof; Rasmuson-Lestander, Åsa; Wang, Xiao-Ru

    2004-01-01

    Wallemia sebi is a deuteromycete fungus commonly found in agricultural environments in many parts of the world and is suspected to be a causative agent of farmer's lung disease. The fungus grows slowly on commonly used culture media and is often obscured by the fast-growing fungi. Thus, its occurrence in different environments has often been underestimated. In this study, we developed two sets of PCR primers specific to W. sebi that can be applied in either conventional PCR or real-time PCR for rapid detection and quantification of the fungus in environmental samples. Both PCR systems proved to be highly specific and sensitive for W. sebi detection even in a high background of other fungal DNAs. These methods were employed to investigate the presence of W. sebi in the aerosols of a farm. The results revealed a high concentration of W. sebi spores, 107 m−3 by real-time PCR and 106 m−3 by cultivation, which indicates the prevalence of W. sebi in farms handling hay and grain and in cow barns. The methods developed in this study could serve as rapid, specific, and sensitive means of detecting W. sebi in aerosol and surface samples and could thus facilitate investigations of its distribution, ecology, clinical diagnosis, and exposure risk assessment. PMID:15574929

  15. 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

  16. Optimized 5-hour multiplex PCR test for the detection of tinea unguium: performance in a routine PCR laboratory.

    PubMed

    Brillowska-Dabrowska, Anna; Nielsen, Sanne Søgaard; Nielsen, Henrik Vedel; Arendrup, Maiken Cavling

    2010-09-01

    We recently reported the development of a 5-hour multiplex PCR test for the detection of tinea unguium and the optimization of this test by the inclusion of an inhibition control. Here we report the performance of this procedure as used in a routine clinical laboratory as compared to conventional microscopy and culture-based techniques performed in a mycology reference laboratory. We found in processing 109 samples that 22 (20.2%) yielded fungi in culture while the suspected etiologic agents were noted microscopically in 15 (13.8%) that were negative in culture. Fungi were detected by PCR in 37 (33.9%) samples, of which only three were positive in culture. Since the majority of PCR positive but culture negative samples were positive in microscopic examinations, the increased sensitivity was not due to contamination. PCR inhibitors were present in 5% of the samples, but this was overcome by re-running the samples with a 50% reduction of sample DNA. In conclusion, the PCR test performance in the routine setting was excellent and provided a markedly reduced time to diagnosis with a higher sensitivity. PMID:20105101

  17. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    SciTech Connect

    Gardner, S. N.

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether there are also TaqMan/Luminex probe matches within predicted amplicons.

  18. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether theremore » are also TaqMan/Luminex probe matches within predicted amplicons.« less

  19. [Do Multiplex PCR techniques displace classical cultures in microbiology?].

    PubMed

    Auckenthaler, Raymond; Risch, Martin

    2015-02-01

    Multiplex PCR technologies progressively find their way in clinical microbiology. This technique allows the simultaneous amplification of multiple DNA targets in a single test run for the identification of pathogens up to the species level. Various pathogens of infectious diseases can be detected by a symptom-oriented approach clearly and quickly with high reliability. Essentially multiplex PCR panels are available for clarification of gastrointestinal, respiratory, sexually transmitted infections and meningitis. Today's offer from industry, university hospitals and large private laboratories of Switzerland is tabulated and commented. PMID:25630288

  20. Use of Treponema pallidum PCR in Testing of Ulcers for Diagnosis of Primary Syphilis1

    PubMed Central

    Sednaoui, Patrice; Lautenschlager, Stephan; Ferry, Tristan; Toutous-Trellu, Laurence; Cavassini, Matthias; Yassir, Fatima; Martinez de Tejada, Begoña; Emonet, Stéphane; Combescure, Christophe; Schrenzel, Jacques; Perneger, Thomas

    2015-01-01

    Treponema pallidum PCR (Tp-PCR) has been noted as a valid method for diagnosing syphilis. We compared Tp-PCR to a combination of darkfield microscopy (DFM), the reference method, and serologic testing in a cohort of 273 patients from France and Switzerland and found the diagnostic accuracy of Tp-PCR was higher than that for DFM. PMID:25531672

  1. Use of Treponema pallidum PCR in testing of ulcers for diagnosis of primary syphilis.

    PubMed

    Gayet-Ageron, Angèle; Sednaoui, Patrice; Lautenschlager, Stephan; Ferry, Tristan; Toutous-Trellu, Laurence; Cavassini, Matthias; Yassir, Fatima; Martinez de Tejada, Begoña; Emonet, Stéphane; Combescure, Christophe; Schrenzel, Jacques; Perneger, Thomas

    2015-01-01

    Treponema pallidum PCR (Tp-PCR) has been noted as a valid method for diagnosing syphilis. We compared Tp-PCR to a combination of darkfield microscopy (DFM), the reference method, and serologic testing in a cohort of 273 patients from France and Switzerland and found the diagnostic accuracy of Tp-PCR was higher than that for DFM. PMID:25531672

  2. Use of Droplet Digital PCR for Estimation of Fish Abundance and Biomass in Environmental DNA Surveys

    PubMed Central

    Doi, Hideyuki; Uchii, Kimiko; Takahara, Teruhiko; Matsuhashi, Saeko; Yamanaka, Hiroki; Minamoto, Toshifumi

    2015-01-01

    An environmental DNA (eDNA) analysis method has been recently developed to estimate the distribution of aquatic animals by quantifying the number of target DNA copies with quantitative real-time PCR (qPCR). A new quantitative PCR technology, droplet digital PCR (ddPCR), partitions PCR reactions into thousands of droplets and detects the amplification in each droplet, thereby allowing direct quantification of target DNA. We evaluated the quantification accuracy of qPCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp. We found that ddPCR quantified the concentration of carp eDNA along with carp abundance and biomass more accurately than qPCR, especially at low eDNA concentrations. In addition, errors in the analysis were smaller in ddPCR than in qPCR. Thus, ddPCR is better suited to measure eDNA concentration in water, and it provides more accurate results for the abundance and biomass of the target species than qPCR. We also found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by using both ddPCR and qPCR; this suggests that abundance can be better estimated by the analysis of eDNA for species with fewer variations in body mass. PMID:25799582

  3. Comparison of PCR/Electron spray Ionization-Time-of-Flight-Mass Spectrometry versus Traditional Clinical Microbiology for active surveillance of organisms contaminating high-use surfaces in a burn intensive care unit, an orthopedic ward and healthcare workers

    PubMed Central

    2012-01-01

    Background Understanding nosocomial pathogen transmission is restricted by culture limitations. Novel platforms, such as PCR-based electron spray ionization-time-of-flight-mass spectrometry (ESI-TOF-MS), may be useful as investigational tools. Methods Traditional clinical microbiology (TCM) and PCR/ESI-TOF-MS were used to recover and detect microorganisms from the hands and personal protective equipment of 10 burn intensive care unit (ICU) healthcare workers providing clinical care at a tertiary care military referral hospital. High-use environmental surfaces were assessed in 9 burn ICU and 10 orthopedic patient rooms. Clinical cultures during the study period were reviewed for pathogen comparison with investigational molecular diagnostic methods. Results From 158 samples, 142 organisms were identified by TCM and 718 by PCR/ESI-TOF-MS. The molecular diagnostic method detected more organisms (4.5 ± 2.1 vs. 0.9 ± 0.8, p < 0.01) from 99% vs. 67% of samples (p < 0.01). TCM detected S. aureus in 13 samples vs. 21 by PCR/ESI-TOF-MS. Gram-negative organisms were less commonly identified than gram-positive by both methods; especially by TCM. Among all detected bacterial species, similar percentages were typical nosocomial pathogens (18-19%) for TCM vs. PCR/ESI-TOF-MS. PCR/ESI-TOF-MS also detected mecA in 112 samples, vanA in 13, and KPC-3 in 2. MecA was associated (p < 0.01) with codetection of coagulase negative staphylococci but not S. aureus. No vanA was codetected with enterococci; one KPC-3 was detected without Klebsiella spp. Conclusions In this pilot study, PCR/ESI-TOF-MS detected more organisms, especially gram-negatives, compared to TCM, but the current assay format is limited by the number of antibiotic resistance determinants it covers. Further large-scale assessments of PCR/ESI-TOF-MS for hospital surveillance are warranted. PMID:23050585

  4. A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples.

    PubMed

    Coudray-Meunier, Coralie; Fraisse, Audrey; Martin-Latil, Sandra; Guillier, Laurent; Delannoy, Sabine; Fach, Patrick; Perelle, Sylvie

    2015-05-18

    Sensitive and quantitative detection of foodborne enteric viruses is classically achieved by quantitative RT-PCR (RT-qPCR). Recently, digital PCR (dPCR) was described as a novel approach to genome quantification without need for a standard curve. The performance of microfluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR for detecting the main viruses responsible for foodborne outbreaks (human Noroviruses (NoV) and Hepatitis A virus (HAV)) in spiked lettuce and bottled water. Two process controls (Mengovirus and Murine Norovirus) were used and external amplification controls (EAC) were added to examine inhibition of RT-qPCR and RT-dPCR. For detecting viral RNA and cDNA, the sensitivity of the RT-dPCR assays was either comparable to that of RT-qPCR (RNA of HAV, NoV GI, Mengovirus) or slightly (around 1 log10) decreased (NoV GII and MNV-1 RNA and of HAV, NoV GI, NoV GII cDNA). The number of genomic copies determined by dPCR was always from 0.4 to 1.7 log10 lower than the expected numbers of copies calculated by using the standard qPCR curve. Viral recoveries calculated by RT-dPCR were found to be significantly higher than by RT-qPCR for NoV GI, HAV and Mengovirus in water, and for NoV GII and HAV in lettuce samples. The RT-dPCR assay proved to be more tolerant to inhibitory substances present in lettuce samples. This absolute quantitation approach may be useful to standardize quantification of enteric viruses in bottled water and lettuce samples and may be extended to quantifying other human pathogens in food samples. PMID:25725459

  5. Methods for producing partially digested restriction DNA fragments and for producing a partially modified PCR product

    DOEpatents

    Wong, Kwong-Kwok

    2000-01-01

    The present invention is an improved method of making a partially modified PCR product from a DNA fragment with a polymerase chain reaction (PCR). In a standard PCR process, the DNA fragment is combined with starting deoxynucleoside triphosphates, a primer, a buffer and a DNA polymerase in a PCR mixture. The PCR mixture is then reacted in the PCR producing copies of the DNA fragment. The improvement of the present invention is adding an amount of a modifier at any step prior to completion of the PCR process thereby randomly and partially modifying the copies of the DNA fragment as a partially modified PCR product. The partially modified PCR product may then be digested with an enzyme that cuts the partially modified PCR product at unmodified sites thereby producing an array of DNA restriction fragments.

  6. Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution.

    PubMed

    Weaver, Suzanne; Dube, Simant; Mir, Alain; Qin, Jian; Sun, Gang; Ramakrishnan, Ramesh; Jones, Robert C; Livak, Kenneth J

    2010-04-01

    This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies, which corresponds to a 1.25-fold difference in relative quantity. Digital PCR is considered as an alternative form of qPCR. For digital PCR, an error model is shown that relates the precision of CNV determination to the number of reaction chambers. The quantitative capability of digital PCR is illustrated with an experiment distinguishing four and five copies of the human gene MRGPRX1. For either real-time qPCR or digital PCR, practical application of these models to achieve enhanced quantitative resolution requires use of a high throughput PCR platform that can simultaneously perform thousands of reactions. Comparing the two methods, real-time qPCR has the advantage of throughput and digital PCR has the advantage of simplicity in terms of the assumptions made for data analysis. PMID:20079846

  7. Use of multiplex PCR and PCR restriction enzyme analysis for detection and exploration of the variability in the free-living amoeba Naegleria in the environment.

    PubMed

    Pélandakis, Michel; Pernin, Pierre

    2002-04-01

    A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites. PMID:11916734

  8. Use of Multiplex PCR and PCR Restriction Enzyme Analysis for Detection and Exploration of the Variability in the Free-Living Amoeba Naegleria in the Environment

    PubMed Central

    Pélandakis, Michel; Pernin, Pierre

    2002-01-01

    A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites. PMID:11916734

  9. PCR detection of bovine herpesviruses from nonbovine ruminants in Hungary.

    PubMed

    Kálmán, Dóra; Egyed, László

    2005-07-01

    Polymerase chain reaction (PCR) was used to test six different nonbovine ruminant species for five bovine herpesviruses including infectious bovine rhinotracheitis virus (BoHV-1), bovine herpes mammillitis virus (BoHV-2), Movar-type herpesvirus (BoHV-4), bovine herpesvirus type 5 (BoHV-5), and alcelaphine herpesvirus 1 (AlHV-1). Species tested included 56 roe deer (Capreolus capreolus), 66 red deer (Cervus elaphus), 20 fallow deer (Dama dama), 16 mouflon (Ovis musimon), 34 domestic sheep, and 44 domestic goats, which were sampled in Hungary in 2003. Tracheal and popliteal lymph nodes collected from these animals were tested for the presence of the five bovine herpesviruses using three nested (two of which were duplex) PCR assays. Three bovine herpesviruses (BoHV-1, -2, and -4) were detected, whereas no evidence of AlHV-1 or BoHV-5 was observed. Prevalence of BoHV-1 ranged from 12% to 47%, and PCR-positive results were observed in all species tested. BoHV-2 was detected from roe deer, red deer, fallow deer, mouflon, and domestic sheep, and the prevalence in these species ranged from 3% to 50%. BoHV-4 was detected in all species, with prevalence ranging from 12% to 69%. Sequenced PCR products were 99-100% identical to bovine herpesviral sequences deposited in the GenBank. PMID:16244057

  10. PCR-BASED IDENTIFICATION OF BACTERIA IN ENDODONTIC INFECTIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR primers, that target the bacterial 16S rRNA genes (or the tuf gene for Enterococcus genus), were used to identify 10 putative bacterial pathogens in root canals with necrotic pulp. In addition, the associations of these microorganisms with symptoms and a history of diabetes mellitus were invest...

  11. Quantitative Detection of Spiroplasma Citri by Real Time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is a need to develop an accurate and rapid method to detect Spiroplasma citri, the causal agent of citrus stubborn disease for use in epidemiology studies. Quantitative real-time PCR was developed for detection of S. citri. Two sets of primers based on sequences from the P58 putative adhesin ...

  12. A Trio of Human Molecular Genetics PCR Assays

    ERIC Educational Resources Information Center

    Reinking, Jeffrey L.; Waldo, Jennifer T.; Dinsmore, Jannett

    2013-01-01

    This laboratory exercise demonstrates three different analytical forms of the polymerase chain reaction (PCR) that allow students to genotype themselves at four different loci. Here, we present protocols to allow students to a) genotype a non-coding polymorphic Variable Number of Tandem Repeat (VNTR) locus on human chromosome 5 using conventional…

  13. MOLD SPECIFIC QUANTITATIVE PCR: THE EMERGING STANDARD IN MOLD ANALYSIS

    EPA Science Inventory

    Today I will talk about the use of quantitative or Real time PCR for the standardized identification and quantification of molds. There are probably at least 100,000 species of molds or fungi. But there are actually about 100 typically found indoors. Some pose a threat to human...

  14. Qualitative PCR method for Roundup Ready soybean: interlaboratory study.

    PubMed

    Kodama, Takashi; Kasahara, Masaki; Minegishi, Yasutaka; Futo, Satoshi; Sawada, Chihiro; Watai, Masatoshi; Akiyama, Hiroshi; Teshima, Reiko; Kurosawa, Yasunori; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

    2011-01-01

    Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots. PMID:21391499

  15. UTILIZATION OF PCR TO DETECT SALMONELLA ON TURKEY CARCASSES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The risk which is presented by food-borne pathogens to the consumer demonstrates the need to utilize rapid methods for the detection of these microbes. This study compared conventional microbiology with the application of PCR assays to detect Salmonella on turkey carcasses at a processing plant in ...

  16. Real-time PCR for Strongyloides stercoralis-associated meningitis.

    PubMed

    Nadir, Eyal; Grossman, Tamar; Ciobotaro, Pnina; Attali, Malka; Barkan, Daniel; Bardenstein, Rita; Zimhony, Oren

    2016-03-01

    Four immunocompromised patients, immigrants from Ethiopia, presented with diverse clinical manifestations of meningitis associated with Strongyloides stercoralis dissemination as determined by identification of intestinal larvae. The cerebrospinal fluid of 3 patients was tested by a validated (for stool) real-time PCR for S. stercoralis and was found positive, establishing this association. PMID:26704620

  17. Multiplex PCR Method for Identifying Recombinant Vaccine-Related Polioviruses

    PubMed Central

    Kilpatrick, David R.; Ching, Karen; Iber, Jane; Campagnoli, Ray; Freeman, Christopher J.; Mishrik, Nada; Liu, Hong-Mei; Pallansch, Mark A.; Kew, Olen M.

    2004-01-01

    The recent discovery of recombinant circulating vaccine-derived poliovirus (recombinant cVDPV) has highlighted the need for enhanced global poliovirus surveillance to assure timely detection of any future cVDPV outbreaks. Six pairs of Sabin strain-specific recombinant primers were designed to permit rapid screening for VDPV recombinants by PCR. PMID:15365031

  18. Detection of major diarrheagenic bacterial pathogens by multiplex PCR panels.

    PubMed

    Sjöling, Åsa; Sadeghipoorjahromi, Leila; Novak, Daniel; Tobias, Joshua

    2015-03-01

    Diarrheal diseases remain a major threat to the youngest population in low- and middle-income countries. The main bacterial pathogens causing diarrhea are diarrheagenic Escherichia coli (DEC) that consists of enteroaggregative (EAEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorrhagic EHEC and enteroinvasive E. coli (EIEC), Salmonella, Shigella spp. (S. dysenteria, S. sonnei, S. flexneri) Campylobacter (C. coli, C. jejuni), Vibrio (V. vulnificus, V. parahaemolyticusm, V. cholerae), Yersinia enterocolitica and Aeromonas hydrophila. The aim of this study was to set up rapid multiplex PCR (mPCR) panels to identify these diarrheagenic pathogens based on their specific virulence genes. Primers against specific target genes were combined into three mPCR panels: one for diarrheal E. coli, one for pathogens causing mainly bloody diarrhea, and the third for the remaining pathogens. The panels were tested against a set of stool samples from Swedish children with diarrhea and controls and the analysis identified bacterial pathogens in 14/54 (26%) of the samples. These results show that our three developed mPCR panels can detect main bacterial diarrheagenic pathogens in clinical samples. PMID:25542594

  19. DETECTION OF FECAL ENTEROCOCCI USING A REAL TIME PCR METHOD

    EPA Science Inventory

    In spite of their importance in public health, the detection of fecal enterococci is performed via culturing methods that are time consuming and that are subject to inaccuracies that relate to their culturable status. In order to address these problems, a real time PCR (TaqMan) ...

  20. Citrus stubborn disease incidence determined by quantitative real time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time (q) PCR was developed for detection of Spiroplasma citri, the causal agent of citrus stubborn disease (CSD), using the DNA binding fluorophore SYBR Green I. The primer pair, P58-3f/4r, developed based on sequences from the P58 putative adhesin multigene of the pathogen result...

  1. Detection of Treponema pallidum by a sensitive reverse transcriptase PCR.

    PubMed Central

    Centurion-Lara, A; Castro, C; Shaffer, J M; Van Voorhis, W C; Marra, C M; Lukehart, S A

    1997-01-01

    Syphilis is diagnosed by serologic testing or by identification of the causative agent, Treponema pallidum. The bacterium has historically been detected in clinical specimens by dark-field microscopy, immunostaining with polyclonal or monoclonal antibodies, or the rabbit inoculation test (RIT). RIT is considered to be very sensitive and specific, although it is available only in research settings and is not clinically useful due to the length of time required to obtain a result. In recent years, several PCR methods have been developed for the detection of T. pallidum, but none of these has shown a clear advantage in sensitivity over RIT. We have developed a specific and highly sensitive reverse transcriptase PCR (RT-PCR) that targets a 366 bp region of the 16S rRNA of T. pallidum. This RT-PCR can detect a single organism by Southern analysis when whole organisms are diluted and 10(-2) to 10(-3) T. pallidum organisms when RNA equivalents are used to make cDNA. The test was demonstrated to detect 10(-2) T. pallidum RNA equivalents in cerebrospinal fluid. Twenty different strains of T. pallidum, isolated from cerebrospinal fluids, aqueous humor, blood, and chancres, were shown to be detectable by this test. This efficient and sensitive technique could be more useful than existing methods for detecting very low numbers of organisms in clinical samples. PMID:9163442

  2. PCR-Based Identification of Bacteria Associated with Endodontic Infections

    PubMed Central

    Fouad, Ashraf F.; Barry, Jody; Caimano, Melissa; Clawson, Michael; Zhu, Qiang; Carver, Rachaele; Hazlett, Karsten; Radolf, Justin D.

    2002-01-01

    PCR primers that target the bacterial 16S rRNA genes (or the tuf gene for the genus Enterococcus) were used to identify 10 putative bacterial pathogens in root canals with necrotic pulp. In addition, the associations of these microorganisms with symptoms and a history of diabetes mellitus were investigated. Microbial samples from the root canals of 24 teeth with necrotic pulp were included in the study. PCR with universal bacterial primers identified bacterial DNA in 22 specimens; the remaining 2 specimens were from intact teeth that had been traumatized 6 months prior to treatment. PCR with specific primers showed that preoperative symptoms were significantly associated with the presence of Streptococcus spp. (P < 0.001 by chi-square analysis). There was also a nonsignificant trend for symptoms to be associated with Fusobacterium nucleatum and Porphyromonas gingivalis (odds ratio, >2) and for diabetes mellitus to be associated with P. gingivalis and Porphyromonas endodontalis (odds ratio, >2). Cloning and sequencing of the universal PCR product in one specimen revealed the presence of an organism related to the genus Olsenella, which has not previously been described in endodontic infections. PMID:12202557

  3. FTA card utility for PCR detection of Mycobacterium leprae.

    PubMed

    Aye, Khin Saw; Matsuoka, Masanori; Kai, Masanori; Kyaw, Kyaw; Win, Aye Aye; Shwe, Mu Mu; Thein, Min; Htoo, Maung Maung; Htoon, Myo Thet

    2011-01-01

    The suitability of the FTA® elute card for the collection of slit skin smear (SSS) samples for PCR detection of Mycobacterium leprae was evaluated. A total of 192 SSS leprosy samples, of bacillary index (BI) 1 to 5, were collected from patients attending two skin clinics in Myanmar and preserved using both FTA® elute cards and 70% ethanol tubes. To compare the efficacy of PCR detection of DNA from each BI class, PCR was performed to amplify an M. leprae-specific repetitive element. Of the 192 samples, 116 FTA® elute card and 112 70% ethanol samples were PCR positive for M. leprae DNA. When correlated with BI, area under the curve (AUC) values of the respective receiver-operating characteristic curves were similar for the FTA® elute card and ethanol collection methods (AUC=0.6). Taken together, our results indicate that the FTA® elute card, which enables the collection, transport, and archiving of clinical samples, is an attractive alternative to ethanol preservation for the detection of M. leprae DNA. PMID:21617312

  4. Facilitated Molecular Typing of Shigella Isolates Using ERIC-PCR

    PubMed Central

    Kosek, Margaret; Yori, Pablo Peñataro; Gilman, Robert H.; Vela, Henry; Olortegui, Maribel Paredes; Chavez, Cesar Banda; Calderon, Maritza; Bao, Juan Perez; Hall, Eric; Maves, Ryan; Burga, Rosa; Sanchez, Graciela Meza

    2012-01-01

    To evaluate the performance of enterobacterial repetitive intergenic sequence-based polymerase chain reaction (ERIC-PCR) typing versus the current standard for the typing of Shigella pulsed gel electrophoresis (PFGE), we typed 116 Shigella isolates from a village in an endemic setting over a 20-month period using both methods. PFGE identified 37 pulse types and had a discrimination index of 0.925 (95% confidence interval = 0.830–1.00), whereas ERIC-PCR identified 42 types and had a discrimination index of 0.961 (95% confidence interval = 0.886–1.00). PFGE and ERIC-PCR showed a 90.4% correlation in the designation of isolates as clonal or non-clonal in pairwise comparisons. Both systems were highly reproducible and provided highly similar and supplementary data compared with serotyping regarding the transmission dynamics of shigellosis in this community. ERIC-PCR is considerably more rapid and inexpensive than PFGE and may have a complementary role to PFGE for initial investigations of hypothesized outbreaks in resource-limited settings. PMID:22665611

  5. Human anaplasmosis: the first Spanish case confirmed by PCR.

    PubMed

    García, J C; Núñez, M J; Castro, B; Fraile, F J; López, A; Mella, M C; Blanco, A; Sieira, C; Loureiro, E; Portillo, A; Oteo, J A

    2006-10-01

    We report a case of human anaplasmosis (HA) fulfilling the confirmation criteria: epidemiologic data and clinical picture compatible with HA; presence of a morulae within polymorphonuclear leukocyte; and positive PCR assay for Anaplasma phagocytophilum: This case report shows the presence of HA in Spain. PMID:17114773

  6. PCR detection of Helicobacter pylori in clinical samples.

    PubMed

    Rimbara, Emiko; Sasatsu, Masanori; Graham, David Y

    2013-01-01

    Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etiologically associated with gastric inflammation (gastritis), peptic ulcer disease, and gastric cancer. Both noninvasive (e.g., urea breath and stool antigen tests) and invasive (gastric biopsy for histology, culture, or PCR) tests are used for diagnosis. PCR detection of H. pylori has been reported using a variety of clinical samples including gastric biopsy, gastric juice, saliva, dental plaque, and stools as well as environmental samples. Whenever possibly, noninvasive tests are preferred over invasive tests. H. pylori are excreted in the stool. Culture from stool is variable whereas stool antigen testing is widely used. Stool consists of a complicated mixture of commensal bacteria and chemicals and often includes inhibitors of PCR. Nevertheless, simple extraction methods are available to efficiently extract DNA from human stools and nested-PCR targeting the 23S rRNA gene have proven to be highly sensitive for the detection of H. pylori. Detection of clarithromycin susceptibility/resistance is important clinically and the mutation of the 23S rRNA gene responsible for resistance can also be detected using stool. This described method can be modified for other clinical samples such as gastric juice or biopsy material. PMID:23104297

  7. Detection of carbapenemases in Enterobacteriaceae by a commercial multiplex PCR.

    PubMed

    Kaase, Martin; Szabados, Florian; Wassill, Lars; Gatermann, Sören G

    2012-09-01

    A commercial multiplex PCR (hyplex SuperBug ID) was tested with a collection of 132 clinical Enterobacteriaceae strains producing different carbapenemases. The sensitivity for the detection of KPC-, VIM-, NDM-, and OXA-48-encoding genes was 100%, whereas two IMP variants were missed. PMID:22785190

  8. Detection of viable Cryptosporidium parvum oocysts by PCR.

    PubMed Central

    Wagner-Wiening, C; Kimmig, P

    1995-01-01

    PCR was used to detect and specifically identify a gene fragment from Cryptosporidium parvum. An 873-bp region of a 2,359-bp DNA fragment encoding a repetitive oocyst protein of C. parvum was shown to be specifically amplified in C. parvum. An excystation protocol before DNA extraction allowed the differentiation between live and dead Cryptosporidium parvum oocysts. PMID:8534121

  9. Halal authenticity of gelatin using species-specific PCR.

    PubMed

    Shabani, Hessam; Mehdizadeh, Mehrangiz; Mousavi, Seyed Mohammad; Dezfouli, Ehsan Ansari; Solgi, Tara; Khodaverdi, Mahdi; Rabiei, Maryam; Rastegar, Hossein; Alebouyeh, Mahmoud

    2015-10-01

    Consumption of food products derived from porcine sources is strictly prohibited in Islam. Gelatin, mostly derived from bovine and porcine sources, has many applications in the food and pharmaceutical industries. To ensure that food products comply with halal regulations, development of valid and reliable analytical methods is very much required. In this study, a species-specific polymerase chain reaction (PCR) assay using conserved regions of mitochondrial DNA (cytochrome b gene) was performed to evaluate the halal authenticity of gelatin. After isolation of DNA from gelatin powders with known origin, conventional PCR using species-specific primers was carried out on the extracted DNA. The amplified expected PCR products of 212 and 271 bp were observed for porcine and bovine gelatin, respectively. The sensitivity of the method was tested on binary gelatin mixtures containing 0.1%, 1%, 10%, and 100% (w/w) of porcine gelatin within bovine gelatin and vice versa. Although most of the DNA is degraded due to the severe processing steps of gelatin production, the minimum level of 0.1% w/w of both porcine and bovine gelatin was detected. Moreover, eight food products labeled as containing bovine gelatin and eight capsule shells were subjected to PCR examination. The results showed that all samples contained bovine gelatin, and the absence of porcine gelatin was verified. This method of species authenticity is very useful to verify whether gelatin and gelatin-containing food products are derived from halal ingredients. PMID:25872445

  10. Novel real-time PCR detection assay for Brucella suis

    PubMed Central

    Hänsel, C.; Mertens, K.; Elschner, M. C.; Melzer, F.

    2015-01-01

    Introduction Brucella suis is the causative agent of brucellosis in suidae and is differentiated into five biovars (bv). Biovars 1 and 3 possess zoonotic potential and can infect humans, whereas biovar 2 represents the main source of brucellosis in feral and domestic pigs in Europe. Both aspects, the zoonotic threat and the economic loss, emphasize the necessity to monitor feral and domestic pig populations. Available serological or PCR based methods lack sensitivity and specificity. Results Here a bioinformatics approach was used to identify a B. suis specific 17 bp repeat on chromosome II (BS1330_II0657 locus). This repeat is common for B. suis bv 1 to 4 and was used to develop a TaqMan probe assay. The average PCR efficiency was determined as 95% and the limit of detection as 12,5 fg/µl of DNA, equally to 3.7 bacterial genomes. This assay has the highest sensitivity of all previously described B. suis specific PCR assays, making it possible to detect 3-4 bacterial genomes per 1 µl of sample. The assay was tested 100% specific for B. suis and negative for other Brucella spp. and closely related non-Brucella species. Conclusions This novel qPCR assay could become a rapid, inexpensive and reliable screening method for large sample pools of B. suis 1 to 4. This method will be applicable for field samples after validation. PMID:26392898

  11. A PCR test for avian malaria in Hawaiian birds.

    PubMed

    Feldman, R A; Freed, L A; Cann, R L

    1995-12-01

    The decline of native Hawaiian forest birds since European contact is attributed to factors ranging from habitat destruction to interactions with introduced species. Remaining populations of Hawaiian honeycreepers (Fringillidae: Drepanidinae) are most abundant and diverse in high elevation refuges above the normal range of disease-carrying mosquitoes. Challenge experiments suggest that honeycreepers are highly susceptible to avian malaria (Plasmodium sp.) but resistance exists in some species. In order to detect low levels of malarial infection and quantify prevalence of Plasmodium in high elevation natural populations of Hawaiian birds, a polymerase chain reaction (PCR) based diagnostic test was developed that identifies rRNA genes of Plasmodium in avian blood samples. Quantitative competitive PCR (QC-PCR) experiments indicate that the detection limit of our test is an order of magnitude greater than that reported for human malaria DNA blot tests. Compared with standard histological methods, the PCR test detected a higher prevalence of diseased birds at mid-elevations. Malaria was detected in three species of native birds living in a high elevation wildlife refuge on the island of Hawaii and in four species from Maui. Our results show that avian malaria is more widespread in Hawaiian forests than previously thought, a finding that has important conservation implications for these threatened species. PMID:8564006

  12. Introducing Undergraduate Students to Real-Time PCR

    ERIC Educational Resources Information Center

    Hancock, Dale; Funnell, Alister; Jack, Briony; Johnston, Jill

    2010-01-01

    An experiment is conducted, which in four 3 h laboratory sessions, introduces third year undergraduate Biochemistry students to the technique of real-time PCR in a biological context. The model used is a murine erythroleukemia cell line (MEL cells). These continuously cycling, immature red blood cells, arrested at an early stage in erythropoiesis,…

  13. QUANTITATIVE PCR OF SELECTED ASPERGILLUS, PENICILLIUM AND PAECILOMYCES SPECIES

    EPA Science Inventory

    A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan®) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of Aspergillus, Penicillium and ...

  14. Genus identification of toxic plant by real-time PCR.

    PubMed

    Matsuyama, Shuji; Nishi, Katsuji

    2011-03-01

    Some plants have toxicities that are dangerous for humans. In the case of poisoning by toxic plants, a rapid and easy screening test is required for accurate medical treatment or forensic investigation. In this study, we designed specific primer pairs for identification of toxic plants, such as subgenus Aconitum, genus Ricinus, genus Illicium, and genus Scopolia, by internal transcribed spacer sequences of nuclear ribosomal DNA. Allied species of target plants, foods, and human DNA were not detected, but each primer pair provided a specific PCR product from the target plant using real-time PCR. This method can detect the subgenus Aconitum, genus Ricinus, and genus Scopolia with template DNA of 10 pg, respectively, and genus Illicium with 1 pg. Furthermore, each primer pair provided the exact PCR product from digested target plants in artificial gastric fluid. When a trace unknown plant sample in forensic investigation is collected from stomach contents, this PCR assay may be useful for screening toxic plants. PMID:20623131

  15. Using qPCR for Water Microbial Risk Assessments

    EPA Science Inventory

    Microbial risk assessment (MRA) has traditionally utilized microbiological data that was obtained by culture-based techniques that are expensive and time consuming. With the advent of PCR methods there is a realistic opportunity to conduct MRA studies economically, in less time,...

  16. Screening ancient tuberculosis with qPCR: challenges and opportunities

    PubMed Central

    Harkins, Kelly M.; Buikstra, Jane E.; Campbell, Tessa; Bos, Kirsten I.; Johnson, Eric D.; Krause, Johannes; Stone, Anne C.

    2015-01-01

    The field of ancient DNA (aDNA) has rapidly accelerated in recent years as a result of new methods in next-generation sequencing, library preparation and targeted enrichment. Such research is restricted, however, by the highly variable DNA preservation within different tissues, especially when isolating ancient pathogens from human remains. Identifying positive candidate samples via quantitative PCR (qPCR) for downstream procedures can reduce reagent costs, increase capture efficiency and maximize the number of sequencing reads of the target. This study uses four qPCR assays designed to target regions within the Mycobacterium tuberculosis complex (MTBC) to examine 133 human skeletal samples from a wide geographical and temporal range, identified by the presence of skeletal lesions typical of chronic disseminated tuberculosis. Given the inherent challenges working with ancient mycobacteria, strict criteria must be used and primer/probe design continually re-evaluated as new data from bacteria become available. Seven samples tested positive for multiple MTBC loci, supporting them as strong candidates for downstream analyses. Using strict and conservative criteria, qPCR remains a fast and effective screening tool when compared with screening by more expensive sequencing and enrichment technologies. PMID:25487341

  17. A Ribeiroia spp. (Class: Trematoda) - Specific PCR-based diagnostic

    USGS Publications Warehouse

    Reinitz, D.M.; Yoshino, T.P.; Cole, R.A.

    2007-01-01

    Increased reporting of amphibian malformations in North America has been noted with concern in light of reports that amphibian numbers and species are declining worldwide. Ribeiroia ondatrae has been shown to cause a variety of types of malformations in amphibians. However, little is known about the prevalence of R. ondatrae in North America. To aid in conducting field studies of Ribeiroia spp., we have developed a polymerase chain reaction (PCR)-based diagnostic. Herein, we describe the development of an accurate, rapid, simple, and cost-effective diagnostic for detection of Ribeiroia spp. infection in snails (Planorbella trivolvis). Candidate oligonucleotide primers for PCR were designed via DNA sequence analyses of multiple ribosomal internal transcribed spacer-2 regions from Ribeiroia spp. and Echinostoma spp. Comparison of consensus sequences determined from both genera identified areas of sequence potentially unique to Ribeiroia spp. The PCR reliably produced a diagnostic 290-base pair (bp) product in the presence of a wide concentration range of snail or frog DNA. Sensitivity was examined with DNA extracted from single R. ondatrae cercaria. The single-tube PCR could routinely detect less than 1 cercariae equivalent, because DNA isolated from a single cercaria could be diluted at least 1:50 and still yield a positive result via gel electrophoresis. An even more sensitive nested PCR also was developed that routinely detected 100 fg of the 290-bp fragment. The assay did not detect furcocercous cercariae of certain Schistosomatidae, Echinostoma sp., or Sphaeridiotrema globulus nor adults of Clinostomum sp. or Cyathocotyle bushiensis. Field testing of 137 P. trivolvis identified 3 positives with no overt environmental cross-reactivity, and results concurred with microscopic examinations in all cases. ?? American Society of Parasitologists 2007.

  18. Application of multiplex PCR, pulsed-field gel electrophoresis (PFGE), and BOX-PCR for molecular analysis of enterococci

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of the study was to use band-based molecular methods including BOX-PCR (Polymerase Chain Reaction) and Pulsed-Field Gel Electrophoresis (PFGE) to determine if genetically related enterococci were found among different stores, food types, or years. Enterococci were also characterized f...

  19. Nested-PCR and TaqMan real-time quantitative PCR assays for human adenoviruses in environmental waters.

    PubMed

    Huang, Wen-Chien; Chou, Yi-Pen; Kao, Po-Min; Hsu, Tsui-Kang; Su, Hung-Chang; Ho, Ying-Ning; Yang, Yi-Chun; Hsu, Bing-Mu

    2016-01-01

    Human adenovirus (HAdV) infections can occur throughout the year. Cases of HAdV-associated respiratory disease have been more common in the late winter, spring, and early summer. In this study, to provide viral pollution data for further epidemiological studies and governmental actions, the presence of HAdV in the aquatic environment was quantitatively surveyed in the summer. This study was conducted to compare the efficiencies of nested-PCR (polymerase chain reaction) and qPCR (quantitative PCR) for detecting HAdV in environmental waters. A total of 73 water samples were collected from Puzi River in Taiwan and subjected to virus concentration methods. In the results, qPCR had much better efficiency for specifying the pathogen in river sample. HAdV41 was detected most frequently in the river water sample (10.9%). The estimated HAdV concentrations ranged between 6.75 × 10(2) and 2.04 × 10(9) genome copies/L. Significant difference was also found in heterotrophic plate counts, conductivity, water temperature, and water turbidity between presence/absence of HAdV. HAdV in the Puzi River may pose a significant health risk. PMID:27120637

  20. Quantitative real-time PCR (qPCR) for Eimeria tenella replication — Implications for experimental refinement and animal welfare

    PubMed Central

    Nolan, Matthew J.; Tomley, Fiona M.; Kaiser, Pete; Blake, Damer P.

    2015-01-01

    The Eimeria species are highly pathogenic parasites of chickens. Research aimed at reducing their impact is hindered by a lack of non-subjective, quantitative, tools to measure parasite replication in the host. The time-consuming, and often time-sensitive, nature of existing approaches precludes their use in large-scale genetic, epidemiological, and evolutionary analyses. We have used quantitative real-time PCR (qPCR) to accurately quantify Eimeria tenella in chicken tissue and shown this to be more efficient and sensitive than traditional methodologies. We tested four chicken-specific reference qPCR assays and found beta-actin (actb) to be optimal for sample normalisation. In an experimental setting, chickens were inoculated with 500, 1500, or 4500 E. tenella oocysts and parasite replication and the impact of infection measured by i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi), ii) faecal oocyst counts (FOCs) on samples taken from six to eight dpi, and iii) lesion scoring on caeca collected post-mortem at five and eight dpi. Quantitative real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups for samples collected five dpi (i.e., prior to gametogony) (R2 = 0.994) (p < 0.002) but not in those from day eight (after most oocyst shedding) (R2 = 0.006) (p > 0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R2 = 0.997) and lesion scoring. Importantly, qPCR offers substantial improvements for animal welfare via improved statistical power and reduced group sizes in experimental studies. The described qPCR method overcomes subjective limitations of coproscopic quantification, allows reproducible medium- to high-throughput examination of tissues, faeces, and oocysts, and is a valuable tool for determining the impact of Eimeria infections in both experimental and field settings

  1. Quantitative real-time PCR (qPCR) for Eimeria tenella replication--Implications for experimental refinement and animal welfare.

    PubMed

    Nolan, Matthew J; Tomley, Fiona M; Kaiser, Pete; Blake, Damer P

    2015-10-01

    The Eimeria species are highly pathogenic parasites of chickens. Research aimed at reducing their impact is hindered by a lack of non-subjective, quantitative, tools to measure parasite replication in the host. The time-consuming, and often time-sensitive, nature of existing approaches precludes their use in large-scale genetic, epidemiological, and evolutionary analyses. We have used quantitative real-time PCR (qPCR) to accurately quantify Eimeria tenella in chicken tissue and shown this to be more efficient and sensitive than traditional methodologies. We tested four chicken-specific reference qPCR assays and found beta-actin (actb) to be optimal for sample normalisation. In an experimental setting, chickens were inoculated with 500, 1500, or 4500 E. tenella oocysts and parasite replication and the impact of infection measured by i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi), ii) faecal oocyst counts (FOCs) on samples taken from six to eight dpi, and iii) lesion scoring on caeca collected post-mortem at five and eight dpi. Quantitative real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups for samples collected five dpi (i.e., prior to gametogony) (R(2)=0.994) (p<0.002) but not in those from day eight (after most oocyst shedding) (R(2)=0.006) (p>0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R(2)=0.997) and lesion scoring. Importantly, qPCR offers substantial improvements for animal welfare via improved statistical power and reduced group sizes in experimental studies. The described qPCR method overcomes subjective limitations of coproscopic quantification, allows reproducible medium- to high-throughput examination of tissues, faeces, and oocysts, and is a valuable tool for determining the impact of Eimeria infections in both experimental and field settings. PMID:26141544

  2. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis

    PubMed Central

    Te, Shu Harn; Chen, Enid Yingru

    2015-01-01

    The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. PMID:26025892

  3. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis.

    PubMed

    Te, Shu Harn; Chen, Enid Yingru; Gin, Karina Yew-Hoong

    2015-08-01

    The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. PMID:26025892

  4. PCR detection and characterization of type-2 porcine circovirus.

    PubMed Central

    Hamel, A L; Lin, L L; Sachvie, C; Grudeski, E; Nayar, G P

    2000-01-01

    A polymerase chain reaction (PCR) assay was developed for detecting porcine circovirus (PCV). The assay readily detected type-2 PCV (PCV-2) and type-1 PCV (PCV-1). The PCR primers were designed based on DNA sequences conserved in all reported PCV genomes. Type 1 PCV and type 2 PCV both produced 438 bp amplification products, which were easily identified and differentiated from one another by restriction fragment length polymorphism (RFLP) analysis. Porcine circovirus was detected in 55% (931/1693) of randomly tested pigs with various clinical signs and lesions, most of which were difficult to differentiate from those associated with porcine reproductive and respiratory syndrome (PRRS). The PCR products from all positive clinical samples were identified by RFLP to be only PCV-2; DNA tested by PCR was extracted directly from one or more of lung, mesenteric or mediastinal lymph nodes, and tonsil. Type 2 PCV was also detected in 6% (2/34) of DNA extracted directly from semen of randomly chosen healthy boars. Positive PCR reactions from 554 diseased pigs were characterized by RFLP and categorized into 5 different profiles (A-E), of which 82.8% were PCV-2A (456/554), 3.0% were PCV-2B (17/554), 9.9% were PCV-2C (55/554), 1.1% were PCV-2D (6/554), and 3.2% were PCV-2E (18/554). The complete genomic nucleotide sequences of PCV-2A, B, C, D, and E were determined and found to have at least 95% homology compared with one another and with all other PCV-2 found in the GenBank database. All PCV-2 had less than 76% homology with PCV-1. This PCR assay will hopefully be useful to veterinary diagnostic laboratories for routine testing and surveillance of infection with PCV-2. The RFLP profiling system might be useful for preliminary characterization and identification of PCV isolates and might also benefit studies on the molecular epidemiology of PCV. Images Figure 1. PMID:10680656

  5. Computational tradeoffs in multiplex PCR assay design for SNP genotyping

    PubMed Central

    Rachlin, John; Ding, Chunming; Cantor, Charles; Kasif, Simon

    2005-01-01

    Background Multiplex PCR is a key technology for detecting infectious microorganisms, whole-genome sequencing, forensic analysis, and for enabling flexible yet low-cost genotyping. However, the design of a multiplex PCR assays requires the consideration of multiple competing objectives and physical constraints, and extensive computational analysis must be performed in order to identify the possible formation of primer-dimers that can negatively impact product yield. Results This paper examines the computational design limits of multiplex PCR in the context of SNP genotyping and examines tradeoffs associated with several key design factors including multiplexing level (the number of primer pairs per tube), coverage (the % of SNP whose associated primers are actually assigned to one of several available tube), and tube-size uniformity. We also examine how design performance depends on the total number of available SNPs from which to choose, and primer stringency criterial. We show that finding high-multiplexing/high-coverage designs is subject to a computational phase transition, becoming dramatically more difficult when the probability of primer pair interaction exceeds a critical threshold. The precise location of this critical transition point depends on the number of available SNPs and the level of multiplexing required. We also demonstrate how coverage performance is impacted by the number of available snps, primer selection criteria, and target multiplexing levels. Conclusion The presence of a phase transition suggests limits to scaling Multiplex PCR performance for high-throughput genomics applications. Achieving broad SNP coverage rapidly transitions from being very easy to very hard as the target multiplexing level (# of primer pairs per tube) increases. The onset of a phase transition can be "delayed" by having a larger pool of SNPs, or loosening primer selection constraints so as to increase the number of candidate primer pairs per SNP, though the latter

  6. A comparative analysis of different molecular targets using PCR for diagnosis of old world leishmaniasis.

    PubMed

    Koltas, Ismail S; Eroglu, Fadime; Uzun, Soner; Alabaz, Derya

    2016-05-01

    The different sensitivity values were obtained in each study conducted for the diagnosis of leishmaniasis with the polymerase chain reaction (PCR). However, a standardized PCR target for the diagnosis of leishmaniasis does not exist. The aim of the current study, the most ideal PCR target was determined for diagnosis of leishmaniasis. A total of 72 smear and 48 bone marrow samples were analyzed with six different molecular targets to determine their potential as a tool for the specific molecular diagnosis of leishmaniasis using PCR. The positivity-negativity value and the sensitivity-specificity of each PCR targets were calculated. The positivity value of PCR targets were sequenced in different levels in the diagnosis of leishmaniasis from highest to lowest in the order of kDNA-PCR > SSU rRNA-PCR > ITS2-PCR > ITS1-PCR > ME-PCR > HSP70-PCR. The sensitivities of PCR targets except ITS1-PCR, ME-PCR and HSP70-PCR were found to be 100% in cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) cases as compared to microscopic examination accepted as a gold standard. The sensitivities of ITS1-PCR, ME-PCR and HSP70-PCR were found 96.6%, 90.0% and 86.6%, respectively, in CL-cases. In addition, the sensitivities of ITS1-PCR, ME-PCR and HSP70-PCR were found 90.0%, 70.0% and 60.0%, respectively, in VL-cases. The kDNA genomic region was the most sensitive for routine diagnosis of leishmaniasis. ITS1-PCR restriction fragment length polymorphism, the alternative method for the identification of Old World Leishmania species, did not require culturing of the parasites. PMID:26896641

  7. Adaptive DNA Computing Algorithm by Using PCR and Restriction Enzyme

    NASA Astrophysics Data System (ADS)

    Kon, Yuji; Yabe, Kaoru; Rajaee, Nordiana; Ono, Osamu

    In this paper, we introduce an adaptive DNA computing algorithm by using polymerase chain reaction (PCR) and restriction enzyme. The adaptive algorithm is designed based on Adleman-Lipton paradigm[3] of DNA computing. In this work, however, unlike the Adleman- Lipton architecture a cutting operation has been introduced to the algorithm and the mechanism in which the molecules used by computation were feedback to the next cycle devised. Moreover, the amplification by PCR is performed in the molecule used by feedback and the difference concentration arisen in the base sequence can be used again. By this operation the molecules which serve as a solution candidate can be reduced down and the optimal solution is carried out in the shortest path problem. The validity of the proposed adaptive algorithm is considered with the logical simulation and finally we go on to propose applying adaptive algorithm to the chemical experiment which used the actual DNA molecules for solving an optimal network problem.

  8. Direct identification and typing of Mycobacterium tuberculosis by PCR.

    PubMed Central

    Neimark, H; Ali Baig, M; Carleton, S

    1996-01-01

    We have developed a rapid PCR assay that types strains of Mycobacterium tuberculosis by generating distinct DNA fingerprints directly from primary cultures. This assay allows strain identification analogous to that achieved by the standard restriction fragment length polymorphism method, and fingerprints are obtained in less than 8 h. This assay does not require subculturing, DNA purification, restriction digestion, Southern blotting, or nucleic acid hybridization. Rapid and precise identification of M. tuberculosis strains permits immediate molecular epidemiologic studies. The assay can be converted to a computer-automated system by employing fluorescently labeled PCR primers and the Perkin-Elmer DNA sequencer so that unknown-specimen fingerprints are identified by computer comparison to a database of M. tuberculosis strain fingerprints. PMID:8880499

  9. Actuation method and apparatus, micropump, and PCR enhancement method

    SciTech Connect

    Ullakko, Kari; Mullner, Peter; Hampikian, Greg; Smith, Aaron

    2015-07-28

    An actuation apparatus includes at least one magnetic shape memory (MSM) element containing a material configured to expand and/or contract in response to exposure to a magnetic field. Among other things, the MSM element may be configured to pump fluid through a micropump by expanding and/or contracting in response to the magnetic field. The magnetic field may rotate about an axis of rotation and exhibit a distribution having a component substantially perpendicular to the axis of rotation. Further, the magnetic field distribution may include at least two components substantially orthogonal to one another lying in one or more planes perpendicular to the axis of rotation. The at least one MSM element may contain nickel, manganese, and gallium. A polymerase chain reaction (PCR) may be enhanced by contacting a PCR reagent and DNA material with the MSM element.

  10. Measuring Digital PCR Quality: Performance Parameters and Their Optimization.

    PubMed

    Lievens, A; Jacchia, S; Kagkli, D; Savini, C; Querci, M

    2016-01-01

    Digital PCR is rapidly being adopted in the field of DNA-based food analysis. The direct, absolute quantification it offers makes it an attractive technology for routine analysis of food and feed samples for their composition, possible GMO content, and compliance with labelling requirements. However, assessing the performance of dPCR assays is not yet well established. This article introduces three straightforward parameters based on statistical principles that allow users to evaluate if their assays are robust. In addition, we present post-run evaluation criteria to check if quantification was accurate. Finally, we evaluate the usefulness of Poisson confidence intervals and present an alternative strategy to better capture the variability in the analytical chain. PMID:27149415

  11. Measuring Digital PCR Quality: Performance Parameters and Their Optimization

    PubMed Central

    Lievens, A.; Jacchia, S.; Kagkli, D.; Savini, C.; Querci, M.

    2016-01-01

    Digital PCR is rapidly being adopted in the field of DNA-based food analysis. The direct, absolute quantification it offers makes it an attractive technology for routine analysis of food and feed samples for their composition, possible GMO content, and compliance with labelling requirements. However, assessing the performance of dPCR assays is not yet well established. This article introduces three straightforward parameters based on statistical principles that allow users to evaluate if their assays are robust. In addition, we present post-run evaluation criteria to check if quantification was accurate. Finally, we evaluate the usefulness of Poisson confidence intervals and present an alternative strategy to better capture the variability in the analytical chain. PMID:27149415

  12. [PCR-based detection of pathogens in clinical rheumatology].

    PubMed

    Ehrenstein, B; Reischl, U

    2016-05-01

    In the differential diagnostics of autoimmune-mediated rheumatic diseases, rheumatologists often have to consider infections (e. g. Lyme arthritis) or reactive diseases (e. g. reactive arthritis after urogenital bacterial infections). Furthermore, infections with an atypical presentation or caused by atypical pathogens (opportunistic infections) can complicate the immunosuppressive therapy of autoimmune diseases. For this purpose not only conventional microbiological culture methods but also PCR-based methods are increasingly being applied for the direct detection of pathogens in clinical specimens. The aim of this overview is to present commonly used PCR methods in the clinical practice of rheumatology and to describe their benefits and limitations compared to culture-based detection methods. PMID:26892924

  13. Multiplex PCR for detection of acquired carbapenemase genes.

    PubMed

    Poirel, Laurent; Walsh, Timothy R; Cuvillier, Vincent; Nordmann, Patrice

    2011-05-01

    A rapid and reliable PCR-based technique was developed for detection of genes encoding carbapenemases belonging to different classes. Primers were designed to amplify the following 11 genes: bla(IMP), bla(VIM), bla(NDM), bla(SPM), bla(AIM), bla(DIM), bla(GIM), bla(SIM)bla(KPC), bla(BIC), and bla(OXA-48). Three different multiplex reaction mixtures were defined and evaluated for the detection of all these 11 genes. Using optimized conditions, each reaction mixture allowed to identify the respective genes, with PCR giving distinct amplicon sizes corresponding to the different genes for each mixture. We reported here a rapid and reliable technique for screening all clinically relevant carbapenemase genes. PMID:21398074

  14. An improved PCR method for gender identification of eagles.

    PubMed

    Chang, Hsueh-Wei; Chou, Ta-Ching; Gu, De-Leung; Cheng, Chun-An; Chang, Chia-Che; Yao, Cheng-Te; Chuang, Li-Yeh; Wen, Cheng-Hao; Chou, Yii-Cheng; Tan, Kock-Yee; Cheng, Chien-Chung

    2008-06-01

    Eagles are sexually monomorphic and therefore it is difficult to determine their gender, which is a crucial need for management purposes. In this study, we have developed an improved gender identification method by exploiting length differences between the Chromo-Helicase-DNA binding protein (CHD)-Z and CHD-W genes of Spilornis cheela hoya. By comparing DNA sequences for CHD-W and CHD-Z from 10 species of Falconiformes eagles we designed universal gender identification PCR primers that exploit differences in product size. Standard agarose gels were shown to easily distinguish between the 148-bp CHD-ZW and the 258-bp CHD-W PCR products. When used with 28 samples of S. cheela hoya, our improved universal primers provided a fast and precise gender identification assay. PMID:18385011

  15. Rapid detection of Serpulina hyodysenteriae in diagnostic specimens by PCR.

    PubMed Central

    Elder, R O; Duhamel, G E; Schafer, R W; Mathiesen, M R; Ramanathan, M

    1994-01-01

    A PCR assay for the detection of Serpulina hyodysenteriae in diagnostic specimens was developed on the basis of sequence analysis of a recombinant clone designated pRED3C6. Clone pRED3C6, which contained a 2.3-kb DNA fragment unique to S. hyodysenteriae, was identified by screening a plasmid library of S. hyodysenteriae isolate B204 genomic DNA in Escherichia coli by colony immunoblot with the mouse monoclonal antibody 10G6/G10, which was produced against cell-free supernatant antigens from the same isolate. Southern blot analysis of HindIII-digested genomic DNA of S. hyodysenteriae serotypes 1 through 7 and of four weakly beta-hemolytic intestinal spirochetes, including Serpulina innocens, with the 2.3-kb DNA fragment of pRED3C6 indicated that the cloned sequence was present exclusively in the seven serotypes of S. hyodysenteriae. An oligonucleotide primer pair for PCR amplification of a 1.55-kb fragment and an internal oligonucleotide probe were designed and synthesized on the basis of sequence analysis of the 2.3-kb DNA fragment of pRED3C6. Purified genomic DNAs from reference isolates of S. hyodysenteriae serotypes 1 through 9, S. innocens, weakly beta-hemolytic intestinal spirochetes belonging to genotypic groups distinct from those of reference Serpulina spp., other cultivable reference isolates of the order Spirochaetales, and enteric bacteria including Escherichia coli, Salmonella spp., Campylobacter spp., and Bacteroides vulgatus were amplified with the oligonucleotide primer pair in a hot-start PCR. The 1.55-kb products were obtained only in the presence of genomic DNA from each of the nine serotypes of S. hyodysenteriae. The specificity of the 1.55-kb products for S. hyodysenteriae was confirmed on the basis of production of a restriction endonuclease pattern of the PCR products identical to the predicted restriction map analysis of pRED3C6 and positive hybridization signal with the S. hyodysenteriae-specific internal oligonucleotide probe. By using

  16. Species identification of cattle and buffalo fat through PCR assay.

    PubMed

    Vaithiyanathan, S; Kulkarni, V V

    2016-04-01

    A method was standardized to isolate quality DNA from cattle and buffalo fat for species identification using QIAamp DNA stool mini kit. The quality of the DNA was sufficient enough to amplify universal primers viz., mt 12S rRNA and mt 16S rRNA, and species specific D loop primers for cattle and buffalo. The sensitivity of the PCR assay in the species specific D loop primer amplification was with a detection level of 0. 47 ng cattle DNA and 0.23 ng buffalo DNA in simplex and, 0. 47 ng cattle DNA and 0.12 ng buffalo DNA in duplex PCR. It is a potentially reliable method for DNA detection to authenticate animal fat. PMID:27413237

  17. Clonal architecture of CXCR4 WHIM-like mutations in Waldenström Macroglobulinaemia.

    PubMed

    Xu, Lian; Hunter, Zachary R; Tsakmaklis, Nicholas; Cao, Yang; Yang, Guang; Chen, Jie; Liu, Xia; Kanan, Sandra; Castillo, Jorge J; Tai, Yu-Tzu; Zehnder, James L; Brown, Jennifer R; Carrasco, Ruben D; Advani, Ranjana; Sabile, Jean M; Argyropoulos, Kimon; Lia Palomba, M; Morra, Enrica; Trojani, Alessandra; Greco, Antonino; Tedeschi, Alessandra; Varettoni, Marzia; Arcaini, Luca; Munshi, Nikhil M; Anderson, Kenneth C; Treon, Steven P

    2016-03-01

    CXCR4(WHIM) somatic mutations are distinctive to Waldenström Macroglobulinaemia (WM), and impact disease presentation and treatment outcome. The clonal architecture of CXCR4(WHIM) mutations remains to be delineated. We developed highly sensitive allele-specific polymerase chain reaction (AS-PCR) assays for detecting the most common CXCR4(WHIM) mutations (CXCR4(S338X C>A and C>G) ) in WM. The AS-PCR assays detected CXCR4(S338X) mutations in WM and IgM monoclonal gammopathy of unknown significance (MGUS) patients not revealed by Sanger sequencing. By combined AS-PCR and Sanger sequencing, CXCR4(WHIM) mutations were identified in 44/102 (43%), 21/62 (34%), 2/12 (17%) and 1/20 (5%) untreated WM, previously treated WM, IgM MGUS and marginal zone lymphoma patients, respectively, but no chronic lymphocytic leukaemia, multiple myeloma, non-IgM MGUS patients or healthy donors. Cancer cell fraction analysis in WM and IgM MGUS patients showed CXCR4(S338X) mutations were primarily subclonal, with highly variable clonal distribution (median 35·1%, range 1·2-97·5%). Combined AS-PCR and Sanger sequencing revealed multiple CXCR4(WHIM) mutations in many individual WM patients, including homozygous and compound heterozygous mutations validated by deep RNA sequencing. The findings show that CXCR4(WHIM) mutations are more common in WM than previously revealed, and are primarily subclonal, supporting their acquisition after MYD88(L265P) in WM oncogenesis. The presence of multiple CXCR4(WHIM) mutations within individual WM patients may be indicative of targeted CXCR4 genomic instability. PMID:26659815

  18. Longitudinal Detection and Persistence of Minority Drug-Resistant Populations and Their Effect on Salvage Therapy

    PubMed Central

    Nishizawa, Masako; Matsuda, Masakazu; Hattori, Junko; Shiino, Teiichiro; Matano, Tetsuro; Heneine, Walid; Johnson, Jeffrey A.; Sugiura, Wataru

    2015-01-01

    Background Drug-resistant HIV are more prevalent and persist longer than previously demonstrated by bulk sequencing due to the ability to detect low-frequency variants. To clarify a clinical benefit to monitoring minority-level drug resistance populations as a guide to select active drugs for salvage therapy, we retrospectively analyzed the dynamics of low-frequency drug-resistant population in antiretroviral (ARV)-exposed drug resistant individuals. Materials and Methods Six HIV-infected individuals treated with ARV for more than five years were analyzed. These individuals had difficulty in controlling viremia, and treatment regimens were switched multiple times guided by standard drug resistance testing using bulk sequencing. To detect minority variant populations with drug resistance, we used a highly sensitive allele-specific PCR (AS-PCR) with detection thresholds of 0.3–2%. According to ARV used in these individuals, we focused on the following seven reverse transcriptase inhibitor-resistant mutations: M41L, K65R, K70R, K103N, Y181C, M184V, and T215F/Y. Results of AS-PCR were compared with bulk sequencing data for concordance and presence of additional mutations. To clarify the genetic relationship between low-frequency and high-frequency populations, AS-PCR amplicon sequences were compared with bulk sequences in phylogenetic analysis. Results The use of AS-PCR enabled detection of the drug-resistant mutations, M41L, K103N, Y181C, M184V and T215Y, present as low-frequency populations in five of the six individuals. These drug resistant variants persisted for several years without ARV pressure. Phylogenetic analysis indicated that pre-existing K103N and T215I variants had close genetic relationships with high-frequency K103N and T215I observed during treatment. Discussion and Conclusion Our results demonstrate the long-term persistence of drug-resistant viruses in the absence of drug pressure. The rapid virologic failures with pre-existing mutant viruses

  19. Underwater application of quantitative PCR on an ocean mooring.

    PubMed

    Preston, Christina M; Harris, Adeline; Ryan, John P; Roman, Brent; Marin, Roman; Jensen, Scott; Everlove, Cheri; Birch, James; Dzenitis, John M; Pargett, Douglas; Adachi, Masao; Turk, Kendra; Zehr, Jonathon P; Scholin, Christopher A

    2011-01-01

    The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions. PMID:21829630

  20. Extraction of PCR-amplifiable genomic DNA from Bacillus anthracisspores

    SciTech Connect

    Torok, Tamas

    2003-05-19

    Bacterial endospore disruption and nucleic acid extractionresulting in DNA of PCR-amplifiable quality and quantity are not trivial.Responding to the needs of the Hazardous Materials Response Unit (HMRU),Laboratory Division, Federal Bureau of Investigation, protocols weredeveloped to close these gaps. Effectiveness and reproducibility of thetechniques were validated with laboratory grown pure spores of Bacillusanthracis and its close phylogenetic neighbors, and with spiked soils anddamaged samples.

  1. Underwater Application of Quantitative PCR on an Ocean Mooring

    PubMed Central

    Preston, Christina M.; Harris, Adeline; Ryan, John P.; Roman, Brent; Marin, Roman; Jensen, Scott; Everlove, Cheri; Birch, James; Dzenitis, John M.; Pargett, Douglas; Adachi, Masao; Turk, Kendra; Zehr, Jonathon P.; Scholin, Christopher A.

    2011-01-01

    The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions. PMID:21829630

  2. Stabilized, Freeze-Dried PCR Mix for Detection of Mycobacteria

    PubMed Central

    Klatser, Paul R.; Kuijper, Sjoukje; van Ingen, Cor W.; Kolk, Arend H. J.

    1998-01-01

    We report here the development of a freeze-drying procedure allowing stabilization at ambient temperature of preoptimized, premixed, and predispensed PCR mixes aimed at the detection of mycobacteria in clinical materials. The freeze-dried mixes retained activity at 4°C and at 20°C for 1 year and for 3 months at 37°C, as judged by their performance with 50 and 500 fg of purified Mycobacterium bovis BCG target DNA. PMID:9620427

  3. Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses.

    PubMed

    Parker, Jayme; Fowler, Nisha; Walmsley, Mary Louise; Schmidt, Terri; Scharrer, Jason; Kowaleski, James; Grimes, Teresa; Hoyos, Shanann; Chen, Jack

    2015-01-01

    Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95-100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2-1280.8 copies/μL (0.08-3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2-8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4-1280.8 copies/μL, 2.50-3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6-94.8 copies/μL, 0.20-1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported. PMID:26569120

  4. Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses

    PubMed Central

    Parker, Jayme; Fowler, Nisha; Walmsley, Mary Louise; Schmidt, Terri; Scharrer, Jason; Kowaleski, James; Grimes, Teresa; Hoyos, Shanann; Chen, Jack

    2015-01-01

    Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95–100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2–1280.8 copies/μL (0.08–3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2–8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4–1280.8 copies/μL, 2.50–3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6–94.8 copies/μL, 0.20–1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported. PMID:26569120

  5. Clinical differences among PCR-proven dengue serotype infections.

    PubMed

    Limkittikul, Kriengsak; Yingsakmongkon, Sangchai; Jittmittraphap, Akanitt; Chuananon, Somchai; Kongphrai, Yuphin; Kowasupathr, Surasak; Rojanawatsirivit, Chaiyaporn; Mammen, Mammen P; Jampangern, Wipawee

    2005-11-01

    The objective of this study was to compare the clinical spectra of the dengue serotypes proven by the PCR technique. This retrospective study reviewed the clinical information of dengue-infected patients who were admitted to northeastern provincial hospitals in Thailand from June to September 2002. Dengue infection and viral serotypes were confirmed by polymerase chain reaction (PCR). Paired anti-dengue immunoglobulin G (IgG) and IgM from paired sera were analyzed by enzyme-linked immunosorbent assay (ELISA). Ninety-nine PCR-proven dengue-infected Thai patients were studied. Their ages ranged from 3-30 years. They were infected with DEN1, DEN2, DEN3 and DEN4 in 21, 55, 12, and 12%, respectively. Twenty-two percent had primary and 78% had secondary infections. Dengue fever was the most common presentation for both primary (77.2%) and secondary infections (46.7%). The ratios of dengue fever:dengue hemorrhagic fever (DF:DHF) and non-dengue shock syndrome:dengue shock syndrome (non-DSS:DSS) for DEN2 was the lowest of the dengue serotypes. There was no difference in the duration of fever, percentage of hepatomegaly and bleeding among the serotypes in both DF and DHF. The trends in the white blood cells, lymphocyte and atypical lymphocyte counts in DEN3 were the highest, while those of DEN1 were the lowest of the dengue serotypes. PMID:16610645

  6. Detection and counting of Nitrobacter populations in soil by PCR.

    PubMed Central

    Degrange, V; Bardin, R

    1995-01-01

    Although the biological conversion of nitrite to nitrate is a well-known process, studies of Nitrobacter populations are hindered by their physiological characteristics. This report describes a new method for detecting and counting Nitrobacter populations in situ with the PCR. Two primers from the 16S rRNA gene were used to generate a 397-bp fragment by amplification of Nitrobacter species DNA. No signal was detected from their phylogenetic neighbors or the common soil bacteria tested. Extraction and purification steps were optimized for minimal loss and maximal purity of soil DNA. The detection threshold and accuracy of the molecular method were determined from soil inoculated with 10, 10(2), or 10(3) Nitrobacter hamburgensis cells per g of soil. Counts were also done by the most-probable-number (MPN)-Griess and fluorescent antibody methods. PCR had a lower detection threshold (10(2) Nitrobacter cells per g of soil) than did the MPN-Griess or fluorescent antibody method. When PCR amplification was coupled with the MPN method, the counting rate reached 65 to 72% of inoculated Nitrobacter cells. Tested on nonsterile soil, this rapid procedure was proved efficient. PMID:7793930

  7. Rat Gene Mapping Using Pcr-Analyzed Microsatellites

    PubMed Central

    Serikawa, T.; Kuramoto, T.; Hilbert, P.; Mori, M.; Yamada, J.; Dubay, C. J.; Lindpainter, K.; Ganten, D.; Guenet, J. L.; Lathrop, G. M.; Beckmann, J. S.

    1992-01-01

    One hundred and seventy-four rat loci which contain short tandem repeat sequences were extracted from the GenBank or EMBL data bases and used to define primers for amplification by the polymerase chain reaction (PCR) of the microsatellite regions, creating PCR-formatted sequence-tagged microsatellite sites (STMSs). One hundred and thirty-four STMSs for 118 loci, including 6 randomly cloned STMSs, were characterized: (i) PCR-analyzed loci were assigned to specific chromosomes using a panel of rat X mouse somatic cell hybrid clones. (ii) Length variation of the STMSs among 8 inbred rat strains could be visualized at 85 of 107 loci examined (79.4%). (iii) A genetic map, integrating biochemical, coat color, mutant and restriction fragment length polymorphism loci, was constructed based on the segregation of 125 polymorphic markers in seven rat backcrosses and in two F(2) crosses. Twenty four linkage groups were identified, all of which were assigned to a defined chromosome. As a reflection of the bias for coding sequences in the public data bases, the STMSs described herein are often associated with genes. Hence, the genetic map we report coincides with a gene map. The corresponding map locations of the homologous mouse and human genes are also listed for comparative mapping purposes. PMID:1628813

  8. [Using inter-SINE-PCR to study mammalian phylogeny].

    PubMed

    Bannikova, A A; Matveev, V A; Kramerov, D A

    2002-06-01

    Results of the use of the fingerprinting method related to short interspersed elements (SINEs), inter-SINE-PCR, in the study of phylogenetic and taxonomic relationship in mammals from orders Chiroptera (family Vespertilionidae) and Lipotyphla (family Erinaceidae) are reported. The inter-SINE-PCR method is based on the amplification of fragments situated between copies of SINEs, which are short retroposons spaced 100 to 1000 bp apart. Specifically selected primers were used, which are complementary to consensus sequences of two short retroposons: the mammalian interspersed repeat (MIR), which is typical of all mammals and some other vertebrates, was used in the cases of bats and Erinaceidae, and the ERI-1 element recently isolated from the genome of the Daurian hedgehog was used in the case of Erinaceidae. The results support the current view on phylogenetic relationship between hedgehogs belonging to genera Erinaceus, Hemiechinus, and Paraechinus (but not the genus Atelerix). In bats, the phylogenetic reconstruction revealed a statistically valid topology only at lower taxonomic levels, whereas the topology for the genus and supragenus ranks was unresolved and fan-shaped. The benefits and limitations of the inter-SINE-PCR method are discussed. PMID:12138785

  9. Clinically Applicable Multiplex PCR for Four Middle Ear Pathogens

    PubMed Central

    Hendolin, Panu H.; Paulin, Lars; Ylikoski, Jukka

    2000-01-01

    The multiplex PCR method for the detection of Alloiococcus otitidis, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae (P. H. Hendolin, A. Markkanen, J. Ylikoski, and J. J. Wahlfors, J. Clin. Microbiol. 35:2854–2858, 1997) in middle ear effusions (MEEs) was modified to be better suited for clinical use. To detect false-negative results, an internal amplification was added to the reaction, and to prevent carryover contamination, the dUTP–uracil-N-glycosidase system was incorporated into the procedure. Labor was minimized by using the heat-activatable AmpliTaq Gold polymerase in order to circumvent manual hot start and by detecting the amplification products on an automated sequencer. The performance of the improved protocol was verified with MEEs from patients with otitis media with effusion. In addition, a ligase detection reaction (LDR) was developed for confirmation of the PCR products. The modifications increased the reliability of the protocol and the hands-off time significantly. However, when two DNA extraction protocols were compared, gram-negative bacteria were detected more often in phenol-treated MEEs (94 versus 46%; P < 0.001), and gram-positive bacteria were detected more often in MEEs dissolved in sodium dodecyl sulfate-NaOH-chaotropic salt (83 versus 27%; P < 0.001). The LDR was found to be 100% specific. In all, the results demonstrate the feasibility of the rapid (7-h) multiplex PCR method for routine laboratory use. PMID:10618075

  10. Sex identification from exfoliated primary teeth--a PCR study.

    PubMed

    Kumar, Manju Gopa; Hegde, Amitha M

    2005-01-01

    Teeth endure postmortem degradation and extreme changes in ambient temperature and pressure better than most human tissues. In the present day scenario the growing number of crime against children in the form of battering, physical/sexual abuse, abduction and kidnapping, the use of exfoliated primary teeth, become many times the only evidence available at the crime scene. Despite exposure of the body to burial, mutilation, explosion or incineration, it is usually possible to extract DNA from pulp tissue of tooth with sufficient quality and quantity. Hence the present study was undertaken to find out the sex of a child from exfoliated/extracted deciduous teeth using a Polymerase Chain reaction (PCR) based analysis. Tooth samples were stored in room temperature after double coding for various periods. Dental pulp tissue was collected from each sample and DNA was isolated by proteinase-k digestion and phenol chloroform extraction methods. PCR amplification was done with two sets of oligonucleiotide primers. Amplification of X (131bp) and Y-specific sequences (172bp) in males and that of the X-specific sequence in females was observed and compared with the template DNA showing male and female controls. Determination of sexes of all freshly collected samples within 24 hours and after 1 month of extraction respectively gave 100% result. However, PCR was not found to be an effective method for sex determination after 6 months post extraction. PMID:16302594

  11. Development of a multiplex PCR for identification of vineyard mealybugs.

    PubMed

    Daane, Kent M; Middleton, Mathew C; Sforza, René; Cooper, Monica L; Walton, Vaughn M; Walsh, Douglas B; Zaviezo, Tania; Almeida, Rodrigo P P

    2011-12-01

    A simple molecular tool was developed and tested to identify seven mealybug species found in North American vineyards: Pseudococcus maritimus Ehrhorn, Pseudococcus viburni (Signoret), Pseudococcus longispinus (Targioni-Tozzeti), Pseudococcus calceolariae (Maskell), Planococcus ficus (Signoret), Planococcus citri (Risso), and Ferrisia gilli Gullan. The developed multiplex PCR is based on the mitochondrial cytochrome c oxidase subunit one gene. In tests, this single-step multiplex PCR correctly identified 95 of 95 mealybug samples, representing all seven species and collected from diverse geographic regions. To test the sensitivity, single specimen samples with different Pl. ficus developmental stages (egg to adult female and adult male) were processed PCR and the resulting output provided consistent positive identification. To test the utility of this protocol for adult males caught in sex baited pheromone traps, Pl. ficus adult males were placed in pheromone traps, aged at a constant temperature of 26±2°C, and processed with the multiplex each day thereafter for 8 d. Results showed consistent positive identification for up to 6 d (range, 6-8 d). Results are discussed with respect to the usefulness of this molecular tool for the identification of mealybugs in pest management programs and biosecurity of invasive mealybugs. PMID:22217778

  12. Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR.

    PubMed Central

    Folgueira, L; Delgado, R; Palenque, E; Aguado, J M; Noriega, A R

    1996-01-01

    A method based on DNA amplification and hybridization has been used for the rapid detection of Mycobacterium tuberculosis in blood samples from 38 hospitalized patients (15 human immunodeficiency virus [HIV] positive and 23 HIV negative) in whom localized or disseminated forms of tuberculosis were suspected. In 32 of these patients, the diagnosis of tuberculosis was eventually confirmed by conventional bacteriological or histological procedures. M. tuberculosis DNA was detected with the PCR technique in the peripheral blood mononuclear cells from 9 of 11 (82%) HIV-infected patients and in 7 of 21 (33%) HIV-negative patients (P < 0.01), while M. tuberculosis blood cultures were positive in 1 of 8 (12.5%) and 1 of 18 (5.5%) patients, respectively. PCR was positive in all cases with disseminated disease in both HIV-negative and HIV-positive patients and also in the HIV-positive patients with extrapulmonary tuberculosis. Seven samples from patients with documented illness other than tuberculosis and 12 specimens from healthy volunteers, including seven volunteers with a recent positive purified protein derivative test, were used as controls and had a negative PCR. These results suggest that detection of M. tuberculosis DNA in peripheral blood mononuclear cells may be a useful tool for rapid diagnosis of disseminated and extrapulmonary forms of tuberculosis, especially in an HIV-positive population. PMID:8904404

  13. Improved purification and PCR amplification of DNA from environmental samples.

    PubMed

    Arbeli, Ziv; Fuentes, Cilia L

    2007-07-01

    Purification and PCR amplification procedures for DNA extracted from environmental samples (soil, compost, and river sediment) were improved by introducing three modifications: precipitation of DNA with 5% polyethylene glycol 8000 (PEG) and 0.6 M NaCl; filtration with a Sepharose 4B-polyvinylpolypyrrolidone (PVPP) spin column; and addition of skim milk (0.3% w/v) to the PCR reaction solution. Humic substances' concentration after precipitation with 5% PEG was 2.57-, 5.3-, and 78.9-fold lower than precipitation with 7.5% PEG, 10% PEG, and isopropanol, respectively. After PEG precipitation, Sepharose, PVPP and the combined (Sepharose-PVPP) column removed 92.3%, 89.5%, and 98%, respectively, of the remaining humic materials. Each of the above-mentioned modifications improved PCR amplification of the 16S rRNA gene. DNA extracted by the proposed protocol is cleaner than DNA extracted by a commercial kit. Nevertheless, the improvement of DNA purification did not improve the detection limit of atrazine degradation gene atzA. PMID:17521406

  14. Electrochemiluminescence-PCR detection of genetically modified organisms

    NASA Astrophysics Data System (ADS)

    Liu, Jinfeng; Xing, Da; Shen, Xingyan; Zhu, Debin

    2005-01-01

    The detection methods for genetically modified (GM) components in foods have been developed recently. But many of them are complicated and time-consuming; some of them need to use the carcinogenic substance, and can"t avoid false-positive results. In this study, an electrochemiluminescence polymerase chain reaction (ECL-PCR) method for detection GM tobaccos is proposed. The Cauliflower mosaic virus 35S (CaMV35S) promoter was amplified by PCR, Then hybridized with a Ru(bpy)32+ (TBR)-labeled and a biotinylated probe. The hybridization products were captured onto streptavidin-coated paramagnetic beads, and detected by measuring the electrochemiluminescence (ECL) signal of the TBR label. Whether the tobaccos contain GM components was discriminated by detecting the ECL signal of CaMV35S promoter. The experiment results show that the detection limit for CaMV35S promoter is 100 fmol, and the GM components can be clearly identified in GM tobaccos. The ECL-PCR method provide a new means in GMOs detection due to its safety, simplicity and high efficiency.

  15. Analysis of copy number variation using quantitative interspecies competitive PCR.

    PubMed

    Williams, Nigel M; Williams, Hywel; Majounie, Elisa; Norton, Nadine; Glaser, Beate; Morris, Huw R; Owen, Michael J; O'Donovan, Michael C

    2008-10-01

    Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls. PMID:18697816

  16. Determination of Arabidopsis thaliana telomere length by PCR.

    PubMed

    Vaquero-Sedas, María I; Vega-Palas, Miguel A

    2014-01-01

    In humans, telomere length studies have acquired great relevance because the length of telomeres has been related to natural processes like disease, aging and cancer. However, very little is known about the influence of telomere length on the biology of wild type plants. The length of plant telomeres has been usually studied by Terminal Restriction Fragment (TRF) analyses. This technique requires high amounts of tissue, including multiple cell types, which might be the reason why very little is known about the influence of telomere length on plant natural processes. In contrast, many of the human telomere length studies have focused on homogenous cell populations. Most of these studies have been performed by PCR, using telomeric degenerated primers, which allow the determination of telomere length from small amounts of human cells. Here, we have adapted the human PCR procedure to analyze the length of Arabidopsis thaliana telomeres. This PCR approach will facilitate the analysis of telomere length from low amounts of tissue. We have used it to determine that CG and non CG DNA methylation positively regulates Arabidopsis telomere length. PMID:24986269

  17. Determination of Arabidopsis thaliana telomere length by PCR

    PubMed Central

    Vaquero-Sedas, María I.; Vega-Palas, Miguel A.

    2014-01-01

    In humans, telomere length studies have acquired great relevance because the length of telomeres has been related to natural processes like disease, aging and cancer. However, very little is known about the influence of telomere length on the biology of wild type plants. The length of plant telomeres has been usually studied by Terminal Restriction Fragment (TRF) analyses. This technique requires high amounts of tissue, including multiple cell types, which might be the reason why very little is known about the influence of telomere length on plant natural processes. In contrast, many of the human telomere length studies have focused on homogenous cell populations. Most of these studies have been performed by PCR, using telomeric degenerated primers, which allow the determination of telomere length from small amounts of human cells. Here, we have adapted the human PCR procedure to analyze the length of Arabidopsis thaliana telomeres. This PCR approach will facilitate the analysis of telomere length from low amounts of tissue. We have used it to determine that CG and non CG DNA methylation positively regulates Arabidopsis telomere length. PMID:24986269

  18. Nested PCR for detection of HSV-1 in oral mucosa

    PubMed Central

    Jalouli, Miranda-Masoumeh; Jalouli, Jamshid; Hasséus, Bengt; Öhman, Jenny; Hirsch, Jan-Michaél

    2015-01-01

    Background It has been estimated that 15%-20% of human tumours are driven by infection and inflammation, and viral infections play an important role in malignant transformation. The evidence that herpes simplex virus type 1 (HSV-1) could be involved in the aetiology of oral cancer varies from weak to persuasive. This study aimed to investigate by nested PCR (NPCR) the prevalence of HSV-1 in samples from normal oral mucosa, oral leukoplakia, and oral squamous cell carcinoma (OSCC). Material and Methods We investigated the prevalence of HSV-1 in biopsies obtained from 26 fresh, normal oral mucosa from healthy volunteers as well as 53 oral leukoplakia and 27 OSCC paraffin-embedded samples. DNA was extracted from the specimens and investigated for the presence of HSV-1 by nested polymerase chain reaction (NPCR) and DNA sequencing. Results HSV-1 was detected in 14 (54%) of the healthy samples, in 19 (36%) of the oral leukoplakia samples, and in 14 (52%) of the OSCC samples. The differences were not statistically significant. Conclusions We observed a high incidence of HSV-1 in healthy oral mucosa, oral leukoplakia, and OSCC tissues. Thus, no connection between OSCC development and presence of HSV-1 was detected. Key words:HSV-1, nested PCR, PCR. PMID:26449432

  19. Ultrasensitive Antibody Detection by Agglutination-PCR (ADAP)

    PubMed Central

    2016-01-01

    Antibodies are widely used biomarkers for the diagnosis of many diseases. Assays based on solid-phase immobilization of antigens comprise the majority of clinical platforms for antibody detection, but can be undermined by antigen denaturation and epitope masking. These technological hurdles are especially troublesome in detecting antibodies that bind nonlinear or conformational epitopes, such as anti-insulin antibodies in type 1 diabetes patients and anti-thyroglobulin antibodies associated with thyroid cancers. Radioimmunoassay remains the gold standard for these challenging antibody biomarkers, but the limited multiplexability and reliance on hazardous radioactive reagents have prevented their use outside specialized testing facilities. Here we present an ultrasensitive solution-phase method for detecting antibodies, termed antibody detection by agglutination-PCR (ADAP). Antibodies bind to and agglutinate synthetic antigen–DNA conjugates, enabling ligation of the DNA strands and subsequent quantification by qPCR. ADAP detects zepto- to attomoles of antibodies in 2 μL of sample with a dynamic range spanning 5–6 orders of magnitude. Using ADAP, we detected anti-thyroglobulin autoantibodies from human patient plasma with a 1000-fold increased sensitivity over an FDA-approved radioimmunoassay. Finally, we demonstrate the multiplexability of ADAP by simultaneously detecting multiple antibodies in one experiment. ADAP’s combination of simplicity, sensitivity, broad dynamic range, multiplexability, and use of standard PCR protocols creates new opportunities for the discovery and detection of antibody biomarkers. PMID:27064772

  20. Direct-to-PCR tissue preservation for DNA profiling.

    PubMed

    Sorensen, Amy; Berry, Clare; Bruce, David; Gahan, Michelle Elizabeth; Hughes-Stamm, Sheree; McNevin, Dennis

    2016-05-01

    Disaster victim identification (DVI) often occurs in remote locations with extremes of temperatures and humidities. Access to mortuary facilities and refrigeration are not always available. An effective and robust DNA sampling and preservation procedure would increase the probability of successful DNA profiling and allow faster repatriation of bodies and body parts. If the act of tissue preservation also released DNA into solution, ready for polymerase chain reaction (PCR), the DVI process could be further streamlined. In this study, we explored the possibility of obtaining DNA profiles without DNA extraction, by adding aliquots of preservative solutions surrounding fresh human muscle and decomposing human muscle and skin tissue samples directly to PCR. The preservatives consisted of two custom preparations and two proprietary solutions. The custom preparations were a salt-saturated solution of dimethyl sulfoxide (DMSO) with ethylenediaminetetraacetic (EDTA) and TENT buffer (Tris, EDTA, NaCl, Tween 20). The proprietary preservatives were DNAgard (Biomatrica(®)) and Tissue Stabilising Kit (DNA Genotek). We obtained full PowerPlex(®) 21 (Promega) and GlobalFiler(®) (Life Technologies) DNA profiles from fresh and decomposed tissue preserved at 35 °C for up to 28 days for all four preservatives. The preservative aliquots removed from the fresh muscle tissue samples had been stored at -80 °C for 4 years, indicating that long-term archival does not diminish the probability of successful DNA typing. Rather, storage at -80 °C seems to reduce PCR inhibition. PMID:26530406

  1. Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses.

    PubMed

    Kwon, Ji Yeon; Hong, Jin Sung; Kim, Min Jea; Choi, Sun Hee; Min, Byeong Eun; Song, Eun Gyeong; Kim, Hyun Hee; Ryu, Ki Hyun

    2014-09-01

    Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10 fg/μl of viral RNA under monoplex conditions and 10 pg/μl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53 °C to 63 °C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed. PMID:24937806

  2. Usefulness of PCR method for detection of Leishmania in Poland.

    PubMed

    Myjak, Przemysław; Szulta, Joanna; de Almeida, Marcos E; da Silva, Alexandre J; Steurer, Francis; Lass, Anna; Pietkiewicz, Halina; Nahorski, Wacław L; Goljan, Jolanta; Knap, Józef; Szostakowska, Beata

    2009-01-01

    Leishmania parasites are the etiological agents of leishmaniosis, with severe course and often fatal prognosis, and the global number of cases has increased in recent decades. The gold standards for the diagnosis of leishmaniosis are microscopic examinations and culture in vitro of the different clinical specimens. The sensitivity of these methods is insufficient. Recent development in specific and sensitive molecular methods (PCR) allows for detection as well as identification of the parasite species (subspecies). The aim of the study was to estimate the usefulness of molecular methods (PCR) for detection of Leishmania species and consequently for the implementation of such methods in routine diagnostics of leishmaniosis in Polish patients returning from endemic areas of the disease. In our investigations we used 54 known Leishmania positive DNA templates (from culture and clinical specimens) received from the CDC (Atlanta, GA, USA). Moreover, 25 samples of bone marrow, blood or other tissues obtained from 18 Polish individuals suspected of leishmaniosis were also examined. In PCR we used two pairs of primers specific to the conserved region of Leishmania kinetoplast DNA (kDNA) minicircle (13A/13B and F/R). Using these primers we obtained amplicons in all DNA templates from the CDC and in three Polish patients suspected for Leishmania infection. In one sample from among these cases we also obtained positive results with DNA isolated from a blood specimen which was previously negative in microscopic examinations. PMID:19899614

  3. Molecular Serogrouping of Listeria monocytogenes from Brazil Using PCR.

    PubMed

    Camargo, Anderson Carlos; Vallim, Deyse Christina; Hofer, Ernesto; Nero, Luís Augusto

    2016-01-01

    We assessed the serotype distribution of Listeria monocytogenes isolates from clinical, beef, and environment samples using two PCR-based protocols for serogrouping. A panel of 134 isolates (22 clinical samples, 79 samples of beef cuts, and 33 samples from the beef processing environment) were subjected to conventional serology and identified as serotypes 1/2a (n = 12), 1/2b (n = 21), 1/2c (n = 71), and 4b (n = 30). Isolates from clinical samples were predominantly serotype 4b, and the most prevalent serotype among the beef cut and environment samples was 1/2c. The protocol described by M. Doumith, C. Buchrieser, P. Glaser, C. Jacquet, and P. Martin (J. Clin. Microbiol. 42:3819-3822, 2004) produced contradictory results for seven 1/2a isolates, which were positive for lmo1118 and had the profile IIc (serotypes 1/2c and 3c). Fifteen serotype 4b isolates amplified the target lmo0737, with the atypical profile IVb variant 1. The results obtained with the protocol described by M. K. Borucki and D. R. Call (J. Clin. Microbiol. 41:5537-5540, 2003) were in full agreement with those of the conventional serology. We recommend using this multiplex PCR approach by adding one pair of the reported primers to the panel to reduce total effort by one PCR while maintaining specificity. We present additional recommendations to improve the efficiency and reproducibility of this serogrouping assay. PMID:26735041

  4. Rapid diagnosis of aneuploidy using segmental duplication quantitative fluorescent PCR.

    PubMed

    Kong, Xiangdong; Li, Lin; Sun, Lei; Fu, Kepeng; Long, Ju; Weng, Xunjin; Ye, Xuehe; Liu, Xinxiong; Wang, Bo; Yan, Shanhuo; Ye, Haiming; Fan, Zuqian

    2014-01-01

    The aim of this study was use a simple and rapid procedure, called segmental duplication quantitative fluorescent polymerase chain reaction (SD-QF-PCR), for the prenatal diagnosis of fetal chromosomal aneuploidies. This method is based on the co-amplification of segmental duplications located on two different chromosomes using a single pair of fluorescent primers. The PCR products of different sizes were subsequently analyzed through capillary electrophoresis, and the aneuploidies were determined based on the relative dosage between the two chromosomes. Each primer set, containing five pairs of primers, was designed to simultaneously detect aneuploidies located on chromosomes 21, 18, 13, X and Y in a single reaction. We applied these two primer sets to DNA samples isolated from individuals with trisomy 21 (n = 36); trisomy 18 (n = 6); trisomy 13 (n = 4); 45, X (n = 5); 47, XXX (n = 3); 48, XXYY (n = 2); and unaffected controls (n = 40). We evaluated the performance of this method using the karyotyping results. A correct and unambiguous diagnosis with 100% sensitivity and 100% specificity, was achieved for clinical samples examined. Thus, the present study demonstrates that SD-QF-PCR is a robust, rapid and sensitive method for the diagnosis of common aneuploidies, and these analyses can be performed in less than 4 hours for a single sample, providing a competitive alternative for routine use. PMID:24625828

  5. An unusual presentation of herpes simplex encephalitis with negative PCR.

    PubMed

    Buerger, Kelly J; Zerr, Kayleigh; Salazar, Richard

    2015-01-01

    A 74-year-old man presented with acute right-sided hemiparesis and epilepsia partialis continua in association with fever and confusion. Initial workup revealed possible cerebritis in the left medial frontal lobe without involvement of the temporal lobes. Cerebrospinal fluid (CSF) analysis revealed minimal lymphocytic pleocytosis but negative real-time herpes simplex virus (HSV) PCR. Acyclovir was discontinued on day 5 due to a negative infectious workup and clinical improvement. On day 9 his condition deteriorated and he was transferred to a higher level of acuity for advanced supportive care. Worsening encephalopathy and refractory status epilepticus ensued despite medical care. Repeat CSF analysis showed mild lymphocytic pleocytosis with negative real-time HSV PCR. Brain MRI revealed progression of cortical enhancement. Immunosuppressive therapy and plasma exchange were attempted without clinical response. On day 24, another lumbar puncture showed only mild lymphocytic pleocytosis. Brain MRI showed involvement of the right medial temporal lobe. Subsequently, acyclovir was resumed. The HSV-1 PCR result was positive on day 30. Unfortunately, the patient expired. PMID:26243746

  6. Primer Extension Reactions for the PCR- based α- complementation Assay

    PubMed Central

    Achuthan, Vasudevan; DeStefano, Jeffrey J.

    2016-01-01

    The PCR- based- α- complementation assay is an effective technique to measure the fidelity of polymerases, especially RNA-dependent RNA polymerases (RDRP) and Reverse Transcriptases (RT). It has been successfully employed to determine the fidelity of the poliovirus polymerase 3D-pol (DeStefano, 2010) as well as the human immunodeficiency virus Reverse Transcriptase (HIV RT) (Achuthan et al., 2014). A major advantage of the assay is that since the PCR step is involved, even the low yield of products obtained after two rounds of low yield of RNA synthesis (for RDRP) or reverse transcription (for RT) can be measured using the assay. The assay also mimics the reverse transcription process, since both RNA- and DNA- directed RT synthesis steps are performed. We recently used this assay to show that the HIV RT, at physiologically relevant magnesium concentration, has accuracy in the same range as other reverse transcriptases (Achuthan et al., 2014). Here, we describe in detail how to prepare the inserts using the primer extension reactions. The prepared inserts are then processed further in the PCR- based- α- complementation assay.

  7. Optimization of Droplet Digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: Clinical implications for quantification of Oseltamivir-resistant subpopulations.

    PubMed

    Taylor, Sean C; Carbonneau, Julie; Shelton, Dawne N; Boivin, Guy

    2015-11-01

    The recent introduction of Droplet Digital PCR (ddPCR) has provided researchers with a tool that permits direct quantification of nucleic acids from a wide range of samples with increased precision and sensitivity versus RT-qPCR. The sample interdependence of RT-qPCR stemming from the measurement of Cq and ΔCq values is eliminated with ddPCR which provides an independent measure of the absolute nucleic acid concentration for each sample without standard curves thereby reducing inter-well and inter-plate variability. Well-characterized RNA purified from H275-wild type (WT) and H275Y-point mutated (MUT) neuraminidase of influenza A (H1N1) pandemic 2009 virus was used to demonstrate a ddPCR optimization workflow to assure robust data for downstream analysis. The ddPCR reaction mix was also tested with RT-qPCR and gave excellent reaction efficiency (between 90% and 100%) with the optimized MUT/WT duplexed assay thus enabling the direct comparison of the two platforms from the same reaction mix and thermal cycling protocol. ddPCR gave a marked improvement in sensitivity (>30-fold) for mutation abundance using a mixture of purified MUT and WT RNA and increased precision (>10 fold, p<0.05 for both inter- and intra-assay variability) versus RT-qPCR from patient samples to accurately identify residual mutant viral population during recovery. PMID:26315318

  8. Single-step PCR in molecular diagnosis of hepatitis C virus infection.

    PubMed Central

    Farma, E; Boeri, E; Bettini, P; Repetto, C M; McDermott, J; Lillo, F B; Varnier, O E

    1996-01-01

    The diagnostic utility of two PCR systems and three PCR detection methods for hepatitis C virus (HCV) RNA was evaluated in serum samples. A nested PCR was considered the reference assay and was compared with two single-step PCR methods: the first is based on the detection of PCR products by liquid hybridization with a 32P-end-labeled probe, and the second is the Roche Amplicor colorimetric assay using microwell plate hybridization with a specific nucleic acid probe. Using the Pelicheck HCV RNA Eurohep genotype 1 proficiency panel, our laboratory achieved medium-high levels of performance with all three methods. The highest sensitivity was, however, observed with the isotopic single-step PCR (ss-PCR) method. The analytical sensitivity of ss-PCR with isotopic detection and ss-PCR with colorimetric detection was identical to that of nested PCR, with a 100% result concordance. Comparison of ss-PCR with enzyme-linked immunosorbent and RIBA assays in the analysis of clinical samples showed a high concordance. ss-PCR methods appear more suitable for diagnostic application. Nevertheless, HCV RNA PCR cannot be considered a screening as