Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

PubMed

Kageyama, Shinji; Shinmura, Kazuya; Yamamoto, Hiroko; Goto, Masanori; Suzuki, Koichi; Tanioka, Fumihiko; Tsuneyoshi, Toshihiro; Sugimura, Haruhiko

2008-04-01

The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

Comparative analyses of genetic/epigenetic diversities and structures in a wild barley species (Hordeum brevisubulatum) using MSAP, SSAP and AFLP.

PubMed

Shan, X H; Li, Y D; Liu, X M; Wu, Y; Zhang, M Z; Guo, W L; Liu, B; Yuan, Y P

2012-08-17

We analyzed genetic diversity and population genetic structure of four artificial populations of wild barley (Hordeum brevisubulatum); 96 plants collected from the Songnen Prairie in northeastern China were analyzed using amplified fragment length polymorphism (AFLP), specific-sequence amplified polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) markers. Indices of (epi-)genetic diversity, (epi-)genetic distance, gene flow, genotype frequency, cluster analysis, PCA analysis and AMOVA analysis generated from MSAP, AFLP and SSAP markers had the same trend. We found a high level of correlation in the artificial populations between MSAP, SSAP and AFLP markers by the Mantel test (r > 0.8). This is incongruent with previous findings showing that there is virtually no correlation between DNA methylation polymorphism and classical genetic variation; the high level of genetic polymorphism could be a result of epigenetic regulation. We compared our results with data from natural populations. The population diversity of the artificial populations was lower. However, different from what was found using AFLP and SSAP, based on MSAP results the methylation polymorphism of the artificial populations was not significantly reduced. This leads us to suggest that the DNA methylation pattern change in H. brevisubulatum populations is not only related to DNA sequence variation, but is also regulated by other controlling systems.

Using microarray analysis to evaluate genetic polymorphisms involved in the metabolism of environmental chemicals.

PubMed

Ban, Susumu; Kondo, Tomoko; Ishizuka, Mayumi; Sasaki, Seiko; Konishi, Kanae; Washino, Noriaki; Fujita, Syoichi; Kishi, Reiko

2007-05-01

The field of molecular biology currently faces the need for a comprehensive method of evaluating individual differences derived from genetic variation in the form of single nucleotide polymorphisms (SNPs). SNPs in human genes are generally considered to be very useful in determining inherited genetic disorders, susceptibility to certain diseases, and cancer predisposition. Quick and accurate discrimination of SNPs is the key characteristic of technology used in DNA diagnostics. For this study, we first developed a DNA microarray and then evaluated its efficacy by determining the detection ability and validity of this method. Using DNA obtained from 380 pregnant Japanese women, we examined 13 polymorphisms of 9 genes, which are associated with the metabolism of environmental chemical compounds found in high frequency among Japanese populations. The ability to detect CYP1A1 I462V, CYP1B1 L432V, GSTP1 I105V and AhR R554K gene polymorphisms was above 98%, and agreement rates when compared with real time PCR analysis methods (kappa values) showed high validity: 0.98 (0.96), 0.97 (0.93), 0.90 (0.81), 0.90 (0.91), respectively. While this DNA microarray analysis should prove important as a method for initial screening, it is still necessary that we find better methods for improving the detection of other gene polymorphisms not part of this study.

A LDR-PCR approach for multiplex polymorphisms genotyping of severely degraded DNA with fragment sizes <100 bp.

PubMed

Zhang, Zhen; Wang, Bao-Jie; Guan, Hong-Yu; Pang, Hao; Xuan, Jin-Feng

2009-11-01

Reducing amplicon sizes has become a major strategy for analyzing degraded DNA typical of forensic samples. However, amplicon sizes in current mini-short tandem repeat-polymerase chain reaction (PCR) and mini-sequencing assays are still not suitable for analysis of severely degraded DNA. In this study, we present a multiplex typing method that couples ligase detection reaction with PCR that can be used to identify single nucleotide polymorphisms and small-scale insertion/deletions in a sample of severely fragmented DNA. This method adopts thermostable ligation for allele discrimination and subsequent PCR for signal enhancement. In this study, four polymorphic loci were used to assess the ability of this technique to discriminate alleles in an artificially degraded sample of DNA with fragment sizes <100 bp. Our results showed clear allelic discrimination of single or multiple loci, suggesting that this method might aid in the analysis of extremely degraded samples in which allelic drop out of larger fragments is observed.

Forensic strategy to ensure the quality of sequencing data of mitochondrial DNA in highly degraded samples.

PubMed

Adachi, Noboru; Umetsu, Kazuo; Shojo, Hideki

2014-01-01

Mitochondrial DNA (mtDNA) is widely used for DNA analysis of highly degraded samples because of its polymorphic nature and high number of copies in a cell. However, as endogenous mtDNA in deteriorated samples is scarce and highly fragmented, it is not easy to obtain reliable data. In the current study, we report the risks of direct sequencing mtDNA in highly degraded material, and suggest a strategy to ensure the quality of sequencing data. It was observed that direct sequencing data of the hypervariable segment (HVS) 1 by using primer sets that generate an amplicon of 407 bp (long-primer sets) was different from results obtained by using newly designed primer sets that produce an amplicon of 120-139 bp (mini-primer sets). The data aligned with the results of mini-primer sets analysis in an amplicon length-dependent manner; the shorter the amplicon, the more evident the endogenous sequence became. Coding region analysis using multiplex amplified product-length polymorphisms revealed the incongruence of single nucleotide polymorphisms between the coding region and HVS 1 caused by contamination with exogenous mtDNA. Although the sequencing data obtained using long-primer sets turned out to be erroneous, it was unambiguous and reproducible. These findings suggest that PCR primers that produce amplicons shorter than those currently recognized should be used for mtDNA analysis in highly degraded samples. Haplogroup motif analysis of the coding region and HVS should also be performed to improve the reliability of forensic mtDNA data. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

[Application of mtDNA polymorphism in species identification of sarcosaphagous insects].

PubMed

Li, Xiang; Cai, Ji-feng

2011-04-01

Species identification of sarcosaphagous insects is one of the important steps in forensic research based on the knowledge of entomology. Recent studies reveal that the application of molecular biology, especially the mtDNA sequences analysis, works well in the species identification of sarcosaphagous insects. The molecular biology characteristics, structures, polymorphism of mtDNA of sarcosaphagous insects, and the recent studies in species identification of sarcosaphagous insects are reviewed in this article.

Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP).

PubMed

Yaish, Mahmoud W; Peng, Mingsheng; Rothstein, Steven J

2014-01-01

DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic DNA digestion.

Fragment length polymorphisms among independent isolates of Epstein-Barr virus from immunocompromised and normal hosts.

PubMed

Katz, B Z; Niederman, J C; Olson, B A; Miller, G

1988-02-01

DNA restriction fragment length polymorphisms of Epstein-Barr virus (EBV) DNA were used as a molecular epidemiological tool to study multiple isolates of virus from the same and different individuals. We studied 35 EBV isolates: 19 from seven immunocompromised children and 16 from seven college students with mononucleosis. Analysis of the fragment length polymorphisms in this collection of isolates permitted several conclusions. Sites of polymorphism were most often encountered in regions with repetitive DNA. Epidemiologically unrelated patients harbored viruses that could be readily distinguished; by contrast, two infants and their mothers harbored similar viruses. Isolates from different sites in the same patient were similar. Variations between different clinical isolates of EBV mimic those found between different laboratory strains of the virus. Fragment length polymorphisms thus provide a useful marker for studying transmission and pathogenesis of EBV infections.

Identification of two new polymorphisms in the manganese superoxide dismutase gene (Mn-SOD); Part of the etiology of Parkinson`s disease?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eggers, B.; Kurth, J.H.; Kurth, M.C.

1994-09-01

Epidemiological studies suggest that several different environmental agents interact with a number of genetic elements to cause Parkinson`s disease (PD), a common neurodegenerative disease. Abnormalities of oxidative metabolism may be central to this process. Specifically, the production and degradation of dopamine may lead to toxic by-products and increased oxidative stress. Toxic by-products include hydrogen peroxide, superoxide, and hydroxyl radicals, all of which are implicated in the aging process of the central nervous system. Superoxide dismutase (SOD) catalyzes superoxide to hydrogen peroxide. Genetic predisposition to PD may be at least partially a result of certain SOD alleles. Using the cDNA sequencemore » of Mn-SOD gene, oligonucleotide primers were designed which span several presumptive splice junction sites. An approximatley 2.4kb PCR product was amplified from gDNA samples that span one or more intron near the 3{prime} end of the Mn-SOD cDNA sequence. The resultant product was screened with a panel of 4-cutters to identify fragments appropriate for SSCP analysis. Twenty-two gDNA samples were screened for SSCP and size differences of these PCR products. After digestion with AluI, two polymorphisms were observed. Two alleles with a size difference of 2-4 bp were observed by denaturing PAGE in one of the fragments. SSCP analysis revealed a polymorphism with 2 alleles in another fragment. Sequence analysis of these polymorphisms is in progress. DNA from several DEPH families was used to confirm Mendelian inheritance of these polymorphisms. Genomic DNA samples have been collected from 265 PD patients and 169 control individuals; allelic frequencies will be determined for these populations, compared by {chi}{sup 2} analysis, and relative risk calculated. These results may support a contribution of Mn-SOD in the genetic predisposition to PD.« less

The XRCC1 Arg194Trp polymorphism is significantly associated with lung adenocarcinoma: a case-control study in an Eastern European Caucasian group

PubMed Central

Cătană, Andreea; Pop, Monica; Hincu, Bianca Domokos; Pop, Ioan V; Petrişor, Felicia M; Porojan, Mihai D; Popp, Radu A

2015-01-01

DNA repair plays an important role in maintaining the integrity of the genome by repairing DNA damage induced by carcinogens. Certain genetic polymorphisms that occur in DNA-repair genes may affect the ability to repair DNA defects, and may represent a risk factor in carcinogenesis. The gene XRCC1 is involved in DNA repair. The purpose of our study was to investigate the association between XRCC1 Arg194Trp and Arg399Gln polymorphisms and the risk of lung cancer in a Romanian population. We recruited 222 healthy controls and 102 patients with lung cancer. Genotypes were determined by multiplex polymerase chain-reaction restriction fragment-length polymorphism. Statistical analysis (odds ratio, recessive model) revealed an increased risk for lung cancer for the homozygous 194Trp genotype (χ2=0.186, odds ratio 10.667, 95% confidence interval 1.309–86.933; P=0.007). Also, we found an association between the 194Trp allele and women with lung adenocarcinoma. In conclusion, the results of the study place the XRCC1 Arg194Trp polymorphism among independent risk factors for developing lung cancer. PMID:26664136

The XRCC1 Arg194Trp polymorphism is significantly associated with lung adenocarcinoma: a case-control study in an Eastern European Caucasian group.

PubMed

Cătană, Andreea; Pop, Monica; Hincu, Bianca Domokos; Pop, Ioan V; Petrişor, Felicia M; Porojan, Mihai D; Popp, Radu A

2015-01-01

DNA repair plays an important role in maintaining the integrity of the genome by repairing DNA damage induced by carcinogens. Certain genetic polymorphisms that occur in DNA-repair genes may affect the ability to repair DNA defects, and may represent a risk factor in carcinogenesis. The gene XRCC1 is involved in DNA repair. The purpose of our study was to investigate the association between XRCC1 Arg194Trp and Arg399Gln polymorphisms and the risk of lung cancer in a Romanian population. We recruited 222 healthy controls and 102 patients with lung cancer. Genotypes were determined by multiplex polymerase chain-reaction restriction fragment-length polymorphism. Statistical analysis (odds ratio, recessive model) revealed an increased risk for lung cancer for the homozygous 194Trp genotype (χ (2)=0.186, odds ratio 10.667, 95% confidence interval 1.309-86.933; P=0.007). Also, we found an association between the 194Trp allele and women with lung adenocarcinoma. In conclusion, the results of the study place the XRCC1 Arg194Trp polymorphism among independent risk factors for developing lung cancer.

High-coverage methylation data of a gene model before and after DNA damage and homologous repair.

PubMed

Pezone, Antonio; Russo, Giusi; Tramontano, Alfonso; Florio, Ermanno; Scala, Giovanni; Landi, Rosaria; Zuchegna, Candida; Romano, Antonella; Chiariotti, Lorenzo; Muller, Mark T; Gottesman, Max E; Porcellini, Antonio; Avvedimento, Enrico V

2017-04-11

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles.

High-coverage methylation data of a gene model before and after DNA damage and homologous repair

PubMed Central

Pezone, Antonio; Russo, Giusi; Tramontano, Alfonso; Florio, Ermanno; Scala, Giovanni; Landi, Rosaria; Zuchegna, Candida; Romano, Antonella; Chiariotti, Lorenzo; Muller, Mark T.; Gottesman, Max E.; Porcellini, Antonio; Avvedimento, Enrico V.

2017-01-01

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles. PMID:28398335

The correlations between DNA methylation and polymorphisms in the promoter region of the human telomerase reverse transcriptase (hTERT) gene with postoperative recurrence in patients with thyroid carcinoma (TC).

PubMed

Li, Jian-Jun; Zheng, Ping Chen Jue-Ru; Wang, Yao-Zong

2017-06-06

This study aims at exploring the correlations between DNA methylation and polymorphisms in the promoter region of the human telomerase reverse transcriptase (hTERT) gene and postoperative recurrence in patients with thyroid carcinoma (TC). A total of 312 patients diagnosed with TC were chosen for the study and categorized into recurrence (n = 75) and non-recurrence (n = 237) groups. The hTERT rs2736100 and rs2736098 polymorphisms were detected by performing polymerase chain reaction-restriction fragment length polymorphism. DNA methylation in the promoter region of hTERT gene was evaluated by pyrosequencing. A telephonic and/or outpatient follow-up was conducted for all patients. The correlations of DNA methylation and polymorphisms in the promoter region of hTERT with postoperative recurrence of TC patients underwent analysis. The patient in the recurrence group showed evidently different pathological types and tumor stages in comparison to the non-recurrence group. The GG genotype of hTERT rs2736100 might increase the recurrence risk of TC patients. No correlations between hTERT rs2736098 polymorphisms and recurrence risk were observed. Compared to the TT + TG genotype frequency, the rs2736100 GG genotype frequency increased in patients without multicentricity, patients with extrathyroidal invasion, patients with lymph node metastasis, patients with undifferentiated carcinoma, and patients in the III + IV stage. The recurrence group showed significantly higher DNA methylation level compared to the non-recurrence group. The DNA methylation level was closely associated to tumor stage and lymph node metastasis of TC patients in the recurrence group. The DNA methylation and rs2736100 polymorphisms in the promoter region of hTERT gene might be in correlation to postoperative recurrence of TC patients.

Association of Leukotrichia in Vitiligo and Asp148Glu Polymorphism of Apurinic/Apyrimidinic Endonuclease 1.

PubMed

Aydin, A Fatih; Aydıngöz, İkbal Esen; Doğru-Abbasoğlu, Semra; Vural, Pervin; Uysal, Müjdat

2017-01-01

Oxidative stress and increased DNA damage have been implicated in the etiopathogenesis of vitiligo. Oxidative DNA damage is mainly repaired by the base excision repair (BER) pathway. We sought to determine whether polymorphisms in DNA repair genes may have a role in the pathogenesis of vitiligo. We conducted a study including 100 patients with vitiligo and age- and sex-matched 193 control subjects to examine the role of single-nucleotide polymorphisms of BER genes, human 8-oxoG DNA N-glycosylase 1 (codon 326), apurinic/apyrimidinic endonuclease 1 (APE1) (codon 148), and X-ray repair cross-complementing group 1 (codon 399) as risk factors for vitiligo. These polymorphisms were determined by quantitative real-time polymerase chain reaction and melting curve analysis. No significant association was observed between the variant alleles of studied genes and vitiligo. However, we showed that the presence of APE1 148Glu variant allele is associated with leukotrichia. This preliminary study suggests that APE1 (codon 148) polymorphism may play a role in vitiligo pathogenesis.

Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus.

PubMed

Lakshmikumaran, M S; D'Ambrosio, E; Laimins, L A; Lin, D T; Furano, A V

1985-09-01

The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this.

Determining Phylogenetic Relationships Among Date Palm Cultivars Using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) Markers.

PubMed

Haider, Nadia

2017-01-01

Investigation of genetic variation and phylogenetic relationships among date palm (Phoenix dactylifera L.) cultivars is useful for their conservation and genetic improvement. Various molecular markers such as restriction fragment length polymorphisms (RFLPs), simple sequence repeat (SSR), representational difference analysis (RDA), and amplified fragment length polymorphism (AFLP) have been developed to molecularly characterize date palm cultivars. PCR-based markers random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) are powerful tools to determine the relatedness of date palm cultivars that are difficult to distinguish morphologically. In this chapter, the principles, materials, and methods of RAPD and ISSR techniques are presented. Analysis of data generated from these two techniques and the use of these data to reveal phylogenetic relationships among date palm cultivars are also discussed.

DNA repair gene polymorphisms and risk of cutaneous melanoma: a systematic review and meta-analysis.

PubMed

Mocellin, Simone; Verdi, Daunia; Nitti, Donato

2009-10-01

Polymorphisms of DNA repair-related genes might modulate cancer predisposition. We performed a systematic review and meta-analysis of the available evidence regarding the relationship between these polymorphisms and the risk of developing cutaneous melanoma. Relevant studies were searched using PubMed, Medline, Embase, Cancerlit, Cochrane and ISI Web of Knowledge databases. Data were gathered according to the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) guidelines. The model-free approach was adopted to perform the meta-analysis of the retrieved data. We identified 20 original reports that describe the relationship between melanoma risk and the single-nucleotide polymorphisms (SNPs) of 16 genes (cases = 4195). For seven SNPs considered in at least two studies, the findings were heterogeneous. Data were suitable for meta-analysis only in the case of the XPD/ERCC2 SNP rs13181 (cases = 2308, controls = 3698) and demonstrated that the variant C allele is associated with increased melanoma risk (odds ratio = 1.12, 95% confidence interval = 1.03-1.21, P = 0.01; population attributable risk = 9.6%). This is the first meta-analysis suggesting that XPD/ERCC2 might represent a low-penetrance melanoma susceptibility gene. Much work is still to be done before definitive conclusions can be drawn on the role of DNA repair alterations in melanomagenesis since for the other genes involved in this highly complex process, the available information is scarce or null.

Molecular characterization of the canine mitochondrial DNA control region for forensic applications.

PubMed

Eichmann, Cordula; Parson, Walther

2007-09-01

The canine mitochondrial DNA (mtDNA) control region of 133 dogs living in the area around Innsbruck, Austria was sequenced. A total of 40 polymorphic sites were observed in the first hypervariable segment and 15 in the second, which resulted in the differentiation of 40 distinct haplotypes. We observed five nucleotide positions that were highly polymorphic within different haplogroups, and they represent good candidates for mtDNA screening. We found five point heteroplasmic positions; all located in HVS-I and a polythymine region in HVS-II, the latter often being associated with length heteroplasmy. In contrast to human mtDNA, the canine control region contains a hypervariable 10 nucleotide repeat region, which is located between the two hypervariable regions. In our population sample, we observed eight different repeat types, which we characterized by direct sequencing and fragment length analysis. The discrimination power of the canine mtDNA control region was 0.93, not taking the polymorphic repeat region into consideration.

Genomic relations among 31 species of Mammillaria haworth (Cactaceae) using random amplified polymorphic DNA.

PubMed

Mattagajasingh, Ilwola; Mukherjee, Arup Kumar; Das, Premananda

2006-01-01

Thirty-one species of Mammillaria were selected to study the molecular phylogeny using random amplified polymorphic DNA (RAPD) markers. High amount of mucilage (gelling polysaccharides) present in Mammillaria was a major obstacle in isolating good quality genomic DNA. The CTAB (cetyl trimethyl ammonium bromide) method was modified to obtain good quality genomic DNA. Twenty-two random decamer primers resulted in 621 bands, all of which were polymorphic. The similarity matrix value varied from 0.109 to 0.622 indicating wide variability among the studied species. The dendrogram obtained from the unweighted pair group method using arithmetic averages (UPGMA) analysis revealed that some of the species did not follow the conventional classification. The present work shows the usefulness of RAPD markers for genetic characterization to establish phylogenetic relations among Mammillaria species.

Screening and Characterization of RAPD Markers in Viscerotropic Leishmania Parasites

PubMed Central

Mkada–Driss, Imen; Talbi, Chiraz; Guerbouj, Souheila; Driss, Mehdi; Elamine, Elwaleed M.; Cupolillo, Elisa; Mukhtar, Moawia M.; Guizani, Ikram

2014-01-01

Visceral leishmaniasis (VL) is mainly due to the Leishmania donovani complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL Leishmania parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 L. donovani, L. archibaldi and L. infantum strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5′ end transversions, and presence of inter– and intra– taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between L. infantum and L. donovani. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents. PMID:25313833

Polymorphism at codon 36 of the p53 gene.

PubMed

Felix, C A; Brown, D L; Mitsudomi, T; Ikagaki, N; Wong, A; Wasserman, R; Womer, R B; Biegel, J A

1994-01-01

A polymorphism at codon 36 in exon 4 of the p53 gene was identified by single strand conformation polymorphism (SSCP) analysis and direct sequencing of genomic DNA PCR products. The polymorphic allele, present in the heterozygous state in genomic DNAs of four of 100 individuals (4%), changes the codon 36 CCG to CCA, eliminates a FinI restriction site and creates a BccI site. Including this polymorphism there are four known polymorphisms in the p53 coding sequence.

Mitochondrial DNA diagnosis for taeniasis and cysticercosis.

PubMed

Yamasaki, Hiroshi; Nakao, Minoru; Sako, Yasuhito; Nakaya, Kazuhiro; Sato, Marcello Otake; Ito, Akira

2006-01-01

Molecular diagnosis for taeniasis and cysticercosis in humans on the basis of mitochondrial DNA analysis was reviewed. Development and application of three different methods, including restriction fragment length polymorphism analysis, base excision sequence scanning thymine-base analysis and multiplex PCR, were described. Moreover, molecular diagnosis of cysticerci found in specimens submitted for histopathology and the molecular detection of taeniasis using copro-DNA were discussed.

The Effect of Polymorphisms in DNA Repair Genes and Carcinogen Metabolizers on Leukocyte Telomere Length: A Cohort of Healthy Spanish Smokers.

PubMed

Verde, Zoraida; Reinoso-Barbero, Luis; Chicharro, Luis; Resano, Pilar; Sánchez-Hernández, Ignacio; Rodríguez González-Moro, Jose Miguel; Bandrés, Fernando; Gómez-Gallego, Félix; Santiago, Catalina

2016-04-01

Smoking implies exposure to carcinogenic agents that causes DNA damage, which could be suspected to enhance telomere attrition. To protect and deal with DNA damage, cells possess mechanisms that repair and neutralize harmful substances. Polymorphisms altering DNA repair capacity or carcinogen metabolism may lead to synergistic effects with tobacco carcinogen-induced shorter telomere length independently of cancer interaction. The aim of this study was to explore the association between leukocyte telomere length (LTL) and several genetic polymorphisms in DNA repair genes and carcinogen metabolizers in a cohort of healthy smokers. We evaluated the effect of six genetic polymorphisms in cytochrome P1A1 (Ile462Val), XRCC1 (Arg399Gln), APEX1 (Asp148Glu), XRCC3 (Thr241Met), and XPD (Asp312Asn; Lys751Gln) on LTL in a cohort of 145 healthy smokers in addition to smoking habits. Logistic regression analysis showed an association between XRCC1 399Gln allele and shorter telomere length (OR = 5.03, 95% CI = 1.08% to 23.36%). There were not association between the rest of polymorphisms analyzed and LTL. Continuous exposure to tobacco could overwhelm the DNA repair machinery, making the effect of the polymorphisms that reduce repair capacity more pronounced. Analyzing the function of smoking-induced DNA-repair genes and LTL is an important goal in order to identify therapeutic targets to treat smoking-induced diseases. © The Author 2015. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes.

PubMed

Masuyama, Kotoka; Shojo, Hideki; Nakanishi, Hiroaki; Inokuchi, Shota; Adachi, Noboru

2017-01-01

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted "bidirectional analysis," which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples.

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes

PubMed Central

Masuyama, Kotoka; Shojo, Hideki; Nakanishi, Hiroaki; Inokuchi, Shota; Adachi, Noboru

2017-01-01

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted “bidirectional analysis,” which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples. PMID:28052096

Genetic mutation underlying orthostatic intolerance and diagnostic and therapeutic methods relating thereto

NASA Technical Reports Server (NTRS)

Blakely, Randy D. (Inventor); Robertson, David (Inventor)

2006-01-01

Isolated polynucleotide molecules and peptides encoded by these molecules are used in the analysis of human norepinephrine (NE) transporter variants, as well as in diagnostic and therapeutic applications, relating to a human NE transporter polymorphism. By analyzing genomic DNA or amplified genomic DNA, or amplified cDNA derived from mRNA, it is possible to type a human NE transporter with regard to the human NE transporter polymorphism, for example, in the context of diagnosing and treating NE transport impairments, and disorders associated with NE transport impairments, such as orthostatic intolerance.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)-A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes.

PubMed

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare . However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop plants with large and complex genomes.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

PubMed Central

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop plants with large and complex genomes. PMID:29250096

Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus.

PubMed Central

Lakshmikumaran, M S; D'Ambrosio, E; Laimins, L A; Lin, D T; Furano, A V

1985-01-01

The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this. Images PMID:3016521

DNA sequence analysis with droplet-based microfluidics

PubMed Central

Abate, Adam R.; Hung, Tony; Sperling, Ralph A.; Mary, Pascaline; Rotem, Assaf; Agresti, Jeremy J.; Weiner, Michael A.; Weitz, David A.

2014-01-01

Droplet-based microfluidic techniques can form and process micrometer scale droplets at thousands per second. Each droplet can house an individual biochemical reaction, allowing millions of reactions to be performed in minutes with small amounts of total reagent. This versatile approach has been used for engineering enzymes, quantifying concentrations of DNA in solution, and screening protein crystallization conditions. Here, we use it to read the sequences of DNA molecules with a FRET-based assay. Using probes of different sequences, we interrogate a target DNA molecule for polymorphisms. With a larger probe set, additional polymorphisms can be interrogated as well as targets of arbitrary sequence. PMID:24185402

Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

DOEpatents

McCutchen-Maloney, Sandra L.

2002-01-01

DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

Single Nucleotide Polymorphism Analysis of European Archaeological M. leprae DNA

PubMed Central

Watson, Claire L.; Lockwood, Diana N. J.

2009-01-01

Background Leprosy was common in Europe eight to twelve centuries ago but molecular confirmation of this has been lacking. We have extracted M. leprae ancient DNA (aDNA) from medieval bones and single nucleotide polymorphism (SNP) typed the DNA, this provides insight into the pattern of leprosy transmission in Europe and may assist in the understanding of M. leprae evolution. Methods and Findings Skeletons have been exhumed from 3 European countries (the United Kingdom, Denmark and Croatia) and are dated around the medieval period (476 to 1350 A.D.). we tested for the presence of 3 previously identified single nucleotide polymorphisms (SNPs) in 10 aDNA extractions. M. leprae aDNA was extracted from 6 of the 10 bone samples. SNP analysis of these 6 extractions were compared to previously analysed European SNP data using the same PCR assays and were found to be the same. Testing for the presence of SNPs in M. leprae DNA extracted from ancient bone samples is a novel approach to analysing European M. leprae DNA and the findings concur with the previously published data that European M. leprae strains fall in to one group (SNP group 3). Conclusions These findings support the suggestion that the M. leprae genome is extremely stable and show that archaeological M. leprae DNA can be analysed to gain detailed information about the genotypic make-up of European leprosy, which may assist in the understanding of leprosy transmission worldwide. PMID:19847306

MULTIPLE ENZYME RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS FOR HIGH RESOLUTION DISTINCTION OF PSEUDOMONAS (SENSU STRICTO) 16S RRNA GENES

EPA Science Inventory

Pseudomonas specific 16S rDNA PCR amplification and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of Alu I, Hinf I, Rsa I, and Tru 9I distinguished 150 published sequences and reference strains of authentic Pseudomonas...

Evaluation of Genetic Variations in Maize Seedlings Exposed to Electric Field Based on Protein and DNA Markers

PubMed Central

AL-Huqail, Asma A.; Abdelhaliem, Ekram

2015-01-01

The current study analyzed proteins and nuclear DNA of electric fields (ELF) exposed and nonexposed maize seedlings for different exposure periods using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isozymes, random amplified polymorphic DNA (RAPD), and comet assay, respectively. SDS-PAGE analysis revealed total of 46 polypeptides bands with different molecular weights ranging from 186.20 to 36.00 KDa. It generated distinctive polymorphism value of 84.62%. Leucine-aminopeptidase, peroxidase, and catalase isozymes showed the highest values of polymorphism (100%) based on zymograms number, relative front (R f), and optical intensity while esterase isozyme generated polymorphism value of 83.33%. Amino acids were analyzed using high-performance liquid chromatography, which revealed the presence of 17 amino acids of variable contents ranging from 22.65% to 28.09%. RAPD revealed that 78 amplified DNA products had highly polymorphism value (95.08%) based on band numbers, with variable sizes ranging from 120 to 992 base pairs and band intensity. Comet assay recorded the highest extent of nuclear DNA damage as percentage of tailed DNA (2.38%) and tail moment unit (5.36) at ELF exposure of maize nuclei for 5 days. The current study concluded that the longer ELF exposing periods had genotoxic stress on macromolecules of maize cells and biomarkers used should be augmented for reliable estimates of genotoxicity after exposure of economic plants to ELF stressors. PMID:26180815

Haplotype data for 23 Y-chromosome markers in a reference sample from Bosnia and Herzegovina.

PubMed

Kovačević, Lejla; Fatur-Cerić, Vera; Hadzic, Negra; Čakar, Jasmina; Primorac, Dragan; Marjanović, Damir

2013-06-01

To detect polymorphisms of 23 Y-chromosomal short tandem repeat (STR) loci, including 6 new loci, in a reference database of male population of Bosnia and Herzegovina, as well as to assess the importance of increasing the number of Y-STR loci utilized in forensic DNA analysis. The reference sample consisted of 100 healthy, unrelated men originating from Bosnia and Herzegovina. Sample collection using buccal swabs was performed in all geographical regions of Bosnia and Herzegovina in the period from 2010 to 2011. DNA samples were typed for 23 Y STR loci, including 6 new loci: DYS576, DYS481, DYS549, DYS533, DYS570, and DYS643, which are included in the new PowerPlex® Y 23 amplification kit. The absolute frequency of generated haplotypes was calculated and results showed that 98 samples had unique Y 23 haplotypes, and that only two samples shared the same haplotype. The most polymorphic locus was DYS418, with 14 detected alleles and the least polymorphic loci were DYS389I, DYS391, DYS437, and DYS393. This study showed that by increasing the number of highly polymorphic Y STR markers, to include those tested in our analysis, leads to a reduction of repeating haplotypes, which is very important in the application of forensic DNA analysis.

Genetic analysis of Apuleia leiocarpa as revealed by random amplified polymorphic DNA markers: prospects for population genetic studies.

PubMed

Lencina, K H; Konzen, E R; Tsai, S M; Bisognin, D A

2016-12-19

Apuleia leiocarpa (Vogel) J.F. MacBride is a hardwood species native to South America, which is at serious risk of extinction. Therefore, it is of prime importance to examine the genetic diversity of this species, information required for developing conservation, sustainable management, and breeding strategies. Although scarcely used in recent years, random amplified polymorphic DNA markers are useful resources for the analysis of genetic diversity and structure of tree species. This study represents the first genetic analysis based on DNA markers in A. leiocarpa that aimed to investigate the levels of polymorphism and to select markers for the precise characterization of its genetic structure. We adapted the original DNA extraction protocol based on cetyltrimethyl ammonium bromide, and describe a simple procedure that can be used to obtain high-quality samples from leaf tissues of this tree. Eighteen primers were selected, revealing 92 bands, from which 75 were polymorphic and 61 were sufficient to represent the overall genetic structure of the population without compromising the precision of the analysis. Some fragments were conserved among individuals, which can be sequenced and used to analyze nucleotide diversity parameters through a wider set of A. leiocarpa individuals and populations. The individuals were separated into 11 distinct groups with variable levels of genetic diversity, which is important for selecting desirable genotypes and for the development of a conservation and sustainable management program. Our results are of prime importance for further investigations concerning the genetic characterization of this important, but vulnerable species.

DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance.

PubMed

Zhao, Jie-hong; Zhang, Ji-shun; Wang, Yi; Wang, Ren-gang; Wu, Chun; Fan, Long-jiang; Ren, Xue-liang

2011-11-01

DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth, development, and polyploidization. However, there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics. We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco, Nicotiana tabacum, using a methylation-sensitive amplified polymorphism (MSAP) technique. The results showed that methylation existed at a high level among tobacco accessions, among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic. A cluster analysis revealed distinct patterns of geography-specific groups. In addition, three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored. This suggests that tobacco breeders should pay more attention to epigenetic traits.

Essentials of Conservation Biotechnology: A mini review

NASA Astrophysics Data System (ADS)

Merlyn Keziah, S.; Subathra Devi, C.

2017-11-01

Equilibrium of biodiversity is essential for the maintenance of the ecosystem as they are interdependent on each other. The decline in biodiversity is a global problem and an inevitable threat to the mankind. Major threats include unsustainable exploitation, habitat destruction, fragmentation, transformation, genetic pollution, invasive exotic species and degradation. This review covers the management strategies of biotechnology which include sin situ, ex situ conservation, computerized taxonomic analysis through construction of phylogenetic trees, calculating genetic distance, prioritizing the group for conservation, digital preservation of biodiversities within the coding and decoding keys, molecular approaches to asses biodiversity like polymerase chain reaction, real time, randomly amplified polymorphic DNA, restriction fragment length polymorphism, amplified fragment length polymorphism, single sequence repeats, DNA finger printing, single nucleotide polymorphism, cryopreservation and vitrification.

Alterations of leaf cell ultrastructures and AFLP DNA profiles in Earth-grown tomato plants propagated from long-term six years Mir-flown seeds

NASA Astrophysics Data System (ADS)

Liu, Min; Xue, Huai; Pan, Yi; Zhang, Chunhua; Lu, Jinying

Leaf cell ultrastructures and DNA variations in the firstand the second-generation of Earthgrown tomato (Lycopersicon esculentun Mill) plants that had been endured a long-term six years spaceflight in the Mir were compared to their ground-based control plants, under observations with a Transmission Electron Microscope and the Amplification Fragment Length Polymorphism (AFLP) analysis. For alterations in the morphological ultrastructures, one plant among the 11 first-generation plants generated from 30 Mir-flown seeds had a three-layered palisade cell structure, while other 10 first-generation plants and all ground-based controls had one-layered palisade cell structure in leaves. Starch grains were larger and in clusters, numbers of starch grains increased in the chloroplasts in the Mir-flown plants. Leaf cells became contracted and deformed, and cell shape patterns were different in the Mir-flown plants. For the leaf genomic DNA alterations, 34 DNA bands were polymorphic with a 1.32% polymorphism among 2582 DNA bands in the first-generation Mir-flown plants. Band types in the spaceflight treated plants were also different from those in the ground-based control. Of 11 survived first-generation plants, 7 spaceflight treated plants (Plant Nos. 1-6 and No. 9) had a same 7 polymorphic bands and a same 0.27%DNA mutation. The DNA mutation rate was greatest in Plants No.10 and No.7 (0.90% and 0.94%), less in Plant No.11 (0.31%) and least in Plant No.8 (0.20%). For the 38 send-generation plants propagated from the No. 5 Mir-flown seed, 6 DNA bands were polymorphic with a 0.23% polymorphism among 2564 amplified DNA bands. Among those 38 second-generation plants amplified by primer pair (E4: ACC, M8: CTT), one DNA band disappeared in 29 second-generation plants and in the original Mir-flown No. 5 plant, compared to the ground-base controls. Among the 38 second-generation plants generated from the Mir-flown No. 5 seed, the DNA band types of 29 second-generation plants were different from that of the ground-base controls and had a same 6 polymorphic bands and a same 0.23% DNA mutation. For the 49 second-generation plants derived from the Mir-flown No. 6 seed, 7 DNA bands were polymorphic with 0.27% polymorphism among 2564 amplified DNA bands. With only one exception among those 49 second-generation plants amplified by primer pair (E3: ACA, M3: CAG), one DNA band disappeared in 48 second-generation plants and in the original Mir-flown No. 6 plant, compared to the ground-based controls. Among the 49 second-generation plants generated from the Mir-flown No. 6 seed, the DNA band types of 48 second-generation plants were different from that of the ground-base controls and had a same 7 polymorphic bands and a same 0.27% DNA mutation. Our results indicated that leaf cell ultrastructures had been altered and heredity variations had been induced by seeds being exposed to a long-term outer-space environment. Further research is needed to elucidate the dynamics and mechanisms resulting in such variations. Plant biology studies in the space environment may open potential approaches to induce mutations and to screen new plant varieties by ground-based selections among spaceflight treated seeds or seedlings.

Analysis of ELA-DQB exon 2 polymorphism in Argentine Creole horses by PCR-RFLP and PCR-SSCP.

PubMed

Villegas-Castagnasso, E E; Díaz, S; Giovambattista, G; Dulout, F N; Peral-García, P

2003-08-01

The second exon of equine leucocyte antigen (ELA)-DQB genes was amplified from genomic DNA of 32 Argentine Creole horses by PCR. Amplified DNA was analysed by PCR-restriction fragment length polymorphism (RFLP) and PCR-single-strand conformation polymorphism (SSCP). The PCR-RFLP analysis revealed two HaeIII patterns, four RsaI patterns, five MspI patterns and two HinfI patterns. EcoRI showed no variation in the analysed sample. Additional patterns that did not account for known exon 2 DNA sequences were observed, suggesting the existence of novel ELA-DQB alleles. PCR-SSCP analysis exhibited seven different band patterns, and the number of bands per animal ranged from four to nine. Both methods indicated that at least two DQB genes are present. The presence of more than two alleles in each animal showed that the primers employed in this work are not specific for a unique DQB locus. The improvement of this PCR-RFLP method should provide a simple and rapid technique for an accurate definition of ELA-DQB typing in horses.

Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive amplification polymorphism (MSAP).

PubMed

Peraza-Echeverria, S; Herrera-Valencia, V A.; Kay, A -J.

2001-07-01

The extent of DNA methylation polymorphisms was evaluated in micropropagated banana (Musa AAA cv. 'Grand Naine') derived from either the vegetative apex of the sucker or the floral apex of the male inflorescence using the methylation-sensitive amplification polymorphism (MSAP) technique. In all, 465 fragments, each representing a recognition site cleaved by either or both of the isoschizomers were amplified using eight combinations of primers. A total of 107 sites (23%) were found to be methylated at cytosine in the genome of micropropagated banana plants. In plants micropropagated from the male inflorescence explant 14 (3%) DNA methylation events were polymorphic, while plants micropropagated from the sucker explant produced 8 (1.7%) polymorphisms. No DNA methylation polymorphisms were detected in conventionally propagated banana plants. These results demonstrated the usefulness of MSAP to detect DNA methylation events in micropropagated banana plants and indicate that DNA methylation polymorphisms are associated with micropropagation.

Identification of Medically Important Yeasts Using PCR-Based Detection of DNA Sequence Polymorphisms in the Internal Transcribed Spacer 2 Region of the rRNA Genes

PubMed Central

Chen, Y. C.; Eisner, J. D.; Kattar, M. M.; Rassoulian-Barrett, S. L.; LaFe, K.; Yarfitz, S. L.; Limaye, A. P.; Cookson, B. T.

2000-01-01

Identification of medically relevant yeasts can be time-consuming and inaccurate with current methods. We evaluated PCR-based detection of sequence polymorphisms in the internal transcribed spacer 2 (ITS2) region of the rRNA genes as a means of fungal identification. Clinical isolates (401), reference strains (6), and type strains (27), representing 34 species of yeasts were examined. The length of PCR-amplified ITS2 region DNA was determined with single-base precision in less than 30 min by using automated capillary electrophoresis. Unique, species-specific PCR products ranging from 237 to 429 bp were obtained from 92% of the clinical isolates. The remaining 8%, divided into groups with ITS2 regions which differed by ≤2 bp in mean length, all contained species-specific DNA sequences easily distinguishable by restriction enzyme analysis. These data, and the specificity of length polymorphisms for identifying yeasts, were confirmed by DNA sequence analysis of the ITS2 region from 93 isolates. Phenotypic and ITS2-based identification was concordant for 427 of 434 yeast isolates examined using sequence identity of ≥99%. Seven clinical isolates contained ITS2 sequences that did not agree with their phenotypic identification, and ITS2-based phylogenetic analyses indicate the possibility of new or clinically unusual species in the Rhodotorula and Candida genera. This work establishes an initial database, validated with over 400 clinical isolates, of ITS2 length and sequence polymorphisms for 34 species of yeasts. We conclude that size and restriction analysis of PCR-amplified ITS2 region DNA is a rapid and reliable method to identify clinically significant yeasts, including potentially new or emerging pathogenic species. PMID:10834993

Assessment of changes in DNA methylation by methylation-sensitive amplification polymorphism in Jatropha curcas L. subjected to salinity stress.

PubMed

Mastan, Shaik G; Rathore, Mangal S; Bhatt, Vacha D; Yadav, P; Chikara, J

2012-10-15

The present study assesses the changes in DNA methylation in leaf and root tissues of Jatropha curcas L., induced by salinity stress using methylation sensitive amplification polymorphism (MSAP) markers. Seedlings of 21 days (d) grown under controlled conditions were subjected to 0–100 mM salinity treatment for 24 h (1 d). Immediate changes in DNA methylation and polymorphism in methylated DNA in whole genome of both leaves and roots were assessed using 10 selective combinations of MSAP primers. In root and leaves 70.06% and 57.89% methylation was observed respectively. Similarly 67.22% and 71.21% polymorphism was observed in methylated DNA from root and leaf tissues respectively. Compared with control, the percentage of methylation and methylation polymorphism in roots of plants under different dosages of salinity was found in the order of 50 mM < 25 mM = 100 mM < 75 mM and 75 mM < 25 mM < 50 mM < 100 mM respectively. Similarly percentage of methylation and methylation polymorphism in leaves of plants treated with different levels of salinity was found in order of 75 mM < 25 mM < 50 mM < 100 mM and 50 mM < 25 mM < 100 mM < 75 mM respectively. The MSAP analysis showed that under salt stress homologous nucleotide sequences in genome from control and salt treated plants of J. curcas showed different patterns of methylation; which suggest that these fragments probably play an important role to induce immediate adaptive responses in Jatropha under salinity stress.

A preliminary report on the genetic variation in pointed gourd (Trichosanthes dioica Roxb.) as assessed by random amplified polymorphic DNA.

PubMed

Adhikari, S; Biswas, A; Bandyopadhyay, T K; Ghosh, P D

2014-06-01

Pointed gourd (Trichosanthes dioica Roxb.) is an economically important cucurbit and is extensively propagated through vegetative means, viz vine and root cuttings. As the accessions are poorly characterized it is important at the beginning of a breeding programme to discriminate among available genotypes to establish the level of genetic diversity. The genetic diversity of 10 pointed gourd races, referred to as accessions was evaluated. DNA profiling was generated using 10 sequence independent RAPD markers. A total of 58 scorable loci were observed out of which 18 (31.03%) loci were considered polymorphic. Genetic diversity parameters [average and effective number of alleles, Shannon's index, percent polymorphism, Nei's gene diversity, polymorphic information content (PIC)] for RAPD along with UPGMA clustering based on Jaccard's coefficient were estimated. The UPGMA dendogram constructed based on RAPD analysis in 10 pointed gourd accessions were found to be grouped in a single cluster and may represent members of one heterotic group. RAPD analysis showed promise as an effective tool in estimating genetic polymorphism in different accessions of pointed gourd.

SNP discovery through de novo deep sequencing using the next generation of DNA sequencers

USDA-ARS?s Scientific Manuscript database

The production of high volumes of DNA sequence data using new technologies has permitted more efficient identification of single nucleotide polymorphisms in vertebrate genomes. This chapter presented practical methodology for production and analysis of DNA sequence data for SNP discovery....

Affordable Hands-On DNA Sequencing and Genotyping: An Exercise for Teaching DNA Analysis to Undergraduates

ERIC Educational Resources Information Center

Shah, Kushani; Thomas, Shelby; Stein, Arnold

2013-01-01

In this report, we describe a 5-week laboratory exercise for undergraduate biology and biochemistry students in which students learn to sequence DNA and to genotype their DNA for selected single nucleotide polymorphisms (SNPs). Students use miniaturized DNA sequencing gels that require approximately 8 min to run. The students perform G, A, T, C…

Comprehensive Survey of Genetic Diversity in Chloroplast Genomes and 45S nrDNAs within Panax ginseng Species

PubMed Central

Kim, Kyunghee; Lee, Sang-Choon; Lee, Junki; Lee, Hyun Oh; Joh, Ho Jun; Kim, Nam-Hoon; Park, Hyun-Seung; Yang, Tae-Jin

2015-01-01

We report complete sequences of chloroplast (cp) genome and 45S nuclear ribosomal DNA (45S nrDNA) for 11 Panax ginseng cultivars. We have obtained complete sequences of cp and 45S nrDNA, the representative barcoding target sequences for cytoplasm and nuclear genome, respectively, based on low coverage NGS sequence of each cultivar. The cp genomes sizes ranged from 156,241 to 156,425 bp and the major size variation was derived from differences in copy number of tandem repeats in the ycf1 gene and in the intergenic regions of rps16-trnUUG and rpl32-trnUAG. The complete 45S nrDNA unit sequences were 11,091 bp, representing a consensus single transcriptional unit with an intergenic spacer region. Comparative analysis of these sequences as well as those previously reported for three Chinese accessions identified very rare but unique polymorphism in the cp genome within P. ginseng cultivars. There were 12 intra-species polymorphisms (six SNPs and six InDels) among 14 cultivars. We also identified five SNPs from 45S nrDNA of 11 Korean ginseng cultivars. From the 17 unique informative polymorphic sites, we developed six reliable markers for analysis of ginseng diversity and cultivar authentication. PMID:26061692

DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance*

PubMed Central

Zhao, Jie-hong; Zhang, Ji-shun; Wang, Yi; Wang, Ren-gang; Wu, Chun; Fan, Long-jiang; Ren, Xue-liang

2011-01-01

DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth, development, and polyploidization. However, there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics. We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco, Nicotiana tabacum, using a methylation-sensitive amplified polymorphism (MSAP) technique. The results showed that methylation existed at a high level among tobacco accessions, among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic. A cluster analysis revealed distinct patterns of geography-specific groups. In addition, three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored. This suggests that tobacco breeders should pay more attention to epigenetic traits. PMID:22042659

APPLICATION OF DNA MICROARRAYS TO REPRODUCTIVE TOXICOLOGY AND THE DEVELOPMENT OF A TESTIS ARRAY

EPA Science Inventory

With the advent of sequence information for entire mammalian genomes, it is now possible to analyze gene expression and gene polymorphisms on a genomic scale. The primary tool for analysis of gene expression is the DNA microarray. We have used commercially available cDNA micro...

Checking of individuality by DNA profiling.

PubMed

Brdicka, R; Nürnberg, P

1993-08-25

A review of methods of DNA analysis used in forensic medicine for identification, paternity testing, etc. is provided. Among other techniques, DNA fingerprinting using different probes and polymerase chain reaction-based techniques such as amplified sequence polymorphisms and minisatellite variant repeat mapping are thoroughly described and both theoretical and practical aspects are discussed.

Comparative analysis of DNA methylation polymorphism in drought sensitive (HPKC2) and tolerant (HPK4) genotypes of horse Gram (Macrotyloma uniflorum).

PubMed

Bhardwaj, Jyoti; Mahajan, Monika; Yadav, Sudesh Kumar

2013-08-01

DNA methylation is known as an epigenetic modification that affects gene expression in plants. Variation in CpG methylation behavior was studied in two natural horse gram (Macrotyloma uniflorum [Lam.] Verdc.) genotypes, HPKC2 (drought-sensitive) and HPK4 (drought-tolerant). The methylation pattern in both genotypes was studied through methylation-sensitive amplified polymorphism. The results revealed that methylation was higher in HPKC2 (10.1%) than in HPK4 (8.6%). Sequencing demonstrated sequence homology with the DRE binding factor (cbf1), the POZ/BTB protein, and the Ty1-copia retrotransposon among some of the polymorphic fragments showing alteration in methylation behavior. Differences in DNA methylation patterns could explain the differential drought tolerance and the epigenetic signature of these two horse gram genotypes.

Randomly amplified polymorphic-DNA analysis for detecting genotoxic effects of Boron on maize (Zea mays L.).

PubMed

Sakcali, M Serdal; Kekec, Guzin; Uzonur, Irem; Alpsoy, Lokman; Tombuloglu, Huseyin

2015-08-01

This study was carried out to investigate the genotoxic effect of boron (B) on maize using randomly amplified polymorphic DNA (RAPD) method. Experimental design was conducted under 0, 5, 10, 25, 50, 100, 125, and 150 ppm B exposures, and physiological changes have revealed a sharp decrease in root growth rates from 28% to 85%, starting from 25 ppm to 150 ppm, respectively. RAPD-polymerase chain reaction (PCR) analysis shows that DNA alterations are clearly observed from beginning to 100 ppm. B-induced inhibition in root growth had a positive correlation with DNA alterations. Total soluble protein, root and stem lengths, and B content analysis in root and leaves encourage these results as a consequence. These preliminary findings reveal that B causes chromosomal aberration and genotoxic effects on maize. Meanwhile, usage of RAPD-PCR technique is a suitable biomarker to detect genotoxic effect of B on maize and other crops for the future. © The Author(s) 2013.

DNA polymorphism identity determination using flow cytometry

DOEpatents

Nolan, John P.; White, P. Scott; Cai, Hong

2001-01-01

DNA polymorphism identity determination using flow cytometry. Primers designed to be immobilized on microspheres are allowed to anneal to the DNA strand under investigation, and are extended by either DNA polymerase using fluorescent dideoxynucleotides or ligated by DNA ligase to fluorescent reporter oligonucleotides. The fluorescence of either the dideoxynucleotide or the reporter oligonucleotide attached to the immobilized primer is measured by flow cytometry, thereby identifying the nucleotide polymorphism on the DNA strand.

Haplotype data for 23 Y-chromosome markers in a reference sample from Bosnia and Herzegovina

PubMed Central

Kovačević, Lejla; Fatur-Cerić, Vera; Hadžić, Negra; Čakar, Jasmina; Primorac, Dragan; Marjanović, Damir

2013-01-01

Aim To detect polymorphisms of 23 Y-chromosomal short tandem repeat (STR) loci, including 6 new loci, in a reference database of male population of Bosnia and Herzegovina, as well as to assess the importance of increasing the number of Y-STR loci utilized in forensic DNA analysis. Methods The reference sample consisted of 100 healthy, unrelated men originating from Bosnia and Herzegovina. Sample collection using buccal swabs was performed in all geographical regions of Bosnia and Herzegovina in the period from 2010 to 2011. DNA samples were typed for 23 Y STR loci, including 6 new loci: DYS576, DYS481, DYS549, DYS533, DYS570, and DYS643, which are included in the new PowerPlex® Y 23 amplification kit. Results The absolute frequency of generated haplotypes was calculated and results showed that 98 samples had unique Y 23 haplotypes, and that only two samples shared the same haplotype. The most polymorphic locus was DYS418, with 14 detected alleles and the least polymorphic loci were DYS389I, DYS391, DYS437, and DYS393. Conclusion This study showed that by increasing the number of highly polymorphic Y STR markers, to include those tested in our analysis, leads to a reduction of repeating haplotypes, which is very important in the application of forensic DNA analysis. PMID:23771760

Cytogenetic and molecular aspects of absolute teratozoospermia: comparison between polymorphic and monomorphic forms.

PubMed

Brahem, Sonia; Elghezal, Hatem; Ghédir, Houda; Landolsi, Hanène; Amara, Abdelbacett; Ibala, Samira; Gribaa, Moez; Saad, Ali; Mehdi, Meriem

2011-12-01

To compare the results of cytogenetic and molecular analysis between absolute polymorphic and monomorphic teratozoospermia. The semen samples from patients with polymorphic teratozoospermia (n = 20), globozoospermia (n = 8), or macrocephalic sperm head syndrome (n = 12), and healthy fertile men (n = 20) were analyzed according to the World Health Organization criteria. The constitutional blood karyotype of the patients was performed on cultured lymphocytes, according to standard techniques. Microdeletion analysis of the Y chromosomes used a sequence tagged site-polymerase chain reaction technique. Triple-color fluorescent in situ hybridization for chromosomes X, Y, and 18 were used to analyze the meiotic segregation. DNA fragmentation was detected using the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling assay. Whatever the type of teratozoospermia, a normal karyotype and an absence of Y chromosome microdeletion were shown for all patients. A significant increase in the sperm aneuploidy rate and DNA fragmentation were shown, regardless of the type of teratozoospermia. Spermatozoa of the patients with globozoospermia carry an abnormal chromosomal constitution and DNA damage rate with the same frequency as that found in the sperm of patients with absolute polymorphic teratozoospermia. However, a greater sperm aneuploidy rate and DNA fragmentation were found in patients whose teratozoospermia was mainly characterized by increased rates of spermatozoa with macrocephalic head and multiple flagella. Our data have demonstrated that DNA fragmentation and sperm aneuploidy are critical tests in teratozoospermic men, because the results could negatively affect the intracytoplasmic sperm injection outcomes and might play an important role in the counseling of couples considering intracytoplasmic sperm injection. Copyright © 2011 Elsevier Inc. All rights reserved.

DNA polymorphism in recombining and non-recombing mating-type-specific loci of the smut fungus Microbotryum

PubMed Central

Votintseva, A A; Filatov, D A

2011-01-01

The population-genetic processes leading to the genetic degeneration of non-recombining regions have mainly been studied in animal and plant sex chromosomes. Here, we report population genetic analysis of the processes in the non-recombining mating-type-specific regions of the smut fungus Microbotryum violaceum. M. violaceum has A1 and A2 mating types, determined by mating-type-specific ‘sex chromosomes' that contain 1–2 Mb long non-recombining regions. If genetic degeneration were occurring, then one would expect reduced DNA polymorphism in the non-recombining regions of this fungus. The analysis of DNA diversity among 19 M. violaceum strains, collected across Europe from Silene latifolia flowers, revealed that (i) DNA polymorphism is relatively low in all 20 studied loci (π∼0.15%), (ii) it is not significantly different between the two mating-type-specific chromosomes nor between the non-recombining and recombining regions, (iii) there is substantial population structure in M. violaceum populations, which resembles that of its host species, S. latifolia, and (iv) there is significant linkage disequilibrium, suggesting that widespread selfing in this species results in a reduction of the effective recombination rate across the genome. We hypothesise that selfing-related reduction of recombination across the M. violaceum genome negates the difference in the level of DNA polymorphism between the recombining and non-recombining regions, and may possibly lead to similar levels of genetic degeneration in the mating-type-specific regions of the non-recombining ‘sex chromosomes' and elsewhere in the genome. PMID:21081967

XRCC3 Thr241Met Polymorphism is not Associated with Lung Cancer Risk in a Romanian Population.

PubMed

Catana, Andreea; Pop, Monica; Marginean, Dragos Horea; Blaga, Ioana Cristina; Porojan, Mihai Dumitru; Popp, Radu Anghel; Pop, Ioan Victor

2016-01-01

Deoxyribonucleic Acid (DNA) repair mechanisms play a critical role in protecting the cellular genome against carcinogens. X-ray cross-complementing gene 3 (XRCC3) is involved in DNA repair and therefore certain genetic polymorphisms that occur in DNA repair genes may affect the ability to repair DNA defects and may represent a risk factor in carcinogenesis. The purpose of our study was to investigate the association between XRCC3 gene substitution of Threonine with Methionine in codon 241 of XRCC3 gene (Thr241Met) polymorphism and the risk of lung cancer, in a Romanian population. We recruited 93 healthy controls and 85 patients with lung cancer, all smokers. Thr241Met, XRCC3 gene genotyping was determined by multiplex Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). Statistical analysis (OR, recessive model), did not revealed an increased risk for lung cancer, for the variant 241Met allele and Thr241Met genotypes (p=0.138, OR=0.634, CI=0.348-1.157; p=0.023, OR=0.257, CI=0.085-6.824). Also, there were no positive statistical associations between Thr241Met polymorphism of XRCC3 gene, gender, tobacco and various histopathological tumor type of lung cancer. In conclusion, the results of the study suggest that the XRCC3 gene Thr241Met polymorphism is not associated with an increased risk for the development of lung cancer in this Romanian group.

Single-strand conformation polymorphism analysis of ribosomal DNA for detection of Phytophthora ramorum directly from plant tissues

Treesearch

Ping Kong; Patricia A. Richardson; Chuanxue Hong; Thomas L. Kubisiak

2006-01-01

At the first Sudden Oak Death Science Symposium, we reported on the use of a single strand conformation polymorphism (SSCP) analysis for rapid identification of Phytophthora ramorum in culture. We have since assessed and improved the fingerprinting technique for detecting this pathogen directly from plant tissues. The improved SSCP protocol uses a...

Molecular Analysis of Date Palm Genetic Diversity Using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSRs).

PubMed

El Sharabasy, Sherif F; Soliman, Khaled A

2017-01-01

The date palm is an ancient domesticated plant with great diversity and has been cultivated in the Middle East and North Africa for at last 5000 years. Date palm cultivars are classified based on the fruit moisture content, as dry, semidry, and soft dates. There are a number of biochemical and molecular techniques available for characterization of the date palm variation. This chapter focuses on the DNA-based markers random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) techniques, in addition to biochemical markers based on isozyme analysis. These techniques coupled with appropriate statistical tools proved useful for determining phylogenetic relationships among date palm cultivars and provide information resources for date palm gene banks.

Genetic Diversity of Pinus Roxburghii Sarg. Collected from Different Himalayan Regions of India Assessed by Random Amplified Polymorphic DNA Analysis

PubMed Central

Sinha, Dwaipayan; Singh, Jyotsna; Tandon, P. K.; Kakkar, Poonam

2013-01-01

Present study was aimed at molecular genetic fingerprint profile of 15 genotypes of three populations of Pinus roxburghii Sarg. from Himalayan regions of India using random amplified polymorphic DNA (RAPD) based markers. Needles of Pinus roxburghii Sarg. were collected from Dharamshala, Himachal Pradesh (HP), Nainital, Uttarakhand (UK) and Darjeeling, West Bengal (WB) regions of India. The samples were subjected to DNA extraction and RAPD analysis using oligonucleotide purification cartridge (OPC) primers. Out of 15 primers tested, nine primers gave scorable bands. Altogether 48 bands were obtained, out of which 43 were found to be polymorphic. Number of amplified fragments with RAPD primers ranged from four to eight with the size of amplicon ranging from 500 to 7,000bp. Investigation of natural diversity at intraspecies level was performed with 15 genotypes. Forty-eight amplification products were scored by RAPD and showed 89.58% polymorphism with a mean intrapopulation genetic diversity (Hpop) of 0.2754. A significant inter- and intrapopulation diversity was observed, with the percentage of polymorphic loci (Pp) ranging from 50.09 to 70.83%, Shannon's information index (I) from 0.3262 to 0.4689 and Nei's gene diversity (h) from 0.2032 to 0.3335 with mean Nei's gene diversity 0.377 and the overall estimate of gene flow being (Nm) 1.3555. Unweighted pair-group method with arithmetic average (UPGMA) analysis based Dendrogram showed single cluster. The variation amongst the samples of the three ecological regions can be attributed to varied climatic conditions and may help in conservation/future cultivation of these species. PMID:24403729

Intragenomic polymorphisms among high-copy loci: a genus-wide study of nuclear ribosomal DNA in Asclepias (Apocynaceae).

PubMed

Weitemier, Kevin; Straub, Shannon C K; Fishbein, Mark; Liston, Aaron

2015-01-01

Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA) across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual's consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the "noncoding" ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming).

Structure, inheritance, and expression of hybrid poplar (Populus trichocarpa x Populus deltoides) phenylalanine ammonia-lyase genes.

PubMed Central

Subramaniam, R; Reinold, S; Molitor, E K; Douglas, C J

1993-01-01

A heterologous probe encoding phenylalanine ammonia-lyase (PAL) was used to identify PAL clones in cDNA libraries made with RNA from young leaf tissue of two Populus deltoides x P. trichocarpa F1 hybrid clones. Sequence analysis of a 2.4-kb cDNA confirmed its identity as a full-length PAl clone. The predicted amino acid sequence is conserved in comparison with that of PAL genes from several other plants. Southern blot analysis of popular genomic DNA from parental and hybrid individuals, restriction site polymorphism in PAL cDNA clones, and sequence heterogeneity in the 3' ends of several cDNA clones suggested that PAL is encoded by at least two genes that can be distinguished by HindIII restriction site polymorphisms. Clones containing each type of PAL gene were isolated from a poplar genomic library. Analysis of the segregation of PAL-specific HindIII restriction fragment-length polymorphisms demonstrated the existence of two independently segregating PAL loci, one of which was mapped to a linkage group of the poplar genetic map. Developmentally regulated PAL expression in poplar was analyzed using RNA blots. Highest expression was observed in young stems, apical buds, and young leaves. Expression was lower in older stems and undetectable in mature leaves. Cellular localization of PAL expression by in situ hybridization showed very high levels of expression in subepidermal cells of leaves early during leaf development. In stems and petioles, expression was associated with subepidermal cells and vascular tissues. PMID:8108506

Genetic relatedness between oral and intestinal isolates of Porphyromonas endodontalis by analysis of random amplified polymorphic DNA.

PubMed

Gonçalves, R B; Väisänen, M L; Van Steenbergen, T J; Sundqvist, G; Mouton, C

1999-01-01

Genomic fingerprints from the DNA of 27 strains of Porphyromonas endodontalis from diverse clinical and geographic origins were generated as random amplified polymorphic DNA (RAPD) using the technique of PCR amplification with a single primer of arbitrary sequence. Cluster analysis of the combined RAPD data obtained with three selected 9- or 10-mer-long primers identified 25 distinct RAPD types which clustered as three main groups identifying three genogroups. Genogroups I and II included exclusively P. endodontalis isolates of oral origin, while 7/9 human intestinal strains of genogroup III which linked at a similarity level of 52% constituted the most homogeneous group in our study. Genotypic diversity within P. endodontalis, as shown by RAPD analysis, suggests that the taxon is composed of two oral genogroups and one intestinal genogroup. This hypothesis remains to be confirmed.

Introducing Human Population Biology through an Easy Laboratory Exercise on Mitochondrial DNA

ERIC Educational Resources Information Center

Pardinas, Antonio F.; Dopico, Eduardo; Roca, Agustin; Garcia-Vazquez, Eva; Lopez, Belen

2010-01-01

This article describes an easy and cheap laboratory exercise for students to discover their own mitochondrial haplogroup. Students use buccal swabs to obtain mucosa cells as noninvasive tissue samples, extract DNA, and with a simple polymerase chain reaction-restriction fragment length polymorphism analysis they can obtain DNA fragments of…

Development of an efficient fungal DNA extraction method to be used in random amplified polymorphic DNA-PCR analysis to differentiate cyclopiazonic acid mold producers.

PubMed

Sánchez, Beatriz; Rodríguez, Mar; Casado, Eva M; Martín, Alberto; Córdoba, Juan J

2008-12-01

A variety of previously established mechanical and chemical treatments to achieve fungal cell lysis combined with a semiautomatic system operated by a vacuum pump were tested to obtain DNA extract to be directly used in randomly amplified polymorphic DNA (RAPD)-PCR to differentiate cyclopiazonic acid-producing and -nonproducing mold strains. A DNA extraction method that includes digestion with proteinase K and lyticase prior to using a mortar and pestle grinding and a semiautomatic vacuum system yielded DNA of high quality in all the fungal strains and species tested, at concentrations ranging from 17 to 89 ng/microl in 150 microl of the final DNA extract. Two microliters of DNA extracted with this method was directly used for RAPD-PCR using primer (GACA)4. Reproducible RAPD fingerprints showing high differences between producer and nonproducer strains were observed. These differences in the RAPD patterns did not differentiate all the strains tested in clusters by cyclopiazonic acid production but may be very useful to distinguish cyclopiazonic acid producer strains from nonproducer strains by a simple RAPD analysis. Thus, the DNA extracts obtained could be used directly without previous purification and quantification for RAPD analysis to differentiate cyclopiazonic acid producer from nonproducer mold strains. This combined analysis could be adaptable to other toxigenic fungal species to enable differentiation of toxigenic and non-toxigenic molds, a procedure of great interest in food safety.

Geographical distribution of genetic polymorphism of the pathogen Histoplasma capsulatum isolated from infected bats, captured in a central zone of Mexico.

PubMed

Taylor, Maria Lucia; Chávez-Tapia, Catalina B; Rojas-Martínez, Alberto; del Rocio Reyes-Montes, Maria; del Valle, Mirian Bobadilla; Zúñiga, Gerardo

2005-09-01

Fourteen Histoplasma capsulatum isolates recovered from infected bats captured in Mexican caves and two human H. capsulatum reference strains were analyzed using random amplification of polymorphic DNA PCR-based and partial DNA sequences of four genes. Cluster analysis of random amplification of polymorphic DNA-patterns revealed differences for two H. capsulatum isolates of one migratory bat Tadarida brasiliensis. Three groups were identified by distance and maximum-parsimony analyses of arf, H-anti, ole, and tub1 H. capsulatum genes. Group I included most isolates from infected bats and one clinical strain from central Mexico; group II included the two isolates from T. brasiliensis; the human G-217B reference strain from USA formed an independent group III. Isolates from group II showed diversity in relation to groups I and III, suggesting a different H. capsulatum population.

Effects of As2O3 on DNA methylation, genomic instability, and LTR retrotransposon polymorphism in Zea mays.

PubMed

Erturk, Filiz Aygun; Aydin, Murat; Sigmaz, Burcu; Taspinar, M Sinan; Arslan, Esra; Agar, Guleray; Yagci, Semra

2015-12-01

Arsenic is a well-known toxic substance on the living organisms. However, limited efforts have been made to study its DNA methylation, genomic instability, and long terminal repeat (LTR) retrotransposon polymorphism causing properties in different crops. In the present study, effects of As2O3 (arsenic trioxide) on LTR retrotransposon polymorphism and DNA methylation as well as DNA damage in Zea mays seedlings were investigated. The results showed that all of arsenic doses caused a decreasing genomic template stability (GTS) and an increasing Random Amplified Polymorphic DNAs (RAPDs) profile changes (DNA damage). In addition, increasing DNA methylation and LTR retrotransposon polymorphism characterized a model to explain the epigenetically changes in the gene expression were also found. The results of this experiment have clearly shown that arsenic has epigenetic effect as well as its genotoxic effect. Especially, the increasing of polymorphism of some LTR retrotransposon under arsenic stress may be a part of the defense system against the stress.

Intragenomic polymorphisms among high-copy loci: a genus-wide study of nuclear ribosomal DNA in Asclepias (Apocynaceae)

PubMed Central

Straub, Shannon C.K.; Fishbein, Mark; Liston, Aaron

2015-01-01

Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA) across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual’s consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the “noncoding” ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming). PMID:25653903

Genetic structure of Pilosocereus gounellei (Cactaceae) as revealed by AFLP marker to guide proposals for improvement and restoration of degraded areas in Caatinga biome.

PubMed

Monteiro, E R; Strioto, D K; Meirelles, A C S; Mangolin, C A; Machado, M F P S

2015-12-15

Amplified fragment length polymorphism (AFLP) analysis was used to evaluate DNA polymorphism in Pilosocereus gounellei with the aim of differentiating samples grown in different Brazilian semiarid regions. Seven primer pairs were used to amplify 703 AFLP markers, of which 700 (99.21%) markers were polymorphic. The percentage of polymorphic markers ranged from 95.3% for the primer combination E-AAG/M-CTT to 100% for E-ACC/M-CAT, E-ACC/M-CAA, E-AGC/M-CAG, E-ACT/M-CTA, and E-AGG/M-CTG. The largest number of informative markers (126) was detected using the primer combination E-AAC/M-CTA. Polymorphism of the amplified DNA fragments ranged from 72.55% (in sample from Piauí State) to 82.79% (in samples from Rio Grande Norte State), with an average of 75.39%. Despite the high genetic diversity of AFLP markers in xiquexique, analysis using the STRUCTURE software identified relatively homogeneous clusters of xiquexique from the same location, indicating a differentiation at the molecular level, among the plant samples from different regions of the Caatinga biome. The AFLP methodology identified genetically homogeneous and contrasting plants, as well as plants from different regions with common DNA markers. Seeds from such plants can be used for further propagation of plants for establishment of biodiversity conservation units and restoration of degraded areas of the Caatinga biome.

Genetic discovery in Xylella fastidiosa through sequence analysis of selected randomly amplified polymorphic DNAs.

PubMed

Chen, Jianchi; Civerolo, Edwin L; Jarret, Robert L; Van Sluys, Marie-Anne; de Oliveira, Mariana C

2005-02-01

Xylella fastidiosa causes many important plant diseases including Pierce's disease (PD) in grape and almond leaf scorch disease (ALSD). DNA-based methodologies, such as randomly amplified polymorphic DNA (RAPD) analysis, have been playing key roles in genetic information collection of the bacterium. This study further analyzed the nucleotide sequences of selected RAPDs from X. fastidiosa strains in conjunction with the available genome sequence databases and unveiled several previously unknown novel genetic traits. These include a sequence highly similar to those in the phage family of Podoviridae. Genome comparisons among X. fastidiosa strains suggested that the "phage" is currently active. Two other RAPDs were also related to horizontal gene transfer: one was part of a broadly distributed cryptic plasmid and the other was associated with conjugal transfer. One RAPD inferred a genomic rearrangement event among X. fastidiosa PD strains and another identified a single nucleotide polymorphism of evolutionary value.

DNApod: DNA polymorphism annotation database from next-generation sequence read archives.

PubMed

Mochizuki, Takako; Tanizawa, Yasuhiro; Fujisawa, Takatomo; Ohta, Tazro; Nikoh, Naruo; Shimizu, Tokurou; Toyoda, Atsushi; Fujiyama, Asao; Kurata, Nori; Nagasaki, Hideki; Kaminuma, Eli; Nakamura, Yasukazu

2017-01-01

With the rapid advances in next-generation sequencing (NGS), datasets for DNA polymorphisms among various species and strains have been produced, stored, and distributed. However, reliability varies among these datasets because the experimental and analytical conditions used differ among assays. Furthermore, such datasets have been frequently distributed from the websites of individual sequencing projects. It is desirable to integrate DNA polymorphism data into one database featuring uniform quality control that is distributed from a single platform at a single place. DNA polymorphism annotation database (DNApod; http://tga.nig.ac.jp/dnapod/) is an integrated database that stores genome-wide DNA polymorphism datasets acquired under uniform analytical conditions, and this includes uniformity in the quality of the raw data, the reference genome version, and evaluation algorithms. DNApod genotypic data are re-analyzed whole-genome shotgun datasets extracted from sequence read archives, and DNApod distributes genome-wide DNA polymorphism datasets and known-gene annotations for each DNA polymorphism. This new database was developed for storing genome-wide DNA polymorphism datasets of plants, with crops being the first priority. Here, we describe our analyzed data for 679, 404, and 66 strains of rice, maize, and sorghum, respectively. The analytical methods are available as a DNApod workflow in an NGS annotation system of the DNA Data Bank of Japan and a virtual machine image. Furthermore, DNApod provides tables of links of identifiers between DNApod genotypic data and public phenotypic data. To advance the sharing of organism knowledge, DNApod offers basic and ubiquitous functions for multiple alignment and phylogenetic tree construction by using orthologous gene information.

DNApod: DNA polymorphism annotation database from next-generation sequence read archives

PubMed Central

Mochizuki, Takako; Tanizawa, Yasuhiro; Fujisawa, Takatomo; Ohta, Tazro; Nikoh, Naruo; Shimizu, Tokurou; Toyoda, Atsushi; Fujiyama, Asao; Kurata, Nori; Nagasaki, Hideki; Kaminuma, Eli; Nakamura, Yasukazu

2017-01-01

With the rapid advances in next-generation sequencing (NGS), datasets for DNA polymorphisms among various species and strains have been produced, stored, and distributed. However, reliability varies among these datasets because the experimental and analytical conditions used differ among assays. Furthermore, such datasets have been frequently distributed from the websites of individual sequencing projects. It is desirable to integrate DNA polymorphism data into one database featuring uniform quality control that is distributed from a single platform at a single place. DNA polymorphism annotation database (DNApod; http://tga.nig.ac.jp/dnapod/) is an integrated database that stores genome-wide DNA polymorphism datasets acquired under uniform analytical conditions, and this includes uniformity in the quality of the raw data, the reference genome version, and evaluation algorithms. DNApod genotypic data are re-analyzed whole-genome shotgun datasets extracted from sequence read archives, and DNApod distributes genome-wide DNA polymorphism datasets and known-gene annotations for each DNA polymorphism. This new database was developed for storing genome-wide DNA polymorphism datasets of plants, with crops being the first priority. Here, we describe our analyzed data for 679, 404, and 66 strains of rice, maize, and sorghum, respectively. The analytical methods are available as a DNApod workflow in an NGS annotation system of the DNA Data Bank of Japan and a virtual machine image. Furthermore, DNApod provides tables of links of identifiers between DNApod genotypic data and public phenotypic data. To advance the sharing of organism knowledge, DNApod offers basic and ubiquitous functions for multiple alignment and phylogenetic tree construction by using orthologous gene information. PMID:28234924

Systematics of gastrointestinal nematodes of domestic ruminants: advances between 1992 and 1995 and proposals for future research.

PubMed

Lichtenfels, J R; Hoberg, E P; Zarlenga, D S

1997-11-01

The systematics of trichostrongyloid nematodes of ruminants provides a foundation for diagnostics and responds to the need to identify eggs in feces, free-living larvae from pastures or fecal cultures and larval or adult nematodes collected from hosts. These needs are associated with diagnostic problems or research projects. Difficulties in identifying all developmental stages of trichostrongyloid nematodes of domestic ruminants still severely limit the effective diagnosis and control of these parasites. Phylogenetic hypotheses as the basis for predictive classifications have been developed only for the subfamilies of the Trichostrongylidae. This report briefly describes recent progress in the development of improved tools for identification, phylogenetic analyses and predictive classifications. It also describes future research needed on the identification and classification of trichostrongyloid nematode parasites of domestic ruminants. Nematodes included are species of the super-family Trichostrongyloidea known to be important pathogens of domestic ruminants. The information summarized is presented by nematode developmental stage and by taxonomic groups. Eggs: While eggs of some trichostrongyloid nematode parasites of ruminants can be readily identified to their genus (Nematodirus), and some to species (e.g. Nematodirus battus), most of the important pathogens (including the Ostertagiinae and Haemonchinae) cannot be identified morphologically or morphometrically even to family level. However, DNA technology has been developed for determining not only the presence of specific pathogens in eggs from fecal samples, but also for estimating the percentage of the total eggs that each pathogen comprises. This new method will make possible a rapid determination of which individual animals in a herd should be treated. Larvae: The most commonly-used method for identifying infective larvae is time-consuming (several weeks), unreliable for estimating intensities of individual species as components of mixed populations and requires highly trained specialists. Available identification keys for larvae are not well illustrated and need to be augmented. Adults: Recent advances in the identification of adult trichostrongyloids and their systematics are organized by taxonomic group. Genera included are Ostertagia, Haemonchus, Cooperia, Trichostrongylus and Nematodirus. Recently, the first phylogenetic analysis of the Trichostrongylidae family established monophyly for the family. A similar analysis of the Molineidae is needed. Ostertagia: Several studies of polymorphism summarized the phenomenon and listed 19 polymorphic species in five genera. Two studies of DNA differences within and among polymorphic species of Ostertagiinae supported earlier hypotheses that the species pairs represent polymorphic species. A phylogenetic analysis of the Ostertagiinae and generic concepts are needed. Haemonchus: A key to three species of Haemonchus provides, for the first time, morphological characteristics for the microscopical identification to species of individual adult nematodes of either sex. The Food and Drug Administration is now requiring that results of drug trials include identification of Haemonchus to species. Cooperia: Studies using random amplified polymorphic DNA methods showed a high degree of variation within and among C. oncophora/C. surnabada, but supported a polymorphic relationship for the species pair. A phylogenetic analysis of the Cooperiinae is needed. Trichostrongylus: Restriction Fragment Length Polymorphisms (RFLPs) of genomic DNA of two strains of T. colubriformis indicated a high degree of intra- and inter-strain DNA polymorphism. However, other studies demonstrated expected species level differences between T. colubriformis and T. vitrinus using Random Amplified Polymorphic DNA (RAPD) methods. Sequences of the second Internal Transcribed Spacer Region (ITS-2) ribosomal repeat showed sequence differences of 1.3-7.6% among five

Assessment of genome origins and genetic diversity in the genus Eleusine with DNA markers.

PubMed

Salimath, S S; de Oliveira, A C; Godwin, I D; Bennetzen, J L

1995-08-01

Finger millet (Eleusine coracana), an allotetraploid cereal, is widely cultivated in the arid and semiarid regions of the world. Three DNA marker techniques, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), and inter simple sequence repeat amplification (ISSR), were employed to analyze 22 accessions belonging to 5 species of Eleusine. An 8 probe--3 enzyme RFLP combination, 18 RAPD primers, and 6 ISSR primers, respectively, revealed 14, 10, and 26% polymorphism in 17 accessions of E. coracana from Africa and Asia. These results indicated a very low level of DNA sequence variability in the finger millets but did allow each line to be distinguished. The different Eleusine species could be easily identified by DNA marker technology and the 16% intraspecific polymorphism exhibited by the two analyzed accessions of E. floccifolia suggested a much higher level of diversity in this species than in E. coracana. Between species, E. coracana and E. indica shared the most markers, while E. indica and E. tristachya shared a considerable number of markers, indicating that these three species form a close genetic assemblage within the Eleusine. Eleusine floccifolia and E. compressa were found to be the most divergent among the species examined. Comparison of RFLP, RAPD, and ISSR technologies, in terms of the quantity and quality of data output, indicated that ISSRs are particularly promising for the analysis of plant genome diversity.

DNA methylation levels analysis in four tissues of sea cucumber Apostichopus japonicus based on fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) during aestivation.

PubMed

Zhao, Ye; Chen, Muyan; Storey, Kenneth B; Sun, Lina; Yang, Hongsheng

2015-03-01

DNA methylation plays an important role in regulating transcriptional change in response to environmental stimuli. In the present study, DNA methylation levels of tissues of the sea cucumber Apostichopus japonicus were analyzed by the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) technique over three stages of the aestivation cycle. Overall, a total of 26,963 fragments were amplified including 9112 methylated fragments among four sea cucumber tissues using 18 pairs of selective primers. Results indicated an average DNA methylation level of 33.79% for A. japonicus. The incidence of DNA methylation was different across tissue types in the non-aestivation stage: intestine (30.16%), respiratory tree (27.61%), muscle (27.94%) and body wall (56.25%). Our results show that hypermethylation accompanied deep-aestivation in A. japonicus, which suggests that DNA methylation may have an important role in regulating global transcriptional suppression during aestivation. Further analysis indicated that the main DNA modification sites were focused on intestine and respiratory tree tissues and that full-methylation but not hemi-methylation levels exhibited significant increases in the deep-aestivation stage. Copyright © 2014 Elsevier Inc. All rights reserved.

A new strategy for complete identification of sea buckthorn cultivars by using random amplified polymorphic DNA markers.

PubMed

Yang, G; Ding, J; Wu, L R; Duan, Y D; Li, A Y; Shan, J Y; Wu, Y X

2015-03-13

DNA fingerprinting is both a popular and important technique with several advantages in plant cultivar identification. However, this technique has not been used widely and efficiently in practical plant identification because the analysis and recording of data generated from fingerprinting and genotyping are tedious and difficult. We developed a novel approach known as a cultivar identification diagram (CID) strategy that uses DNA markers to separate plant individuals in a more efficient, practical, and referable manner. A CID was manually constructed and a polymorphic marker was generated from each polymerase chain reaction for sample separation. In this study, 67 important sea buckthorn cultivars cultivated in China were successfully separated with random amplified polymorphic DNA markers using the CID analysis strategy, with only seven 11-nucleotide primers employed. The utilization of the CID of these 67 sea buckthorn cultivars was verified by identifying 2 randomly chosen groups of cultivars among the 67 cultivars. The main advantages of this identification strategy include fewer primers used and separation of all cultivars using the corresponding primers. This sea buckthorn CID was able to separate any sea buckthorn cultivars among the 67 studied, which is useful for sea buckthorn cultivar identification, cultivar-right-protection, and for the sea buckthorn nursery industry in China.

Inducing mutations through γ-irradiation in seeds of Mucuna pruriens for developing high L-DOPA-yielding genotypes.

PubMed

Singh, Susheel Kumar; Yadav, Deepti; Lal, Raj Kishori; Gupta, Madan M; Dhawan, Sunita Singh

2017-04-01

To develop elite genotypes in Mucuna pruriens (L.) DC with high L-DOPA (L-3, 4 dihydroxyphenylalanine) yields, with non-itching characteristics and better adaptability by applying γ-irradiation. Molecular and chemical analysis was performed for screening based on specific characteristics desired for developing suitable genotypes. Developed, mutant populations were analyzed for L-DOPA % in seeds through TLC (thin layer chromatography), and the results obtained were validated with the HPLC (High performance liquid chromatography). The DNA (Deoxyribonucleic acid) was isolated from the leaf at the initial stage and used for DNA polymorphism. RNA (Ribonucleic acid) was isolated from the leaf during maturity and used for expression analysis. The selected mutant T-I-7 showed 5.7% L-DOPA content compared to 3.18% of parent CIM-Ajar. The total polymorphism obtained was 57% with the molecular marker analysis. The gene expression analysis showed higher fold change expression of the dopadecarboxylase gene (DDC) in control compared to selected mutants (T-I-7, T-II-23, T-IV-9, T-VI-1). DNA polymorphism was used for the screening of mutants for efficient screening at an early stage. TLC was found suitable for the large-scale comparative chemical analysis of L-DOPA. The expression profile of DDC clearly demonstrated the higher yields of L-DOPA in selected mutants developed by γ-irradiation in the seeds of the control.

Analysis on the DNA Fingerprinting of Aspergillus Oryzae Mutant Induced by High Hydrostatic Pressure

NASA Astrophysics Data System (ADS)

Wang, Hua; Zhang, Jian; Yang, Fan; Wang, Kai; Shen, Si-Le; Liu, Bing-Bing; Zou, Bo; Zou, Guang-Tian

2011-01-01

The mutant strains of aspergillus oryzae (HP300a) are screened under 300 MPa for 20 min. Compared with the control strains, the screened mutant strains have unique properties such as genetic stability, rapid growth, lots of spores, and high protease activity. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) are used to analyze the DNA fingerprinting of HP300a and the control strains. There are 67.9% and 51.3% polymorphic bands obtained by these two markers, respectively, indicating significant genetic variations between HP300a and the control strains. In addition, comparison of HP300a and the control strains, the genetic distances of random sequence and simple sequence repeat of DNA are 0.51 and 0.34, respectively.

Single nucleotide polymorphism analysis using different colored dye dimer probes

NASA Astrophysics Data System (ADS)

Marmé, Nicole; Friedrich, Achim; Denapaite, Dalia; Hakenbeck, Regine; Knemeyer, Jens-Peter

2006-09-01

Fluorescence quenching by dye dimer formation has been utilized to develop hairpin-structured DNA probes for the detection of a single nucleotide polymorphism (SNP) in the penicillin target gene pbp2x, which is implicated in the penicillin resistance of Streptococcus pneumoniae. We designed two specific DNA probes for the identification of the pbp2x genes from a penicillin susceptible strain R6 and a resistant strain Streptococcus mitis 661 using green-fluorescent tetramethylrhodamine (TMR) and red-fluorescent DY-636, respectively. Hybridization of each of the probes to its respective target DNA sequence opened the DNA hairpin probes, consequently breaking the nonfluorescent dye dimers into fluorescent species. This hybridization of the target with the hairpin probe achieved single nucleotide specific detection at nanomolar concentrations via increased fluorescence.

Assay for identification of heterozygous single-nucleotide polymorphism (Ala67Thr) in human poliovirus receptor gene.

PubMed

Nandi, Shyam Sundar; Sharma, Deepa Kailash; Deshpande, Jagadish M

2016-07-01

It is important to understand the role of cell surface receptors in susceptibility to infectious diseases. CD155 a member of the immunoglobulin super family, serves as the poliovirus receptor (PVR). Heterozygous (Ala67Thr) polymorphism in CD155 has been suggested as a risk factor for paralytic outcome of poliovirus infection. The present study pertains to the development of a screening test to detect the single nucleotide (SNP) polymorphism in the CD155 gene. New primers were designed for PCR, sequencing and SNP analysis of Exon2 of CD155 gene. DNAs extracted from either whole blood (n=75) or cells from oral cavity (n=75) were used for standardization and validation of the SNP assay. DNA sequencing was used as the gold standard method. A new SNP assay for detection of heterozygous Ala67Thr genotype was developed and validated by testing 150 DNA samples. Heterozygous CD155 was detected in 27.33 per cent (41/150) of DNA samples tested by both SNP detection assay and sequencing. The SNP detection assay was successfully developed for identification of Ala67Thr polymorphism in human PVR/CD155 gene. The SNP assay will be useful for large scale screening of DNA samples.

Polymorphisms and allele frequencies of the ABO blood group gene among the Jomon, Epi-Jomon and Okhotsk people in Hokkaido, northern Japan, revealed by ancient DNA analysis.

PubMed

Sato, Takehiro; Kazuta, Hisako; Amano, Tetsuya; Ono, Hiroko; Ishida, Hajime; Kodera, Haruto; Matsumura, Hirofumi; Yoneda, Minoru; Dodo, Yukio; Masuda, Ryuichi

2010-10-01

To investigate the genetic characteristics of the ancient populations of Hokkaido, northern Japan, polymorphisms of the ABO blood group gene were analyzed for 17 Jomon/Epi-Jomon specimens and 15 Okhotsk specimens using amplified product-length polymorphism and restriction fragment length polymorphism analyses. Five ABO alleles were identified from the Jomon/ Epi-Jomon and Okhotsk people. Allele frequencies of the Jomon/Epi-Jomon and Okhotsk people were compared with those of the modern Asian, European and Oceanic populations. The genetic relationships inferred from principal component analyses indicated that both Jomon/Epi-Jomon and Okhotsk people are included in the same group as modern Asian populations. However, the genetic characteristics of these ancient populations in Hokkaido were significantly different from each other, which is in agreement with the conclusions from mitochondrial DNA and ABCC11 gene analyses that were previously reported.

Determination of genotoxic effects of boron and zinc on Zea mays using protein and random amplification of polymorphic DNA analyses.

PubMed

Erturk, Filiz Aygun; Nardemir, Gokce; Hilal, A Y; Arslan, Esra; Agar, Guleray

2015-11-01

In this research, we aimed to determine genotoxic effects of boron (B) and zinc (Zn) on Zea mays by using total soluble protein content and random amplification of polymorphic DNA (RAPD) analyses. For the RAPD analysis, 16 RAPD primers were found to produce unique polymorphic band profiles on treated maize seedlings. With increased Zn and B concentrations, increased polymorphism rate was observed, while genomic template stability and total soluble protein content decreased. The treatment with Zn was more effective than that of B groups on the levels of total proteins. The obtained results from this study revealed that the total soluble protein levels and RAPD profiles were performed as endpoints of genotoxicity and these analyses can offer useful biomarker assays for the evaluation of genotoxic effects on Zn and B polluted plants. © The Author(s) 2013.

Effects of different preservation methods on inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) molecular markers in botanic samples.

PubMed

Wang, Xiaolong; Li, Lin; Zhao, Jiaxin; Li, Fangliang; Guo, Wei; Chen, Xia

2017-04-01

To evaluate the effects of different preservation methods (stored in a -20°C ice chest, preserved in liquid nitrogen and dried in silica gel) on inter simple sequence repeat (ISSR) or random amplified polymorphic DNA (RAPD) analyses in various botanical specimens (including broad-leaved plants, needle-leaved plants and succulent plants) for different times (three weeks and three years), we used a statistical analysis based on the number of bands, genetic index and cluster analysis. The results demonstrate that methods used to preserve samples can provide sufficient amounts of genomic DNA for ISSR and RAPD analyses; however, the effect of different preservation methods on these analyses vary significantly, and the preservation time has little effect on these analyses. Our results provide a reference for researchers to select the most suitable preservation method depending on their study subject for the analysis of molecular markers based on genomic DNA. Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

Characterization of early follicular cDNA library suggests evidence for genetic polymorphisms in the inbred strain C108 of Bombyx mori.

PubMed

Mills, D R; Goldsmith, M R

2000-04-01

Recent work towards the completion of a saturated molecular genetic linkage map for the lepidopteran silkworm, Bombyx mori (n = 28), has provided evidence for existing polymorphisms in the inbred strain C108. Two inbred parental strains, p50 and C108, were crossed to produce the F1 (P/C) hybrid offspring. The populations used in this project were comprised of a combination of 29 F2 (F1 x F1) and 31 reciprocal backcross (P/C x C/C, P/C x P/P) progeny. All restriction fragment length polymorphisms (RFLPs) for the initial analysis were hybridized with anonymous probes derived from a random early follicular cDNA (Rcf) library from Bombyx. A total of 19 Rcf probes were selected as showing scorable codominant polymorphic patterns when screened against F2 and backcross DNAs digested with the restriction enzymes EcoRI, HindIII, or PstI, and Southern blotted to nylon membranes for hybridization. Of the newly reported Rcf probes, 7 (37%) were characterized as producing 'simple' polymorphic patterns, while 12 (63%) were characterized as producing 'complex' polymorphic patterns. Further characterization of the complex patterns subdivided this group into two general classes: polymorphisms that contained an additional allele, and multiple bands that contained an easily scored two banded polymorphism. Because the extra allele class was limited to the (P/C x C/C) backcross progeny, it is suggested that the inbred parental strain C108 harbors polymorphic loci that are inherited in a simple Mendelian fashion. A genetic analysis discussing plausible origins and maintenance of these polymorphisms is presented.

[Multilocus genotyping of polymorphous STR-loci of chromosomal DNA in individual cells: technical difficulties].

PubMed

Ivanov, P L; Leonov, S N; Zemskova, E Iu; Kobylianskiĭ, A G; Dziubenko, E V

2013-01-01

This study was designed to estimate the effectiveness of special technical procedures for the enhancement of sensitivity of multiplex analysis of DNA, such as the use of low-plexity PCR systems and the whole genome preamplification technology, and the possibility of their application for the purpose of forensic medical genotyping of polymorphous STR-loci of chromosomal DNA in individual cells. The authors refused to use the imitation model (equivalent DNA dilutions) for the sake of obtaining the maximally informative data and chose to work with real preparations of solitary buccal epithelial cells isolated by the laser microdissection technique. It was shown that neither the use of the low-plexity multilocus PCR systems nor the whole genome pre-amplification technology makes possible reliable genotyping of STR-loci of chromosomal DNA in individual cells. The proposed techniques allow for DNA genotyping in preparations consisting of 10 diploid cells whereas the methods for reliable genotyping of STR-loci of chromosomal DNA in individual cells remains to be developed.

Methylation-sensitive amplified polymorphism analysis of Verticillium wilt-stressed cotton (Gossypium).

PubMed

Wang, W; Zhang, M; Chen, H D; Cai, X X; Xu, M L; Lei, K Y; Niu, J H; Deng, L; Liu, J; Ge, Z J; Yu, S X; Wang, B H

2016-10-06

In this study, a methylation-sensitive amplification polymorphism analysis system was used to analyze DNA methylation level in three cotton accessions. Two disease-sensitive near-isogenic lines, PD94042 and IL41, and one disease-resistant Gossypium mustelinum accession were exposed to Verticillium wilt, to investigate molecular disease resistance mechanisms in cotton. We observed multiple different DNA methylation types across the three accessions following Verticillium wilt exposure. These included hypomethylation, hypermethylation, and other patterns. In general, the global DNA methylation level was significantly increased in the disease-resistant accession G. mustelinum following disease exposure. In contrast, there was no significant difference in the disease-sensitive accession PD94042, and a significant decrease was observed in IL41. Our results suggest that disease-resistant cotton might employ a mechanism to increase methylation level in response to disease stress. The differing methylation patterns, together with the increase in global DNA methylation level, might play important roles in tolerance to Verticillium wilt in cotton. Through cloning and analysis of differently methylated DNA sequences, we were also able to identify several genes that may contribute to disease resistance in cotton. Our results revealed the effect of DNA methylation on cotton disease resistance, and also identified genes that played important roles, which may shed light on the future cotton disease-resistant molecular breeding.

[Effect of mitochondrial DNA 5178 C/A polymorphism on risks for type 2 diabetes mellitus and its complications].

PubMed

Yang, Xunjun; Zhang, Yuning; Ma, Yin; Zhao, Qiongya; Lyu, Jianxin

2015-12-01

To explore the role of mitochondrial DNA 5178 C/A (Mt5178) polymorphism of NADH-dehydrogenase subunit 2 (ND2) gene in type-2 diabetes mellitus (T2DM) among ethnic Han Chinese through a case-control study. The Mt5178C/A polymorphism was determined by sequencing 1103 T2DM patients and 791 healthy controls. Logistic regression analysis was conducted to estimate odds ratios (OR) and 95% confidence intervals (CI). To confirm the results, a meta-analysis was conducted based on published literature on the association of Mt5178 variant with T2DM. No significant association was found between the Mt5178C/A variant and T2DM either by our study or the meta-analysis which included eight published studies. Nevertheless, it was found that the T2DM patients with 5178C genotype were at a higher risk for nephropathy complication (OR=1.49, 95%CI: 1.005-2.197, P<0.05) and at significantly lower risk for hypertension complication (OR=0.744, 95%CI: 0.556-0.996, P<0.05) compared with those carrying a 5178A genotype. No association was found between the Mt5178C/A polymorphism of mitochondrial ND2 gene with the increased risk of T2DM. However, the polymorphism may affect the development of nephropathy and hypertension complications among T2DM patients.

Single-Cell Quantitative PCR: Advances and Potential in Cancer Diagnostics.

PubMed

Ok, Chi Young; Singh, Rajesh R; Salim, Alaa A

2016-01-01

Tissues are heterogeneous in their components. If cells of interest are a minor population of collected tissue, it would be difficult to obtain genetic or genomic information of the interested cell population with conventional genomic DNA extraction from the collected tissue. Single-cell DNA analysis is important in the analysis of genetics of cell clonality, genetic anticipation, and single-cell DNA polymorphisms. Single-cell PCR using Single Cell Ampligrid/GeXP platform is described in this chapter.

Extracellular DNA in single- and multiple-species unsaturated biofilms.

PubMed

Steinberger, R E; Holden, P A

2005-09-01

The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.

Applicability of SCAR markers to food genomics: olive oil traceability.

PubMed

Pafundo, Simona; Agrimonti, Caterina; Maestri, Elena; Marmiroli, Nelson

2007-07-25

DNA analysis with molecular markers has opened a shortcut toward a genomic comprehension of complex organisms. The availability of micro-DNA extraction methods, coupled with selective amplification of the smallest extracted fragments with molecular markers, could equally bring a breakthrough in food genomics: the identification of original components in food. Amplified fragment length polymorphisms (AFLPs) have been instrumental in plant genomics because they may allow rapid and reliable analysis of multiple and potentially polymorphic sites. Nevertheless, their direct application to the analysis of DNA extracted from food matrixes is complicated by the low quality of DNA extracted: its high degradation and the presence of inhibitors of enzymatic reactions. The conversion of an AFLP fragment to a robust and specific single-locus PCR-based marker, therefore, could extend the use of molecular markers to large-scale analysis of complex agro-food matrixes. In the present study is reported the development of sequence characterized amplified regions (SCARs) starting from AFLP profiles of monovarietal olive oils analyzed on agarose gel; one of these was used to identify differences among 56 olive cultivars. All the developed markers were purposefully amplified in olive oils to apply them to olive oil traceability.

Development and application of a 6.5 million feature Affymetrix Genechip® for massively parallel discovery of single position polymorphisms in lettuce (Lactuca spp.)

PubMed Central

2012-01-01

Background High-resolution genetic maps are needed in many crops to help characterize the genetic diversity that determines agriculturally important traits. Hybridization to microarrays to detect single feature polymorphisms is a powerful technique for marker discovery and genotyping because of its highly parallel nature. However, microarrays designed for gene expression analysis rarely provide sufficient gene coverage for optimal detection of nucleotide polymorphisms, which limits utility in species with low rates of polymorphism such as lettuce (Lactuca sativa). Results We developed a 6.5 million feature Affymetrix GeneChip® for efficient polymorphism discovery and genotyping, as well as for analysis of gene expression in lettuce. Probes on the microarray were designed from 26,809 unigenes from cultivated lettuce and an additional 8,819 unigenes from four related species (L. serriola, L. saligna, L. virosa and L. perennis). Where possible, probes were tiled with a 2 bp stagger, alternating on each DNA strand; providing an average of 187 probes covering approximately 600 bp for each of over 35,000 unigenes; resulting in up to 13 fold redundancy in coverage per nucleotide. We developed protocols for hybridization of genomic DNA to the GeneChip® and refined custom algorithms that utilized coverage from multiple, high quality probes to detect single position polymorphisms in 2 bp sliding windows across each unigene. This allowed us to detect greater than 18,000 polymorphisms between the parental lines of our core mapping population, as well as numerous polymorphisms between cultivated lettuce and wild species in the lettuce genepool. Using marker data from our diversity panel comprised of 52 accessions from the five species listed above, we were able to separate accessions by species using both phylogenetic and principal component analyses. Additionally, we estimated the diversity between different types of cultivated lettuce and distinguished morphological types. Conclusion By hybridizing genomic DNA to a custom oligonucleotide array designed for maximum gene coverage, we were able to identify polymorphisms using two approaches for pair-wise comparisons, as well as a highly parallel method that compared all 52 genotypes simultaneously. PMID:22583801

Development and application of a 6.5 million feature Affymetrix Genechip® for massively parallel discovery of single position polymorphisms in lettuce (Lactuca spp.).

PubMed

Stoffel, Kevin; van Leeuwen, Hans; Kozik, Alexander; Caldwell, David; Ashrafi, Hamid; Cui, Xinping; Tan, Xiaoping; Hill, Theresa; Reyes-Chin-Wo, Sebastian; Truco, Maria-Jose; Michelmore, Richard W; Van Deynze, Allen

2012-05-14

High-resolution genetic maps are needed in many crops to help characterize the genetic diversity that determines agriculturally important traits. Hybridization to microarrays to detect single feature polymorphisms is a powerful technique for marker discovery and genotyping because of its highly parallel nature. However, microarrays designed for gene expression analysis rarely provide sufficient gene coverage for optimal detection of nucleotide polymorphisms, which limits utility in species with low rates of polymorphism such as lettuce (Lactuca sativa). We developed a 6.5 million feature Affymetrix GeneChip® for efficient polymorphism discovery and genotyping, as well as for analysis of gene expression in lettuce. Probes on the microarray were designed from 26,809 unigenes from cultivated lettuce and an additional 8,819 unigenes from four related species (L. serriola, L. saligna, L. virosa and L. perennis). Where possible, probes were tiled with a 2 bp stagger, alternating on each DNA strand; providing an average of 187 probes covering approximately 600 bp for each of over 35,000 unigenes; resulting in up to 13 fold redundancy in coverage per nucleotide. We developed protocols for hybridization of genomic DNA to the GeneChip® and refined custom algorithms that utilized coverage from multiple, high quality probes to detect single position polymorphisms in 2 bp sliding windows across each unigene. This allowed us to detect greater than 18,000 polymorphisms between the parental lines of our core mapping population, as well as numerous polymorphisms between cultivated lettuce and wild species in the lettuce genepool. Using marker data from our diversity panel comprised of 52 accessions from the five species listed above, we were able to separate accessions by species using both phylogenetic and principal component analyses. Additionally, we estimated the diversity between different types of cultivated lettuce and distinguished morphological types. By hybridizing genomic DNA to a custom oligonucleotide array designed for maximum gene coverage, we were able to identify polymorphisms using two approaches for pair-wise comparisons, as well as a highly parallel method that compared all 52 genotypes simultaneously.

Molecular genetic studies in Saudi population; identified variants from GWAS and meta-analysis in stroke.

PubMed

Alharbi, Khalid Khalaf; Ali Khan, Imran; Alotaibi, Mohammad Abdullah; Saud Aloyaid, Abdullah; Al-Basheer, Haifa Abdulaziz; Alghamdi, Naelah Abdullah; Al-Baradie, Raid Saleem; Al-Sulaiman, A M

2018-01-01

Stroke is a multifactorial and heterogeneous disorder, correlates with heritability and considered as one of the major diseases. The prior reports performed the variable models such as genome-wide association studies (GWAS), replication, case-control, cross-sectional and meta-analysis studies and still, we lack diagnostic marker in the global world. There are limited studies were carried out in Saudi population, and we aim to investigate the molecular association of single nucleotide polymorphisms (SNPs) identified through GWAS and meta-analysis studies in stroke patients in the Saudi population. In this case-control study, we have opted gender equality of 207 cases and 207 controls from the capital city of Saudi Arabia in King Saud University Hospital. The peripheral blood (5 ml) sample will be collected in two different vacutainers, and three mL of the coagulated blood will be used for lipid analysis (biochemical tests) and two mL will be used for DNA analysis (molecular tests). Genomic DNA will be extracted with the collected blood samples, and specific primers will be designed for the opted SNPs ( SORT1 -rs646218 and OLR1 -rs11053646 polymorphisms) and PCR-RFLP will be performed and randomly DNA sequencing will be carried out to cross check the results. The rs646218 and rs11053646 polymorphisms were significantly associated with allele, genotype and dominant models with and without crude odds ratios (OR's) and Multiple logistic regression analysis (p < 0.05). Correlation between lipid profile and genotypes has confirmed the significant relation between triglycerides and rs646218 and rs1105364 6polymorphisms. However, rs11053646 polymorphism was correlated with HDLC (p = 0.04). Genotypes were examined in both males' vs. males and females' vs. females in cases and control and we concluded that in rs11053646 polymorphisms with male subjects compared between cases and controls found to be associated with dominant model heterozygote genotypes (p < 0.05). The results of the current study confirmed the SORT1 and OLR1  SNPs were associated in the Saudi population. The current results were in the association with the prior study results documented through GWAS and meta-analysis association. However, other ethnic population studies should be performed to rule out in the human hereditary diseases.

[Analysis of methylation-sensitive amplified polymorphism in wheat genome under the wheat leaf rust stress].

PubMed

Fu, Sheng-Jie; Wang, Hui; Feng, Li-Na; Sun, Yi; Yang, Wen-Xiang; Liu, Da-Qun

2009-03-01

Intrinsic DNA methylation pattern is an integral component of the epigenetic network in many eukaryotes. DNA methylation plays an important role in regulating gene expression in eukaryotes. Biological stress in plant provides an inherent epigenetic driving force of evolution. Study of DNA methylation patterns arising from biological stress will help us fully understand the epigenetic regulation of gene expression and DNA methylation of biological functions. The wheat near-isogenic lines TcLr19 and TcLr41 were resistant to races THTT and TKTJ, respectively, and Thatcher is compatible in the interaction with Puccinia triticina THTT and TKTJ, respectively. By means of methylation-sensitive amplified polymorphism (MSAP) analysis, the patterns of cytosine methylation in TcLr19, TcLr41, and Thatcher inoculated with P. triticina THTT and TKTJ were compared with those of the untreated samples. All the DNA fragments, each representing a recognition site cleaved by each or both of isoschizomers, were amplified using 60 pairs of selective primers. The results indicated that there was no significant difference between the challenged and unchallenged plants at DNA methylation level. However, epigenetic difference between the near-isogenic line for wheat leaf rust resistance gene Lr41 and Thatcher was present.

An analysis on DNA fingerprints of thirty papaya cultivars (Carica papaya L.), grown in Thailand with the use of amplified fragment length polymorphisms technique.

PubMed

Ratchadaporn, Janthasri; Sureeporn, Katengam; Khumcha, U

2007-09-15

The experiment was carried out at the Department of Horticulture, Ubon Ratchathani University, Ubon Ratchathani province, Northeast Thailand during June 2002 to May 2003 aims to identify DNA fingerprints of thirty papaya cultivars with the use of Amplified Fragment Length Polymorphisms (AFLP) technique. Papaya cultivars were collected from six different research centers in Thailand. Papaya plants of each cultivar were grown under field conditions up to four months then leaf numbers 2 and 3 of each cultivar (counted from top) were chosen for DNA extraction and the samples were used for AFLP analysis. Out of 64 random primers being used, 55 pairs gave an increase in DNA bands but only 12 pairs of random primers were randomly chosen for the final analysis of the experiment. The results showed that AFLP markers gave Polymorphic Information Contents (PIC) of three ranges i.e., AFLP markers of 235 lied on a PIC range of 0.003-0.05, 47 for a PIC range of 0.15-0.20 and 12 for a PIC range of 0.35-0.40. The results on dendrogram cluster analysis revealed that the thirty papaya cultivars were classified into six groups i.e., (1) Kaeg Dum and Malador (2) Kaeg Nuan (3) Pakchong and Solo (4) Taiwan (5) Co Coa Hai Nan and (6) Sitong. Nevertheless, in spite of the six papaya groups all papaya cultivars were genetically related to each other where diversity among the cultivars was not significantly found.

Variations in the Phytophthora infestans Population in Nepal as Revealed by Nuclear and Mitochondrial DNA Polymorphisms.

PubMed

Ghimire, S R; Hyde, K D; Hodgkiss, I J; Shaw, D S; Liew, E C Y

2003-02-01

ABSTRACT Phytophthora infestans isolates collected from potato and tomato crops from various parts of Nepal during the 1999 and 2000 crop seasons were characterized for nuclear and mitochondrial DNA polymorphisms using restriction fragment length polymorphism markers. The nuclear DNA probe RG57 detected 11 multilocus genotypes among 280 isolates. Three genotypes were detected 21 times or more, constituting 94% of the total population, whereas frequencies of other genotypes ranged from 0.004 to 0.014. The overall genotypic diversity as estimated by the Gleason index was 1.78. Most of the overall diversity was present at the highest level (i.e., interregional, 46%), indicating limited gene flow among regions. Cluster analysis of multilocus genotypes derived from RG57 and mating type data for Nepalese isolates and representative isolates worldwide showed Nepalese isolates grouping into four clusters. Characterization of 67 isolates for mitochondrial DNA polymorphisms revealed the presence of two mt-haplotypes, Ia and Ib with the proportions of 0.88 and 0.12, respectively. Polymorphisms in nuclear and mitochondrial DNA revealed a moderate level of diversity in this population. Genotype NP3 had an identical RG57 fingerprint to US1 and had mt-haplotype Ib, confirming the presence of an old population in Nepal. Most of the genotypes had a different RG57 fingerprint than that of US1 and mt-haplotype Ia, the common characteristics of new populations. The presence of a new population at high proportions in Nepal was consistent with the global trend of mt-haplotype distribution, and suggests the displacement of old populations. This study indicates at least three possible introductions of P. infestans to Nepal.

Associations between sperm quality, DNA damage, and CYP1A1, GSTT1 and GSTM1 polymorphisms with 1-hydroxypyrene urinary levels in men occupationally exposed to polycyclic aromatic hydrocarbons.

PubMed

Recio-Vega, Rogelio; Olivas-Calderon, Edgar; Michel-Ramirez, Gladis; Martinez-Salinas, Rebeca Isabel; Gallegos-Arreola, Martha Patricia; Ocampo-Gomez, Guadalupe Leticia; Perez-Morales, Rebeca

2018-05-29

During recent decades, several reports have suggested a decrease in semen quality and DNA damage due in part to environmental toxicants and industrial chemicals. Among these xenobiotics, polycyclic aromatic hydrocarbons (PAHs) are of particular concern because of their remarkable mutagenic and carcinogenic properties and because several experimental and epidemiological studies have reported adverse effects of PAHs on male reproductive health and DNA structure. The aim of the study was to evaluate the association between 1-hydroxypyrene (1-OHP) urinary levels and sperm quality, DNA damage and the frequency of CYP1A1, GSTT1, and GSTM1 polymorphisms. Semen, urine and blood samples were taken for sperm-quality assessment, 1-OHP urinary level measurement, DNA damage evaluation and polymorphism frequency analysis of three genes implicated in PAH metabolism in a total of 70 Mexican subjects exposed and nonexposed to PAHs. A significant decrease in sperm quality and increased DNA damage were registered in occupationally exposed volunteers. Polymorphisms modified the 1-OHP urinary levels; however, no associations were found between them. Inverse associations were registered between the sperm concentration/mL and 1-OHP levels and between tail lengths and the GSMT1 null genotype. Our data showed an inverse association between 1-OHP urinary levels and both sperm quality and the DNA integrity. Additionally, the heterozygote variants of CYP1A1-m1 and CYP1A1-m2 significantly increased the urinary excretion of 1-OHP, and the GSTM1 null variant was inversely associated with the comet parameters evaluated.

Development of a Multiplex Single Base Extension Assay for Mitochondrial DNA Haplogroup Typing

PubMed Central

Nelson, Tahnee M.; Just, Rebecca S.; Loreille, Odile; Schanfield, Moses S.; Podini, Daniele

2007-01-01

Aim To provide a screening tool to reduce time and sample consumption when attempting mtDNA haplogroup typing. Methods A single base primer extension assay was developed to enable typing, in a single reaction, of twelve mtDNA haplogroup specific polymorphisms. For validation purposes a total of 147 samples were tested including 73 samples successfully haplogroup typed using mtDNA control region (CR) sequence data, 21 samples inconclusively haplogroup typed by CR data, 20 samples previously haplogroup typed using restriction fragment length polymorphism (RFLP) analysis, and 31 samples of known ancestral origin without previous haplogroup typing. Additionally, two highly degraded human bones embalmed and buried in the early 1950s were analyzed using the single nucleotide polymorphisms (SNP) multiplex. Results When the SNP multiplex was used to type the 96 previously CR sequenced specimens, an increase in haplogroup or macrohaplogroup assignment relative to conventional CR sequence analysis was observed. The single base extension assay was also successfully used to assign a haplogroup to decades-old, embalmed skeletal remains dating to World War II. Conclusion The SNP multiplex was successfully used to obtain haplogroup status of highly degraded human bones, and demonstrated the ability to eliminate possible contributors. The SNP multiplex provides a low-cost, high throughput method for typing of mtDNA haplogroups A, B, C, D, E, F, G, H, L1/L2, L3, M, and N that could be useful for screening purposes for human identification efforts and anthropological studies. PMID:17696300

Gene analysis of steroid 5 alpha-reductase 1 in hyperandrogenic women.

PubMed

Eminović, Izet; Komel, Radovan; Prezelj, Janez; Karamehić, Jasenko; Gavrankapetanović, Faris; Heljić, Becir

2005-08-01

To examine the gene encoding for 5alpha-reductase type 1 in hyperandrogenic women, and assess the association of its eventual mutations or polymorphisms with the development of the hyperandrogenic female pattern. Sixteen hyperandrogenic women were included in the study. Single-stranded conformation polymorphism analysis (SSCP) and DNA sequencing were performed after polymerase chain reaction amplification of each of the 5 exons of the SRD5A1 gene in both hyperandrogenic and control group (16 participants). Sequence analysis identified the existence of many polymorphisms; in codon 24 of exon 1, GGC (Gly) into GAC (Asp); in codon 30 of exon 1, CGG (Arg) into CGC (Arg); in exon 3 codon 169, ACA to ACG (both encoding for threonine); in exon 5, AGA to AGG (both encoding for arginine, codon 260); and T/C polymorphism in intron 2. Polymorphisms were found in both groups. Polymorphisms of SRD5A1 gene were the same in both hyperandrogenic and healthy women, indicating no significant associations of genetic polymorphisms/variations of SRD5A1 gene with clinical manifestations of hyperandrogenic disorders in women.

Sequence analysis of the mitochondrial DNA control region of ciscoes (genus Coregonus): taxonomic implications for the Great Lakes species flock.

PubMed

Reed, K M; Dorschner, M O; Todd, T N; Phillips, R B

1998-09-01

Sequence variation in the control region (D-loop) of the mitochondrial DNA (mtDNA) was examined to assess the genetic distinctiveness of the shortjaw cisco (Coregonus zenithicus). Individuals from within the Great Lakes Basin as well as inland lakes outside the basin were sampled. DNA fragments containing the entire D-loop were amplified by PCR from specimens of C. zenithicus and the related species C. artedi, C. hoyi, C. kiyi, and C. clupeaformis. DNA sequence analysis revealed high similarity within and among species and shared polymorphism for length variants. Based on this analysis, the shortjaw cisco is not genetically distinct from other cisco species.

New polymorphic markers in the vicinity of the pearl locus on mouse chromosome 13.

PubMed

Xu, H P; Yanak, B L; Wigler, M H; Gorin, M B

1996-01-01

We have used a Mus domesticus/-Mus spretus congenic animal that was selected for retention of Mus spretus DNA around the pearl locus to create a highly polymorphic region suitable for screening new markers. Representation difference analysis (RDA) was performed with either DNA from the congenic animal or C57BL/6J as the driver for subtraction. Four clones were identified, characterized, and converted to PCR-based polymorphic markers. Three of the four markers equally subdivide a 10-cM interval containing the pearl locus, with the fourth located centromeric to it. These markers have been placed on the mouse genetic map by use of an interspecific backcross panel between Mus domesticus (C57BL/6J) and Mus spretus generated by The Jackson Laboratory.

[Association between genetic polymorphisms of DNA repair genes XRCC1, XPD, XRCC3 and the capacity of DNA repair induce by benzene].

PubMed

Xu, Jianning; Yang, Min; Huang, Huilong; Wang, Quankai

2007-09-01

To explore the correlation between genetic polymorphisms of XRCC1, XPD, XRCC3 and DNA repair capacity induced by benzene. Eighty patients suffered from chronic benzene poisoning were investigated. PCR-RFLP was applied to detect the single nucleotide polymorphisms on C26304T, G27466A, G28152A, G36189A of XRCC1, C22541A, C23591T, A35931C of XPD, C18067T of XRCC3. Cytokinesis-block micronucleus (CBMN) and alkaline comet were applied to detect the DNA repair capacity. The DNA repair capacity of the subjects carrying XPD 35931C variant allele or carrying XRCC3 18067 C/T variant genotype were higher than those carrying corresponding mild genotype. There could be a correlation between polymorphisms of XRCC3 and DNA repair capacity of DNA damage induced by benzene.

DNA repair polymorphisms in B-cell chronic lymphocytic leukemia in sufferers of Chernobyl Nuclear Power Plant accident.

PubMed

Abramenko, Iryna; Bilous, Nadiia; Chumak, Anatolyi; Kostin, Alexey; Martina, Zoya; Dyagil, Iryna

2012-01-01

An association between DNA repair gene polymorphisms, environmental factors, and development of some types of cancer has been suggested by several studies. Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the clean-up workers of the Chernobyl Nuclear Power Plant (NPP) accident and it has some specific features. Therefore, we have studied the possible differences in DNA repair gene polymorphisms in CLL patients depending on ionizing radiation (IR) exposure history and their clinical characterictics. Arg399Gln XRCC1, Thr241Met XRCC3, and Lys751Gln XPD polymorphisms were studied in 64 CLL patients, exposed to IR due to the Chernobyl NPP accident, 114 IR-non-exposed CLL patients, and 103 sex- and age-matched IR-exposed controls using polymerase chain reaction-restriction fragment-length polymorphism analysis. All investigated polymorphisms were equally distributed between two groups of CLL patients and IR-exposed controls, except that that there was a significant reduction of the common homozygous Lys/Lys XPD genotype among IR-exposed CLL patients (23.7%) compared with IR-exposed controls (45.6%), OR = 0.37; 95% CI = 0.18-0.75; (P = 0.005). The number of IR-non-exposed CLL patients (37.4%) with the Lys/Lys XPD genotype was also decreased compared to IR-exposed controls, although this difference was not significant (P = 0.223). These preliminary data suggest a possible modifying role of Lys751Gln XPD polymorphism for the development of CLL, expecially in radiation-exposed persons.

[Mitochondrial DNA polymorphisms shared between modern humans and neanderthals: adaptive convergence or evidence for interspecific hybridization?].

PubMed

Maliarchuk, B A

2013-09-01

An analysis of the variability of the nucleotide sequences in the mitochondrial genome of modern humans, neanderthals, Denisovans, and other primates has shown that there are shared polymorphisms at positions 2758 and 7146 between modern Homo sapiens (in phylogenetic cluster L2'3'4'5'6) and Homo neanderthalensis (in the group of European neanderthals younger than 48000 years). It is suggested that the convergence may be due to adaptive changes in the mitochondrial genomes of modern humans and neanderthals or interspecific hybridization associated with mtDNA recombination.

Phylogenetic Relationships in Actinidia as Revealed by RAPD Analysis

Treesearch

Hongwen Huang; Zuozhou Li; Jianqiang Li; Thomas L. Kubiisiak; Desmond R. Lavne

2002-01-01

Phylogenetic relationships within the Actinidia were investigated using randomly amplified polymorphic DNA (RAPD) markers. DNAs from 10 taxa, including31 species encompassing all four sections and four series of the traditional subdivisions within the genus, were amplified using 22 preselected 10-mer oligonucieotide primers. A total 204 DNA bands...

Transforming growth factor-β and toll-like receptor-4 polymorphisms are not associated with fibrosis in haemochromatosis

PubMed Central

Wood, Marnie J; Powell, Lawrie W; Dixon, Jeannette L; Subramaniam, V Nathan; Ramm, Grant A

2013-01-01

AIM: To investigate the role of genetic polymorphisms in the progression of hepatic fibrosis in hereditary haemochromatosis. METHODS: A cohort of 245 well-characterised C282Y homozygous patients with haemochromatosis was studied, with all subjects having liver biopsy data and DNA available for testing. This study assessed the association of eight single nucleotide polymorphisms (SNPs) in a total of six genes including toll-like receptor 4 (TLR4), transforming growth factor-beta (TGF-β), oxoguanine DNA glycosylase, monocyte chemoattractant protein 1, chemokine C-C motif receptor 2 and interleukin-10 with liver disease severity. Genotyping was performed using high resolution melt analysis and sequencing. The results were analysed in relation to the stage of hepatic fibrosis in multivariate analysis incorporating other cofactors including alcohol consumption and hepatic iron concentration. RESULTS: There were significant associations between the cofactors of male gender (P = 0.0001), increasing age (P = 0.006), alcohol consumption (P = 0.0001), steatosis (P = 0.03), hepatic iron concentration (P < 0.0001) and the presence of hepatic fibrosis. Of the candidate gene polymorphisms studied, none showed a significant association with hepatic fibrosis in univariate or multivariate analysis incorporating cofactors. We also specifically studied patients with hepatic iron loading above threshold levels for cirrhosis and compared the genetic polymorphisms between those with no fibrosis vs cirrhosis however there was no significant effect from any of the candidate genes studied. Importantly, in this large, well characterised cohort of patients there was no association between SNPs for TGF-β or TLR4 and the presence of fibrosis, cirrhosis or increasing fibrosis stage in multivariate analysis. CONCLUSION: In our large, well characterised group of haemochromatosis subjects we did not demonstrate any relationship between candidate gene polymorphisms and hepatic fibrosis or cirrhosis. PMID:24409064

Transforming growth factor-β and toll-like receptor-4 polymorphisms are not associated with fibrosis in haemochromatosis.

PubMed

Wood, Marnie J; Powell, Lawrie W; Dixon, Jeannette L; Subramaniam, V Nathan; Ramm, Grant A

2013-12-28

To investigate the role of genetic polymorphisms in the progression of hepatic fibrosis in hereditary haemochromatosis. A cohort of 245 well-characterised C282Y homozygous patients with haemochromatosis was studied, with all subjects having liver biopsy data and DNA available for testing. This study assessed the association of eight single nucleotide polymorphisms (SNPs) in a total of six genes including toll-like receptor 4 (TLR4), transforming growth factor-beta (TGF-β), oxoguanine DNA glycosylase, monocyte chemoattractant protein 1, chemokine C-C motif receptor 2 and interleukin-10 with liver disease severity. Genotyping was performed using high resolution melt analysis and sequencing. The results were analysed in relation to the stage of hepatic fibrosis in multivariate analysis incorporating other cofactors including alcohol consumption and hepatic iron concentration. There were significant associations between the cofactors of male gender (P = 0.0001), increasing age (P = 0.006), alcohol consumption (P = 0.0001), steatosis (P = 0.03), hepatic iron concentration (P < 0.0001) and the presence of hepatic fibrosis. Of the candidate gene polymorphisms studied, none showed a significant association with hepatic fibrosis in univariate or multivariate analysis incorporating cofactors. We also specifically studied patients with hepatic iron loading above threshold levels for cirrhosis and compared the genetic polymorphisms between those with no fibrosis vs cirrhosis however there was no significant effect from any of the candidate genes studied. Importantly, in this large, well characterised cohort of patients there was no association between SNPs for TGF-β or TLR4 and the presence of fibrosis, cirrhosis or increasing fibrosis stage in multivariate analysis. In our large, well characterised group of haemochromatosis subjects we did not demonstrate any relationship between candidate gene polymorphisms and hepatic fibrosis or cirrhosis.

Screening of Israeli Holstein-Friesian cattle for restriction fragment length polymorphisms using homologous and heterologous deoxyribonucleic acid probes.

PubMed

Hallerman, E M; Nave, A; Soller, M; Beckmann, J S

1988-12-01

Genomic DNA of Israeli Holstein-Friesian dairy cattle were screened with a battery of 17 cloned or subcloned DNA probes in an attempt to document restriction fragment length polymorphisms at a number of genetic loci. Restriction fragment length polymorphisms were observed at the chymosin, oxytocin-neurophysin I, lutropin beta, keratin III, keratin VI, keratin VII, prolactin, and dihydrofolate reductase loci. Use of certain genomic DNA fragments as probes produced hybridization patterns indicative of satellite DNA at the respective loci. Means for distinguishing hybridizations to coding sequences for unique genes from those to satellite DNA were developed. Results of this study are discussed in terms of strategy for the systematic development of large numbers of bovine genomic polymorphisms.

Genetic alterations of hepatocellular carcinoma by random amplified polymorphic DNA analysis and cloning sequencing of tumor differential DNA fragment

PubMed Central

Xian, Zhi-Hong; Cong, Wen-Ming; Zhang, Shu-Hui; Wu, Meng-Chao

2005-01-01

AIM: To study the genetic alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC), and to find the tumor related DNA fragments. METHODS: DNA isolated from tumors and corresponding noncancerous liver tissues of 56 HCC patients was amplified by random amplified polymorphic DNA (RAPD) with 10 random 10-mer arbitrary primers. The RAPD bands showing obvious differences in tumor tissue DNA corresponding to that of normal tissue were separated, purified, cloned and sequenced. DNA sequences were analyzed and compared with GenBank data. RESULTS: A total of 56 cases of HCC were demonstrated to have genetic alterations, which were detected by at least one primer. The detestability of genetic alterations ranged from 20% to 70% in each case, and 17.9% to 50% in each primer. Serum HBV infection, tumor size, histological grade, tumor capsule, as well as tumor intrahepatic metastasis, might be correlated with genetic alterations on certain primers. A band with a higher intensity of 480 bp or so amplified fragments in tumor DNA relative to normal DNA could be seen in 27 of 56 tumor samples using primer 4. Sequence analysis of these fragments showed 91% homology with Homo sapiens double homeobox protein DUX10 gene. CONCLUSION: Genetic alterations are a frequent event in HCC, and tumor related DNA fragments have been found in this study, which may be associated with hepatocarcin-ogenesis. RAPD is an effective method for the identification and analysis of genetic alterations in HCC, and may provide new information for further evaluating the molecular mechanism of hepatocarcinogenesis. PMID:15996039

Extensive sequence-influenced DNA methylation polymorphism in the human genome

PubMed Central

2010-01-01

Background Epigenetic polymorphisms are a potential source of human diversity, but their frequency and relationship to genetic polymorphisms are unclear. DNA methylation, an epigenetic mark that is a covalent modification of the DNA itself, plays an important role in the regulation of gene expression. Most studies of DNA methylation in mammalian cells have focused on CpG methylation present in CpG islands (areas of concentrated CpGs often found near promoters), but there are also interesting patterns of CpG methylation found outside of CpG islands. Results We compared DNA methylation patterns on both alleles between many pairs (and larger groups) of related and unrelated individuals. Direct observation and simulation experiments revealed that around 10% of common single nucleotide polymorphisms (SNPs) reside in regions with differences in the propensity for local DNA methylation between the two alleles. We further showed that for the most common form of SNP, a polymorphism at a CpG dinucleotide, the presence of the CpG at the SNP positively affected local DNA methylation in cis. Conclusions Taken together with the known effect of DNA methylation on mutation rate, our results suggest an interesting interdependence between genetics and epigenetics underlying diversity in the human genome. PMID:20497546

DNA polymorphism analysis of candidate genes for type 2 diabetes mellitus in a Mexican ethnic group.

PubMed

Flores-Martínez, S E; Islas-Andrade, S; Machorro-Lazo, M V; Revilla, M C; Juárez, R E; Mújica-López, K I; Morán-Moguel, M C; López-Cardona, M G; Sánchez-Corona, J

2004-01-01

Type 2 diabetes mellitus is a complex metabolic disorder resulting from the action and interaction of many genetic and environmental factors. It has been reported that polymorphisms in genes involved in the metabolism of glucose are associated with the susceptibility to develop type 2 diabetes mellitus. Although the risk of developing type 2 diabetes mellitus increases with age, as well as with obesity and hypertension, its prevalence and incidence are different among geographical regions and ethnic groups. In Mexico, a higher prevalence and incidence has been described in the south of the country, and differences between urban and rural communities have been observed. We studied 73 individuals from Santiago Jamiltepec, a small indigenous community from Oaxaca State, Mexico. This population has shown a high prevalence of type 2 diabetes mellitus, and the aim of this study was to analyze the relationship between the Pst I (insulin gene), Nsi I (insulin receptor gene) and Gly972Arg (insulin receptor substrate 1 gene) polymorphisms and type 2 diabetes mellitus, obesity and hypertension in this population. Clinical evaluation consisted of BMI and blood pressure measurements, and biochemical assays consisted of determination of fasting plasma insulin and glucose levels. PCR and restriction enzyme digestion analysis were applied to genomic DNA to identify the three polymorphisms. From statistical analysis carried out here, individually, the Pst I, Nsi I and Gly972Arg polymorphisms were not associated with the type 2 diabetes, obese or hypertensive phenotypes in this population. Nevertheless, there was an association between the Nsi I and Pst I polymorphisms and increased serum insulin levels.

Assessment of genetic diversity among Indian potato (Solanum tuberosum L.) collection using microsatellite and retrotransposon based marker systems.

PubMed

Sharma, Vishakha; Nandineni, Madhusudan R

2014-04-01

Potato (Solanum tuberosum) is an important non-cereal crop throughout the world and is highly recommended for ensuring global food security. Owing to the complexities in genetics and inheritance pattern of potato, the conventional method of cross breeding for developing improved varieties has been difficult. Identification and tagging of desirable traits with informative molecular markers would aid in the development of improved varieties. Insertional polymorphism of copia-like and gypsy-like long terminal repeat retrotransposons (RTN) were investigated among 47 potato varieties from India using Inter-Retrotransposon Amplified Polymorphism (IRAP) and Retrotransposon Microsatellite Amplified Polymorphism (REMAP) marker techniques and were compared with the DNA profiles obtained with simple sequence repeats (SSRs). The genetic polymorphism, efficiency of polymorphism and effectiveness of marker systems were evaluated to assess the extent of genetic diversity among Indian potato varieties. A total of 139 polymorphic SSR alleles, 270 IRAP and 98 REMAP polymorphic bands, showing polymorphism of 100%, 87.9% and 68.5%, respectively, were used for detailed characterization of the genetic relationships among potato varieties by using cluster analysis and principal coordinate analysis (PCoA). IRAP analysis resulted in the highest number of polymorphic bands with an average of 15 polymorphic bands per assay unit when compared to the other two marker systems. Based on pair-wise comparison, the genetic similarity was calculated using Dice similarity coefficient. The SSRs showed a wide range in genetic similarity values (0.485-0.971) as compared to IRAP (0.69-0.911) and REMAP (0.713-0.947). A Mantel's matrix correspondence test showed a high positive correlation (r=0.6) between IRAP and REMAP, an intermediate value (r=0.58) for IRAP and SSR and the lowest value (r=0.17) for SSR and REMAP. Statistically significant cophenetic correlation coefficient values, of 0.961, 0.941 and 0.905 were observed for REMAP, IRAP and SSR, respectively. The widespread presence and distinct DNA profiles for copia-like and gypsy-like RTNs in the examined genotypes indicate that these elements are active in the genome and may have even contributed to the potato genome organization. Although the three marker systems were capable of distinguishing all the 47 varieties; high reproducibility, low cost and ease of DNA profiling data collection make IRAP and REMAP markers highly efficient whole-genome scanning molecular probes for population genetic studies. Information obtained from the present study regarding the genetic association and distinctiveness provides an useful guide for selection of germplasm for plant breeding and conservation efforts. Copyright © 2014. Published by Elsevier Inc.

[Polymorphism of genes encoding proteins of DNA repair vs. occupational and environmental exposure to lead, arsenic and pesticides].

PubMed

Bukowski, Karol; Woźniak, Katarzyna

2018-03-09

Genetic polymorphism is associated with the occurrence of at least 2 different alleles in the locus with a frequency higher than 1% in the population. Among polymorphisms we can find single nucleotide polymorphism (SNP) and polymorphism of variable number of tandem repeats. The presence of certain polymorphisms in genes encoding DNA repair enzymes is associated with the speed and efficiency of DNA repair and can protect or expose humans to the effects provoked by xenobiotics. Chemicals, such as lead, arsenic pesticides are considered to exhibit strong toxicity. There are many different polymorphisms in genes encoding DNA repair enzymes, which determine the speed and efficiency of DNA damage repair induced by these xenobiotics. In the case of lead, the influence of various polymorphisms, such as APE1 (apurinic/apyrimidinic endonuclease 1) (rs1130409), hOGG1 (human 8-oxoguanine glycosylase) (rs1052133), XRCC1 (X-ray repair cross-complementing protein group 1) (rs25487), XRCC1 (rs1799782) and XRCC3 (X-ray repair cross-complementing protein group 3) (rs861539) were described. For arsenic polymorphisms, such as ERCC2 (excision repair cross-complementing) (rs13181), XRCC3 (rs861539), APE1 (rs1130409) and hOGG1 (rs1052133) were examined. As to pesticides, separate and combined effects of polymorphisms in genes encoding DNA repair enzymes, such as XRCC1 (rs1799782), hOGG1 (rs1052133), XRCC4 (X-ray repair cross-complementing protein group 4) (rs28360135) and the gene encoding the detoxification enzyme PON1 paraoxonase (rs662) were reported. Med Pr 2018;69(2):225-235. This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.

Comparison of semi-automated commercial rep-PCR fingerprinting, spoligotyping, 12-locus MIRU-VNTR typing and single nucleotide polymorphism analysis of the embB gene as molecular typing tools for Mycobacterium bovis.

PubMed

Armas, Federica; Camperio, Cristina; Coltella, Luana; Selvaggini, Serena; Boniotti, Maria Beatrice; Pacciarini, Maria Lodovica; Di Marco Lo Presti, Vincenzo; Marianelli, Cinzia

2017-08-04

Highly discriminatory genotyping strategies are essential in molecular epidemiological studies of tuberculosis. In this study we evaluated, for the first time, the efficacy of the repetitive sequence-based PCR (rep-PCR) DiversiLab Mycobacterium typing kit over spoligotyping, 12-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and embB single nucleotide polymorphism (SNP) analysis for Mycobacterium bovis typing. A total of 49 M. bovis animal isolates were used. DNA was extracted and genomic DNA was amplified using the DiversiLab Mycobacterium typing kit. The amplified fragments were separated and detected using a microfluidics chip with Agilent 2100. The resulting rep-PCR-based DNA fingerprints were uploaded to and analysed using web-based DiversiLab software through Pearson's correlation coefficient. Rep-PCR DiversiLab grouped M. bovis isolates into ten different clusters. Most isolates sharing identical spoligotype, MIRU-VNTR profile or embB gene polymorphism were grouped into different rep-PCR clusters. Rep-PCR DiversiLab displayed greater discriminatory power than spoligotyping and embB SNP analysis but a lower resolution power than the 12-locus MIRU-VNTR analysis. MIRU-VNTR confirmed that it is superior to the other PCR-based methods tested here. In combination with spoligotyping and 12-locus MIRU-VNTR analysis, rep-PCR improved the discriminatory power for M. bovis typing.

Effects of Yangtze River source water on genomic polymorphisms of male mice detected by RAPD.

PubMed

Zhang, Xiaolin; Zhang, Zongyao; Zhang, Xuxiang; Wu, Bing; Zhang, Yan; Yang, Liuyan; Cheng, Shupei

2010-02-01

In order to evaluate the environmental health risk of drinking water from Yangtze River source, randomly amplified polymorphic DNA (RAPD) markers were used to detect the effects of the source water on genomic polymorphisms of hepatic cell of male mice (Mus musculus, ICR). After the mice were fed with source water for 90 days, RAPD-polymerase chain reactions (PCRs) were performed on hepatic genomic DNA using 20 arbitrary primers. Totally, 189 loci were generated, including 151 polymorphic loci. On average, one PCR primer produced 5.3, 4.9 and 4.8 bands for each mouse in the control, the groups fed with source water and BaP solution, respectively. Compared with the control, feeding mice with Yangtze River source water caused 33 new loci to appear and 19 to disappear. Statistical analysis of RAPD printfingers revealed that Yangtze River source water exerted a significant influence on the hepatic genomic polymorphisms of male mice. This study suggests that RAPD is a reliable and sensitive method for the environmental health risk of Yangtze River source water.

Environmental polycyclic aromatic hydrocarbon (PAH) exposure and DNA damage in Mexican children.

PubMed

Sánchez-Guerra, Marco; Pelallo-Martínez, Nadia; Díaz-Barriga, Fernando; Rothenberg, Stephen J; Hernández-Cadena, Leticia; Faugeron, Sylvain; Oropeza-Hernández, Luis F; Guaderrama-Díaz, Margarita; Quintanilla-Vega, Betzabet

2012-02-18

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants presenting a public health risk, particularly to children, a vulnerable population. PAHs have genotoxic and carcinogenic properties, which depend on their metabolism. Many enzymes involved in PAH metabolism, including CYP1A1, CYP1B1, GSTM and GSTT are polymorphic, which may modulate the activation/deactivation of these compounds. We evaluated PAH exposure and DNA damage in children living in the vicinity of the main petrochemical complex located in the Gulf of Mexico, and explored the modulation by genetic polymorphisms of PAH excretion and related DNA damage. The participants (n=82) were children aged 6-10y attending schools near the industrial area. Urinary 1-hydroxypyrene (1-OHP; a biomarker of PAH exposure) was determined by reverse-phase-HPLC; DNA damage by the comet assay (Olive Tail Moment (OTM) parameter); CYP1A1*2C and CYP1B1*3 polymorphisms by real time-PCR; and GSTM1*0 and GSTT1*0 by multiplex PCR. The median value of 1-OHP was 0.37μmol/mol creatinine; 59% of children had higher 1-OHP concentrations than those reported in environmentally exposed adults (0.24μmol/mol creatinine). A stratified analysis showed increased DNA damage in children with 1-OHP concentrations greater than the median value. We observed higher 1-OHP concentrations in children with CYP1A1*2C or GSTM1*0 polymorphisms, and a positive influence of CYP1A1*2C on OTM values in children with the highest PAH exposure. The data indicate that children living in the surroundings of petrochemical industrial areas are exposed to high PAH levels, contributing to DNA damage and suggesting an increased health risk; furthermore, data suggest that polymorphisms affecting activation enzymes may modulate PAH metabolism and toxicity. Copyright © 2012 Elsevier B.V. All rights reserved.

Genetic characterization of hybridization and introgression between anadromous rainbow trout (oncorhynchus mykiss irideus) and coastal cutthroat trout (o. clarki clarki)

USGS Publications Warehouse

Young, W.P.; Ostberg, C.O.; Keim, P.; Thorgaard, G.H.

2001-01-01

Interspecific hybridization represents a dynamic evolutionary phenomenon and major conservation problem in salmonid fishes. In this study we used amplified fragment length polymorphisms (AFLP) and mitochondrial DNA (mtDNA) markers to describe the extent and characterize the pattern of hybridization and introgression between coastal rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki). Hybrid individuals were initially identified using principle coordinate analysis of 133 polymorphic AFLP markers. Subsequent analysis using 23 diagnostic AFLP markers revealed the presence of F1, rainbow trout backcross, cutthroat trout backcross and later-generation hybrids. mtDNA analysis demonstrated equal numbers of F1 hybrids with rainbow and cutthroat trout mtDNA indicating reciprocal mating of the parental types. In contrast, rainbow and cutthroat trout backcross hybrids always exhibited the mtDNA from the recurrent parent, indicating a male hybrid mating with a pure female. This study illustrates the usefulness of the AFLP technique for generating large numbers of species diagnostic markers. The pattern of hybridization raises many questions concerning the existence and action of reproductive isolating mechanisms between these two species. Our findings are consistent with the hypothesis that introgression between anadromous populations of coastal rainbow and coastal cutthroat trout is limited by an environment-dependent reduction in hybrid fitness.

Effect of Gamma Rays on Sophora davidii and Detection of DNA Polymorphism through ISSR Marker

PubMed Central

Wang, Puchang; Mo, Bentian; Luo, Tianqiong

2017-01-01

Sophora davidii (Franch.) Kom. ex Pavol is an important medicinal plant and a feeding scrub with ecological value. The effects of different gamma irradiation doses (20–140 Kr) on seed germination and seedling morphology were investigated in S. davidii, and intersimple sequence repeat (ISSR) markers were used to identify the DNA polymorphism among mutants. Significant variations were observed for seed germination, stem diameter, and number of branches per plant. The improved agronomic traits, such as stem diameter and number of branches per plant, were recorded at 80 Kr dose and 20 Kr dose for seed germination. ISSR analysis generated in total 183 scorable fragments, of which 94 (51.37%) were polymorphic. The percentage of polymorphism ranged from 14.29 to 93.33 with an average of 45.69%. Jaccard's coefficients of dissimilarity varied from 0.6885 to 1.000, indicative of the level of genetic variation among the mutants. The constructed dendrogram grouped the entities into five clusters. Consequently, it was concluded that gamma rays irradiation of seeds generates a sufficient number of induced mutations and that ISSR analysis offered a useful molecular marker for the identification of mutants. PMID:28612030

Influence of XRCC1 Genetic Polymorphisms on Ionizing Radiation-Induced DNA Damage and Repair.

PubMed

Sterpone, Silvia; Cozzi, Renata

2010-07-25

It is well known that ionizing radiation (IR) can damage DNA through a direct action, producing single- and double-strand breaks on DNA double helix, as well as an indirect effect by generating oxygen reactive species in the cells. Mammals have evolved several and distinct DNA repair pathways in order to maintain genomic stability and avoid tumour cell transformation. This review reports important data showing a huge interindividual variability on sensitivity to IR and in susceptibility to developing cancer; this variability is principally represented by genetic polymorphisms, that is, DNA repair gene polymorphisms. In particular we have focussed on single nucleotide polymorphisms (SNPs) of XRCC1, a gene that encodes for a scaffold protein involved basically in Base Excision Repair (BER). In this paper we have reported and presented recent studies that show an influence of XRCC1 variants on DNA repair capacity and susceptibility to breast cancer.

A polymorphic DNA marker that represents a conserved expressed sequence in the region of the Huntington disease gene.

PubMed Central

Hayden, M R; Hewitt, J; Wasmuth, J J; Kastelein, J J; Langlois, S; Conneally, M; Haines, J; Smith, B; Hilbert, C; Allard, D

1988-01-01

A polymorphic marker (D4S62) that is genetically closely linked to D4S10 and is in the region of the gene for Huntington disease is described. A four-allele polymorphism is detected when HincII-digested DNA is hybridized with D4S62. D4S62 maps, by Southern blot analysis using somatic-cell hybrids, to 4p16.1 closer to the centromere than does D4S10. The use of the polymorphisms detected by D4S62 increases the informativeness of markers close to the gene for Huntington disease and will be useful for preclinical diagnosis. D4S62 detects transcripts of approximately 6,000 nucleotides in rat, mouse, and monkey liver and brain. This represents the first demonstration of conserved expressed sequences close to the gene for Huntington disease. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 PMID:2892395

Cadmium-induced genomic instability in Arabidopsis: Molecular toxicological biomarkers for early diagnosis of cadmium stress.

PubMed

Wang, Hetong; He, Lei; Song, Jie; Cui, Weina; Zhang, Yanzhao; Jia, Chunyun; Francis, Dennis; Rogers, Hilary J; Sun, Lizong; Tai, Peidong; Hui, Xiujuan; Yang, Yuesuo; Liu, Wan

2016-05-01

Microsatellite instability (MSI) analysis, random-amplified polymorphic DNA (RAPD), and methylation-sensitive arbitrarily primed PCR (MSAP-PCR) are methods to evaluate the toxicity of environmental pollutants in stress-treated plants and human cancer cells. Here, we evaluate these techniques to screen for genetic and epigenetic alterations of Arabidopsis plantlets exposed to 0-5.0 mg L(-1) cadmium (Cd) for 15 d. There was a substantial increase in RAPD polymorphism of 24.5, and in genomic methylation polymorphism of 30.5-34.5 at CpG and of 14.5-20 at CHG sites under Cd stress of 5.0 mg L(-1) by RAPD and of 0.25-5.0 mg L(-1) by MSAP-PCR, respectively. However, only a tiny increase of 1.5 loci by RAPD occurred under Cd stress of 4.0 mg L(-1), and an additional high dose (8.0 mg L(-1)) resulted in one repeat by MSI analysis. MSAP-PCR detected the most significant epigenetic modifications in plantlets exposed to Cd stress, and the patterns of hypermethylation and polymorphisms were consistent with inverted U-shaped dose responses. The presence of genomic methylation polymorphism in Cd-treated seedlings, prior to the onset of RAPD polymorphism, MSI and obvious growth effects, suggests that these altered DNA methylation loci are the most sensitive biomarkers for early diagnosis and risk assessment of genotoxic effects of Cd pollution in ecotoxicology. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genetic diversity study of Chromobacterium violaceum isolated from Kolli Hills by amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD).

PubMed

Ponnusamy, K; Jose, S; Savarimuthu, I; Michael, G P; Redenbach, M

2011-09-01

Chromobacterium are saprophytes that cause highly fatal opportunistic infections. Identification and strain differentiation were performed to identify the strain variability among the environmental samples. We have evaluated the suitability of individual and combined methods to detect the strain variations of the samples collected in different seasons. Amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD) profiles were obtained using four different restriction enzyme digestions (AluI, HaeIII, MspI and RsaI) and five random primers. A matrix of dice similarity coefficients was calculated and used to compare these restriction patterns. ARDRA showed rapid differentiation of strains based on 16S rDNA, but the combined RAPD and ARDRA gave a more reliable differentiation than when either of them was analysed individually. A high level of genetic diversity was observed, which indicates that the Kolli Hills' C. violaceum isolates would fall into at least three new clusters. Results showed a noteworthy bacterial variation and genetic diversity of C. violaceum in the unexplored, virgin forest area. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

Use of DNA markers in forest tree improvement research

Treesearch

D.B. Neale; M.E. Devey; K.D. Jermstad; M.R. Ahuja; M.C. Alosi; K.A. Marshall

1992-01-01

DNA markers are rapidly being developed for forest trees. The most important markers are restriction fragment length polymorphisms (RFLPs), polymerase chain reaction- (PCR) based markers such as random amplified polymorphic DNA (RAPD), and fingerprinting markers. DNA markers can supplement isozyme markers for monitoring tree improvement activities such as; estimating...

Development of polymorphic genic-SSR markers by cDNA library sequencing in boxwood, Buxus spp. (Buxaceae)

USDA-ARS?s Scientific Manuscript database

Genic microsatellites or simple sequence repeat (genic-SSR) markers were developed in boxwood (Buxus taxa) for genetic diversity analysis, identification of taxa, and to facilitate breeding. cDNA libraries were developed from mRNA extracted from leaves of Buxus sempervirens ‘Vardar Valley’ and seque...

RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF PCR-AMPLIFIED NIFH SEQUENCES FROM WETLAND PLANT RHIZOSPHERE COMMUNITIES

EPA Science Inventory

We describe a method to assess the community structure of N2-fixing bacteria in the rhizosphere. Total DNA was extracted from Spartina alterniflora and Sesbania macrocarpa root zones by bead-beating and purified by CsCl-EtBr gradient centrifugation. The average DNA yield was 5.5 ...

A functional polymorphism in the reduced folate carrier gene and DNA hypomethylation in mothers of children with autism

PubMed Central

James, S. Jill; Melnyk, Stepan; Jernigan, Stefanie; Lehman, Sara; Seidel, Lisa; Gaylor, David .W.; Cleves, Mario A.

2010-01-01

The biologic basis of autism is complex and is thought to involve multiple and variable gene-environment interactions. While the logical focus has been on the affected child, the impact of maternal genetics on intrauterine microenvironment during pivotal developmental windows could be substantial. Folate-dependent one carbon metabolism is a highly polymorphic pathway that regulates the distribution of one-carbon derivatives between DNA synthesis (proliferation) and DNA methylation (cell-specific gene expression and differentiation). These pathways are essential to support the programmed shifts between proliferation and differentiation during embryogenesis and organogenesis. Maternal genetic variants that compromise intrauterine availability of folate derivatives could alter fetal cell trajectories and disrupt normal neurodevelopment. In this investigation, the frequency of common functional polymorphisms in the folate pathway was investigated in a large population-based sample of autism case-parent triads. In case-control analysis, a significant increase in the reduced folate carrier (RFC1) G allele frequency was found among case mothers, but not among fathers or affected children. Subsequent log linear analysis of the RFC1 A80G genotype within family trios revealed that the maternal G allele was associated with a significant increase in risk of autism whereas the inherited genotype of the child was not. Further, maternal DNA from the autism mothers was found to be significantly hypomethylated relative to reference control DNA. Metabolic profiling indicated that plasma homocysteine, adenosine, and S-adenosylhomocyteine were significantly elevated among autism mothers consistent with reduced methylation capacity and DNA hypomethylation. Together, these results suggest that the maternal genetics/epigenetics may influence fetal predisposition to autism. PMID:20468076

A functional polymorphism in the reduced folate carrier gene and DNA hypomethylation in mothers of children with autism.

PubMed

James, S Jill; Melnyk, Stepan; Jernigan, Stefanie; Pavliv, Oleksandra; Trusty, Timothy; Lehman, Sara; Seidel, Lisa; Gaylor, David W; Cleves, Mario A

2010-09-01

The biologic basis of autism is complex and is thought to involve multiple and variable gene-environment interactions. While the logical focus has been on the affected child, the impact of maternal genetics on intrauterine microenvironment during pivotal developmental windows could be substantial. Folate-dependent one carbon metabolism is a highly polymorphic pathway that regulates the distribution of one-carbon derivatives between DNA synthesis (proliferation) and DNA methylation (cell-specific gene expression and differentiation). These pathways are essential to support the programmed shifts between proliferation and differentiation during embryogenesis and organogenesis. Maternal genetic variants that compromise intrauterine availability of folate derivatives could alter fetal cell trajectories and disrupt normal neurodevelopment. In this investigation, the frequency of common functional polymorphisms in the folate pathway was investigated in a large population-based sample of autism case-parent triads. In case-control analysis, a significant increase in the reduced folate carrier (RFC1) G allele frequency was found among case mothers, but not among fathers or affected children. Subsequent log linear analysis of the RFC1 A80G genotype within family trios revealed that the maternal G allele was associated with a significant increase in risk of autism whereas the inherited genotype of the child was not. Further, maternal DNA from the autism mothers was found to be significantly hypomethylated relative to reference control DNA. Metabolic profiling indicated that plasma homocysteine, adenosine, and S-adenosylhomocyteine were significantly elevated among autism mothers consistent with reduced methylation capacity and DNA hypomethylation. Together, these results suggest that the maternal genetics/epigenetics may influence fetal predisposition to autism. (c) 2010 Wiley-Liss, Inc.

Typing and Subtyping of 83 Clinical Isolates Purified from Surgically Implanted Silicone Feeding Tubes by Random Amplified Polymorphic DNA Amplification

PubMed Central

Dautle, Melanie P.; Ulrich, Ricky L.; Hughes, Thomas A.

2002-01-01

In this study, 83 clinical isolates purified from biofilms colonizing 18 silicone gastrostomy devices (12 “buttons” and six tubes converted to skin level devices) were selected for subtype characterization utilizing genetic analysis. The tubes, previously used for feeding, remained in place for 3 to 47 months (mean, 20.0 months) in children ranging in age from 6 months to 17 years. Classification of specific microbes using random amplified polymorphic DNA (RAPD) analysis revealed genetic similarities and differences among isolates belonging to the same genus. Both gram-positive and -negative bacteria were investigated, including 2 isolates of Bacillus brevis, 4 isolates of Bacillus licheniformis, 2 isolates of Bacillus pumilus, 3 isolates of Enterococcus durans, 19 isolates of Enterococcus faecalis, 8 isolates of Enterococcus faecium, 2 isolates of Enterococcus hirae, 7 isolates of Escherichia coli, 8 isolates of Lactobacillus plantarum, 19 isolates of Staphylococcus aureus, 2 isolates of Staphylococcus epidermidis, and 7 isolates of Staphylococcus saprophyticus. Amplified DNA fragments (amplicons) provided species-specific fingerprints for comparison by agarose gel electrophoresis. A total of 62 distinct RAPD types were categorized from the five genera studied. Typing analysis suggested cross acquisition of E. coli, E. faecalis, and S. aureus in three patient pairs. Genomic polymorphism detection proved efficient and reliable for classifying bacterial subtypes isolated from biofilms adhering to various portions of commonly employed enteral access tubes. PMID:11825951

Efficiency of mitochondrial DNA restriction analysis and RAPD-PCR to characterize yeasts growing on dry-cured Iberian ham at the different geographic areas of ripening.

PubMed

Andrade, María J; Rodríguez, Mar; Casado, Eva; Córdoba, Juan J

2010-03-01

The efficiency of mitochondrial DNA (mtDNA) restriction analysis and random amplification of polymorphic DNA (RAPD)-PCR to characterize yeasts growing on dry-cured Iberian ham was evaluated. Besides, the distribution of the main species and biotypes of yeasts in the different ripening areas of this product was investigated. MtDNA restriction analysis allowed yeast characterization at species and strain level. RAPD-PCR with the primers (GACA)(4) and (GAC)(5) was inappropriate for characterization at species level. Most of the mtDNA restriction patterns detected in dry-cured Iberian ham were consistent with Debaryomyces hansenii. Several yeasts biotypes were associated to specific geographic areas of dry-cured Iberian ham ripening. Copyright 2009 Elsevier Ltd. All rights reserved.

Sequence analysis of the mitochondrial DNA control region of ciscoes (genus Coregonus): Taxonomic implications for the Great Lakes species flock

USGS Publications Warehouse

Reed, Kent M.; Dorschner, Michael O.; Todd, Thomas N.; Phillips, Ruth B.

1998-01-01

Sequence variation in the control region (D-loop) of the mitochondrial DNA (mtDNA) was examined to assess the genetic distinctiveness of the shortjaw cisco (Coregonus zenithicus). Individuals from within the Great Lakes Basin as well as inland lakes outside the basin were sampled. DNA fragments containing the entire D-loop were amplified by PCR from specimens ofC. zenithicus and the related species C. artedi, C. hoyi, C. kiyi, and C. clupeaformis. DNA sequence analysis revealed high similarity within and among species and shared polymorphism for length variants. Based on this analysis, the shortjaw cisco is not genetically distinct from other cisco species.

Leishmania major: genetic heterogeneity of Iranian isolates by single-strand conformation polymorphism and sequence analysis of ribosomal DNA internal transcribed spacer.

PubMed

Tashakori, Mahnaz; Mahnaz, Tashakori; Kuhls, Katrin; Katrin, Kuhls; Al-Jawabreh, Amer; Amer, Al-Jawabreh; Mauricio, Isabel L; Isabel, Mauricio; Schönian, Gabriele; Gabriele, Schönian; Farajnia, Safar; Safar, Farajnia; Alimohammadian, Mohammad Hossein; Hossein, Alimohammadian Mohammad

2006-04-01

Protozoan parasites of Leishmania major are the causative agents of cutaneous leishmaniasis in different parts of Iran. We applied PCR-based methods to analyze L. major parasites isolated from patients with active lesions from different geographic areas in Iran in order to understand DNA polymorphisms within L. major species. Twenty-four isolates were identified as L. major by RFLP analysis of the ribosomal internal transcribed spacer 1 (ITS1) amplicons. These isolates were further studied by single-strand conformation polymorphism (SSCP) analysis and sequencing of ITS1 and ITS2. Data obtained from SSCP analysis of the ITS1 and ITS2 loci revealed three and four different patterns among all studied samples, respectively. Sequencing of ITS1 and ITS2 confirmed the results of SSCP analysis and showed the potential of the PCR-SSCP method for assessing genetic heterogeneity within L. major. Different patterns in ITS1 were due to substitution of one nucleotide, whereas in ITS2 the changes were defined by variation in the number of repeats in two polymorphic microsatellites. In total five genotypic groups LmA, LmB, LmC, LmD and LmE were identified among L. major isolates. The most frequent genotype, LmA, was detected in isolates collected from different endemic areas of cutaneous leishmaniasis in Iran. Genotypes LmC, LmD and LmE were found only in the new focus of CL in Damghan (Semnan province) and LmB was identified exclusively among isolates of Kashan focus (Isfahan province). The distribution of genetic polymorphisms suggests the existence of distinct endemic regions of L. major in Iran.

Interaction of methylation-related genetic variants with circulating fatty acids on plasma lipids: a meta-analysis of 7 studies & methylation analysis of 3 studies in the Cohorts for Heart & Aging Research

USDA-ARS?s Scientific Manuscript database

Background: DNA methylation is influenced by diet and single nucleotide polymorphisms (SNPs), and methylation modulates gene expression. Objective: We aimed to explore whether the gene-by-diet interactions on blood lipids act through DNA methylation. Design: We selected 7 SNPs on the basis of predic...

An Authentic RFLP Lab for High School or College Biology Students.

ERIC Educational Resources Information Center

Guilfoile, Patrick G.; Plum, Stephen

1998-01-01

Explains how students can perform an alternative authentic DNA fingerprinting analysis. Presents restriction fragment length polymorphism (RFLP) analysis and can serve as a simulated molecular epidemiology laboratory or as a simulated forensic laboratory exercise. (DDR)

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs

PubMed Central

Kakuda, Tsuneo; Shojo, Hideki; Tanaka, Mayumi; Nambiar, Phrabhakaran; Minaguchi, Kiyoshi; Umetsu, Kazuo; Adachi, Noboru

2016-01-01

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10−13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs. PMID:27355212

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

PubMed

Kakuda, Tsuneo; Shojo, Hideki; Tanaka, Mayumi; Nambiar, Phrabhakaran; Minaguchi, Kiyoshi; Umetsu, Kazuo; Adachi, Noboru

2016-01-01

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.

A pseudoautosomal random amplified polymorphic DNA marker for the sex chromosomes of Silene dioica.

PubMed Central

Di Stilio, V S; Kesseli, R V; Mulcahy, D L

1998-01-01

The segregation pattern of an 810-bp random amplified polymorphic DNA (RAPD) band in the F1 and backcross generations of a Silene dioica (L.) Clairv. family provides evidence that this molecular marker is located in the pseudoautosomal region (PAR) of the X and Y chromosomes. The marker was found through a combination of bulked segregant analysis (BSA) and RAPD techniques. Recombination rates between this pseudoautosomal marker and the differentiating portion of the Y chromosome are 15% in both generations. Alternative explanations involving nondisjunction or autosomal inheritance are presented and discussed. Chromosome counts provide evidence against the nondisjunction hypothesis, and probability calculations argue against the possibility of autosomal inheritance. This constitutes the first report of a pseudoautosomal DNA marker for plant sex chromosomes. PMID:9691057

Multiplex fluorescent PCR for noninvasive prenatal detection of fetal-derived paternally inherited diseases using circulatory fetal DNA in maternal plasma.

PubMed

Tang, Dong-ling; Li, Yan; Zhou, Xin; Li, Xia; Zheng, Fang

2009-05-01

To develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma and use the established multiplex in noninvasive prenatal genetic diagnosis and its further applications in forensic casework. The DNA template was extracted from 47 pregnant women and the whole blood samples from the stated biological fathers were used to detect genotype. Using multiplex fluorescent PCR at 16 different polymorphic short tandem repeat (STR) loci, maternal DNA extracted from plasma samples at early pregnancy, medium pregnancy and late pregnancy were used to detect genotype. Their husbands' DNA was also used for fetal genotype ascertainment. Multiplex fluorescent PCR with 16 polymorphic short tandem repeats revealed the presence of fetal DNA in all cases. Every pregnant women/husband pair was informative in at least 3 of 16 loci. The chances of detecting paternally inherited fetal alleles ranged from 66.67 to 94.12%. They are 66.67% in early pregnancy, 85.71% in medium pregnancy and 94.12% in late pregnancy. The accuracy of Multiplex PCR assay to detect fetal DNA was 100%. Circulating fetal DNA analysis can be used as a possible alternative tool in routine laboratory prenatal diagnosis in the near future; this highly polymorphic STR multiplex has greatly improved the chances of detecting paternally inherited fetal alleles compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma. Our proposed technique can be applied to both female and male fetuses, which provides a sensitive, accurate and efficient method for noninvasive prenatal genetic diagnosis and forensic casework.

[Gene polymorphisms in patients with Down's syndrome].

PubMed

Kuz'mina, N S; Ushenkova, L I; Shagirova, Zh M; Sheĭkhaev, G O; Mikhaĭlov, V F; Kurbatova, L A; Mazurik, V K; Rubanovich, A V; Zasukhina, G D

2009-01-01

Polymorphisms of glutation-S-transferase (GSTM1, GSTT1 GSTP1) and methylentetrahydrofolate reductase (MTHFR) genes have been studied in DNA from blood lymphocytes of 18 patients with Down's syndrome and 61 controls. Frequencies of normal alleles of GST genotypes were lower in patients as compared to the controls. A DNA analysis of 11 patients and 17 controls revealed the presence of mutations in region 246-250 of exon 7 of the p53 gene in 4 patients. Mutations were not found in the control group. Due to the small sample size, the results of this study should be interpreted with caution and need replication in larger studies.

[New data on the phylogeography and genetic diversity of the brown bear Ursus arctos Linnaeus, 1758 of northeastern Eurasia (mtDNA control region polymorphism analysis)].

PubMed

Salomashkina, V V; Kholodova, M V; Tiuten'kov, O Iu; Moskvitina, N S; Erokhin, N G

2014-01-01

An analysis of polymorphism of the fragment of the control region of mitochondrial DNA of 53 tissue samples of the brown bear Ursus arctos from several regions of the eastern part of Russia was carried out. It was found that most of the described haplotypes belong to cluster 3a, the most common in Eurasia, and do not form regionally specific haplogroups. However, among the bears from Western and Eastern Siberia, as well as the island of Kunashir, three haplotypes were identified, which are close to the haplogroup typical of Eastern Hokkaido bears. The assumption was made of the existence in Siberia and the Far East of one or more Pleistocene refugia.

Functional analysis of regulatory single-nucleotide polymorphisms.

PubMed

Pampín, Sandra; Rodríguez-Rey, José C

2007-04-01

The identification of regulatory polymorphisms has become a key problem in human genetics. In the past few years there has been a conceptual change in the way in which regulatory single-nucleotide polymorphisms are studied. We revise the new approaches and discuss how gene expression studies can contribute to a better knowledge of the genetics of common diseases. New techniques for the association of single-nucleotide polymorphisms with changes in gene expression have been recently developed. This, together with a more comprehensive use of the old in-vitro methods, has produced a great amount of genetic information. When added to current databases, it will help to design better tools for the detection of regulatory single-nucleotide polymorphisms. The identification of functional regulatory single-nucleotide polymorphisms cannot be done by the simple inspection of DNA sequence. In-vivo techniques, based on primer-extension, and the more recently developed 'haploChIP' allow the association of gene variants to changes in gene expression. Gene expression analysis by conventional in-vitro techniques is the only way to identify the functional consequences of regulatory single-nucleotide polymorphisms. The amount of information produced in the last few years will help to refine the tools for the future analysis of regulatory gene variants.

CCAAT/enhancer-binding protein alpha (CEBPA) polymorphisms and mutations in healthy individuals and in patients with peripheral artery disease, ischaemic heart disease and hyperlipidaemia.

PubMed

Fuchs, O; Kostecka, A; Provazníková, D; Krásná, B; Kotlín, R; Stanková, M; Kobylka, P; Dostálová, G; Zeman, M; Chochola, M

2010-01-01

The CCAAT/enhancer-binding protein alpha, encoded by the intronless CEBPA gene, is a transcription factor that induces expression of genes involved in differentiation of granulocytes, monocytes, adipocytes and hepatocytes. Both mono- and bi-allelic CEBPA mutations were detected in acute myeloid leukaemia and myelodysplastic syndrome. In this study we also identified CEBPA mutations in healthy individuals and in patients with peripheral artery disease, ischaemic heart disease and hyperlipidaemia. We found 16 various deletions with the presence of two direct repeats in CEBPA by analysis of 431 individuals. Three most frequent repeats included in these deletions in CEBPA gene are CGCGAG (493- 498_865-870), GG (486-487_885-886), and GCCAAGCAGC (508-517_907-916), all according to GenBank Accession No. NM_004364.2. In one case we identified that a father with ischaemic heart disease and his healthy son had two identical deletions (493_864del and 508_906del, both according to GenBank Accession No. NM_004364.2) in CEBPA. The occurrence of deletions between two repetitive sequences may be caused by recombination events in the repair process. A double-stranded cut in DNA may initiate these recombination events in adjacent DNA sequences. Four types of polymorphisms in the CEBPA gene were also detected in the screened individuals. Polymorphism in CEBPA gene 690 G>T according to GenBank Accession No. NM_004364.2 is the most frequent type in our analysis. Statistical analysis did not find significant differences in the frequency of polymorphisms in CEBPA in patients and in healthy individuals with the exception of P4 polymorphism (580_585dup according to GenBank Accesion No. NM_004364.2). P4 polymorphism was significantly increased in ischaemic heart disease patients.

Individual specific DNA fingerprints from a hypervariable region probe: alpha-globin 3'HVR.

PubMed

Fowler, S J; Gill, P; Werrett, D J; Higgs, D R

1988-06-01

A probe detecting a hypervariable region (HVR) 3' to the alpha globin locus on chromosome 16 has been used to produce DNA fingerprints. Segregation analysis has revealed multiple, randomly dispersed DNA fragments inherited in a Mendelian fashion with minimal allelism and linkage. The fingerprints are highly polymorphic (probability of chance association between random individuals much less than 10(-14]. The probe is, therefore, a powerful discriminating tool: it is envisaged that this probe will have forensic applications, including paternity cases, and will be informative in linkage analysis.

New polymorphisms of Xeroderma Pigmentosum DNA repair genes in myelodysplastic syndrome.

PubMed

Santiago, Sabrina Pinheiro; Junior, Howard Lopes Ribeiro; de Sousa, Juliana Cordeiro; de Paula Borges, Daniela; de Oliveira, Roberta Taiane Germano; Farias, Izabelle Rocha; Costa, Marília Braga; Maia, Allan Rodrigo Soares; da Nóbrega Ito, Mayumi; Magalhães, Silvia Maria Meira; Pinheiro, Ronald Feitosa

2017-07-01

The association between Xeroderma Pigmentosum DNA repair genes (XPA rs1800975, XPC rs2228000, XPD rs1799793 and XPF rs1800067) polymorphisms and myelodysplastic syndrome (MDS) have not been reported. To assess the functional role between these polymorphisms and MDS, we evaluated 189 samples stratified in two groups: 95 bone marrow samples from MDS patients and 94 from healthy elderly volunteers used as controls. Genotypes for all polymorphisms were identified in DNA samples in an allelic discrimination experiment by real-time polymerase chain reaction (qPCR). We also studied the mRNA expression of XPA and XPC genes to evaluate if its polymorphisms were functional in 53 RNAm MDS patients by qPCR methodologies. To the rs2228000 polymorphism, the CT and TT polymorphic genotype were associated with increased odds ratio (OR) of more profound cytopenia (hemoglobin and neutrophils count). To the rs1799793 polymorphism, we found that the GG homozygous wild-type genotype was associated with a decreased chance of developing MDS. We observed low expression of XPA in younger patients, in hypoplastic MDS and patients with abnormal karyotype when presented AG or AA polymorphic genotypes. We also found that there was a statistically significant interaction between the presence of micromegakaryocyte on down regulation of XPC regarding the CT heterozygous genotype of the rs1800975 polymorphism. Our results suggest that new functional polymorphisms of Xeroderma Pigmentosum DNA repair genes in MDS are related to its pathogenesis and prognosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Single nucleotide polymorphisms in the mitochondrial displacement loop and outcome of esophageal squamous cell carcinoma.

PubMed

Zhang, Ruixing; Wang, Rui; Zhang, Fengbin; Wu, Chensi; Fan, Haiyan; Li, Yan; Wang, Cuiju; Guo, Zhanjun

2010-11-26

Accumulation of single nucleotide polymorphisms (SNPs) in the displacement loop (D-loop) of mitochondrial DNA (mtDNA) has been described for different types of cancers and might be associated with cancer risk and disease outcome. We used a population-based series of esophageal squamous cell carcinoma (ESCC) patients for investigating the prediction power of SNPs in mitochondrial D-loop. The D-loop region of mtDNA was sequenced for 60 ESCC patients recorded in the Fourth Hospital of Hebei Medical University between 2003 and 2004. The 5 year survival curve were calculated with the Kaplan-Meier method and compared by the log-rank test at each SNP site, a multivariate survival analysis was also performed with the Cox proportional hazards method. The SNP sites of nucleotides 16274G/A, 16278C/T and 16399A/G were identified for prediction of post-operational survival by the log-rank test. In an overall multivariate analysis, the 16278 and 16399 alleles were identified as independent predictors of ESCC outcome. The length of survival of patients with the minor allele 16278T genotype was significantly shorter than that of patients with 16278C at the 16278 site (relative risk, 3.001; 95% CI, 1.029 - 8.756; p = 0.044). The length of survival of patients with the minor allele 16399G genotype was significantly shorter than that of patients with the more frequent allele 16399A at the 16399 site in ESCC patients (relative risk, 3.483; 95% CI, 1.068 - 11.359; p = 0.039). Genetic polymorphisms in the D-loop are independent prognostic markers for patients with ESCC. Accordingly, the analysis of genetic polymorphisms in the mitochondrial D-loop can help identify patient subgroups at high risk of a poor disease outcome.

A comparative genetic analysis of the Irish greyhound population using multilocus DNA fingerprinting, canine single locus minisatellites and canine microsatellites.

PubMed

Sutton, M D; Holmes, N G; Brennan, F B; Binns, M M; Kelly, E P; Duke, E J

1998-06-01

Pairwise analysis of HinfI/33.6 DNA fingerprints from a total of one hundred and fifty-three Irish greyhounds of known pedigree were used to determine band-share estimates of unrelated, first-degree and second-degree relationships. Forty-eight unrelated Irish greyhounds were used to determine allele frequencies for three single-locus minisatellites, and following a preliminary screen, eight of the most polymorphic tetra-nucleotide microsatellites from a panel of 15. The results indicated that both band-share estimates by DNA fingerprinting and microsatellite allele frequencies are highly effective in resolving parentage in this greyhound population, while single-locus minisatellites showed limited polymorphism and could not be used alone for routine parentage testing in this breed. The present study also demonstrated that, to obtain optimal resolution of parentage, sample sets of known pedigree status are required to determine the band-share distribution and/or microsatellite allele frequencies.

DNA methylation changes detected by methylation-sensitive amplified polymorphism in two contrasting rice genotypes under salt stress.

PubMed

Wang, Wensheng; Zhao, Xiuqin; Pan, Yajiao; Zhu, Linghua; Fu, Binying; Li, Zhikang

2011-09-20

DNA methylation, one of the most important epigenetic phenomena, plays a vital role in tuning gene expression during plant development as well as in response to environmental stimuli. In the present study, a methylation-sensitive amplified polymorphism (MSAP) analysis was performed to profile DNA methylation changes in two contrasting rice genotypes under salt stress. Consistent with visibly different phenotypes in response to salt stress, epigenetic markers classified as stable inter-cultivar DNA methylation differences were determined between salt-tolerant FL478 and salt-sensitive IR29. In addition, most tissue-specific DNA methylation loci were conserved, while many of the growth stage-dependent DNA methylation loci were dynamic between the two genotypes. Strikingly, salt stress induced a decrease in DNA methylation specifically in roots at the seedling stage that was more profound in IR29 than in the FL478. This result may indicate that demethylation of genes is an active epigenetic response to salt stress in roots at the seedling stage, and helps to further elucidate the implications of DNA methylation in crop growth and development. Copyright © 2011. Published by Elsevier Ltd.

Single-molecule dilution and multiple displacement amplification for molecular haplotyping.

PubMed

Paul, Philip; Apgar, Josh

2005-04-01

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.

An association analysis between mitochondrial DNA content, G10398A polymorphism, HPV infection, and the prognosis of cervical cancer in the Chinese Han population.

PubMed

Feng, Dali; Xu, Hui; Li, Xin; Wei, Yuehua; Jiang, Huangang; Xu, Hong; Luo, Aihua; Zhou, Fuxiang

2016-04-01

The aim was to analyze quantitative (mitochondrial DNA (mtDNA) content) and qualitative (G10398A polymorphism) mtDNA alterations as well as human papillomavirus (HPV) infection in cervical cancer prognosis. One hundred and twenty-two cases of formalin-fixed paraffin-embedded cervical carcinoma specimens were collected from the Yichang Tumor Hospital and Zhongnan Hospital of Wuhan University in the recent 10 years together with medical records. A quantitative real-time PCR (RT-PCR) was used to determine the copy number of the mitochondrial DNA and HPV expression levels. G10398A polymorphism was determined by PCR-RFLP assay. The overall survival of patients with higher mtDNA content was significantly reduced compared with lower mtDNA content patients (P = 0.029). But there was no difference of prognosis between the mtDNA 10398 A allele and G allele. However, the Kaplan-Meier survival curve illustrated a significantly reduced overall survival in the patients with 10398A plus high mtDNA copy number compared with the other groups (P < 0.05). Although no association between HPV expression level and cervical cancer prognosis was observed, 10398A got increased mtDNA content compared with 10398G (P < 0.05) and 10398G displayed an increased HPV-positive rate compared with 10398A. Furthermore, HPV-18 and mtDNA content were positively related in the younger subgroup (≤45 years) (correlation coefficient = 0.456, P = 0.022). This study indicated that mtDNA content and HPV infection status are associated with cervical cancer prognosis. High mitochondrial DNA content plus 10398 A may be a marker of poor prognosis in cervical cancer. And mtDNA variation may potentially influence the predisposition to HPV infection and cervical carcinogenesis.

Isolation of human simple repeat loci by hybridization selection.

PubMed

Armour, J A; Neumann, R; Gobert, S; Jeffreys, A J

1994-04-01

We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

Rapid estimation of microbial populations in fish samples by using terminal restriction fragment length polymorphism analysis of 16S rDNA.

PubMed

Tanaka, Yuichiro; Takahashi, Hajime; Kitazawa, Nao; Kimura, Bon

2010-01-01

A rapid system using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting 16S rDNA is described for microbial population analysis in edible fish samples. The defined terminal restriction fragment database was constructed by collecting 102 strains of bacteria representing 53 genera that are associated with fish. Digestion of these 102 strains with two restriction enzymes, HhaI and MspI, formed 54 pattern groups with discrimination to the genus level. This T-RFLP system produced results comparable to those from a culture-based method in six natural fish samples with a qualitative correspondence of 71.4 to 92.3%. Using the T-RFLP system allowed an estimation of the microbial population within 7 h. Rapid assay of the microbial population is advantageous for food manufacturers and testing laboratories; moreover, the strategy presented here allows adaptation to specific testing applications.

Multiplexed Microsphere Suspension-Array Assay for Urine Mitochondrial DNA Typing by C-Stretch Length in Hypervariable Regions.

PubMed

Aoki, Kimiko; Tanaka, Hiroyuki; Kawahara, Takashi

2018-07-01

The standard method for personal identification and verification of urine samples in doping control is short tandem repeat (STR) analysis using nuclear DNA (nDNA). The DNA concentration of urine is very low and decreases under most conditions used for sample storage; therefore, the amount of DNA from cryopreserved urine samples may be insufficient for STR analysis. We aimed to establish a multiplexed assay for urine mitochondrial DNA typing containing only trace amounts of DNA, particularly for Japanese populations. A multiplexed suspension-array assay using oligo-tagged microspheres (Luminex MagPlex-TAG) was developed to measure C-stretch length in hypervariable region 1 (HV1) and 2 (HV2), five single nucleotide polymorphisms (SNPs), and one polymorphic indel. Based on these SNPs and the indel, the Japanese population can be classified into five major haplogroups (D4, B, M7a, A, D5). The assay was applied to DNA samples from urine cryopreserved for 1 - 1.5 years (n = 63) and fresh blood (n = 150). The assay with blood DNA enabled Japanese subjects to be categorized into 62 types, exhibiting a discriminatory power of 0.960. The detection limit for cryopreserved urine was 0.005 ng of nDNA. Profiling of blood and urine pairs revealed that 5 of 63 pairs showed different C-stretch patterns in HV1 or HV2. The assay described here yields valuable information in terms of the verification of urine sample sources employing only trace amounts of recovered DNA. However, blood cannot be used as a reference sample.

The Ins and Outs of DNA Fingerprinting the Infectious Fungi

PubMed Central

Soll, David R.

2000-01-01

DNA fingerprinting methods have evolved as major tools in fungal epidemiology. However, no single method has emerged as the method of choice, and some methods perform better than others at different levels of resolution. In this review, requirements for an effective DNA fingerprinting method are proposed and procedures are described for testing the efficacy of a method. In light of the proposed requirements, the most common methods now being used to DNA fingerprint the infectious fungi are described and assessed. These methods include restriction fragment length polymorphisms (RFLP), RFLP with hybridization probes, randomly amplified polymorphic DNA and other PCR-based methods, electrophoretic karyotyping, and sequencing-based methods. Procedures for computing similarity coefficients, generating phylogenetic trees, and testing the stability of clusters are then described. To facilitate the analysis of DNA fingerprinting data, computer-assisted methods are described. Finally, the problems inherent in the collection of test and control isolates are considered, and DNA fingerprinting studies of strain maintenance during persistent or recurrent infections, microevolution in infecting strains, and the origin of nosocomial infections are assessed in light of the preceding discussion of the ins and outs of DNA fingerprinting. The intent of this review is to generate an awareness of the need to verify the efficacy of each DNA fingerprinting method for the level of genetic relatedness necessary to answer the epidemiological question posed, to use quantitative methods to analyze DNA fingerprint data, to use computer-assisted DNA fingerprint analysis systems to analyze data, and to file data in a form that can be used in the future for retrospective and comparative studies. PMID:10756003

Effects of DNA Extraction Procedures on Bacteroides Profiles in Fecal Samples From Various Animals Determined by Terminal Restriction Fragment Length Polymorphism Analysis

EPA Science Inventory

A major assumption in microbial source tracking is that some fecal bacteria are specific to a host animal, and thus provide unique microbial fingerprints that can be used to differentiate hosts. However, the DNA information obtained from a particular sample may be biased dependi...

Chloroplast and mitochondrial DNA are paternally inherited in Sequoia sempervirens D. Don Endl

PubMed Central

Neale, David B.; Marshall, Kimberly A.; Sederoff, Ronald R.

1989-01-01

Restriction fragment length polymorphisms in controlled crosses were used to infer the mode of inheritance of chloroplast DNA and mitochondrial DNA in coast redwood (Sequoia sempervirens D. Don Endl.). Chloroplast DNA was paternally inherited, as is true for all other conifers studied thus far. Surprisingly, a restriction fragment length polymorphism detected by a mitochondrial probe was paternally inherited as well. This polymorphism could not be detected in hybridizations with chloroplast probes covering the entire chloroplast genome, thus providing evidence that the mitochondrial probe had not hybridized to chloroplast DNA on the blot. We conclude that mitochondrial DNA is paternally inherited in coast redwood. To our knowledge, paternal inheritance of mitochondrial DNA in sexual crosses of a multicellular eukaryotic organism has not been previously reported. Images PMID:16594091

Haemophilia A: carrier detection and prenatal diagnosis by linkage analysis using DNA polymorphism.

PubMed Central

Tuddenham, E G; Goldman, E; McGraw, A; Kernoff, P B

1987-01-01

Restriction fragment length polymorphisms (RFLPs) within or close to the factor VIII locus are very useful for genetic linkage analysis. Such RFLPs allow a mutant allele to be tracked in a family, segregating haemophilia A even when, as is usually the case, the precise mutation causing failure to synthesise factor VIII is unknown. To date two markers tightly linked to the factor VIII locus have been described, one of which is highly polymorphic and therefore informative in most kindreds. A significant crossover rate, however, does not make diagnosis absolute. Three intragenic RFLPs have been defined, which, taken together, are informative in about 70% of women, providing virtually deterministic genetic diagnosis. PMID:2889753

Unraveling the sequence-dependent polymorphic behavior of d(CpG) steps in B-DNA.

PubMed

Dans, Pablo Daniel; Faustino, Ignacio; Battistini, Federica; Zakrzewska, Krystyna; Lavery, Richard; Orozco, Modesto

2014-10-01

We have made a detailed study of one of the most surprising sources of polymorphism in B-DNA: the high twist/low twist (HT/LT) conformational change in the d(CpG) base pair step. Using extensive computations, complemented with database analysis, we were able to characterize the twist polymorphism in the d(CpG) step in all the possible tetranucleotide environment. We found that twist polymorphism is coupled with BI/BII transitions, and, quite surprisingly, with slide polymorphism in the neighboring step. Unexpectedly, the penetration of cations into the minor groove of the d(CpG) step seems to be the key element in promoting twist transitions. The tetranucleotide environment also plays an important role in the sequence-dependent d(CpG) polymorphism. In this connection, we have detected a previously unexplored intramolecular C-H···O hydrogen bond interaction that stabilizes the low twist state when 3'-purines flank the d(CpG) step. This work explains a coupled mechanism involving several apparently uncorrelated conformational transitions that has only been partially inferred by earlier experimental or theoretical studies. Our results provide a complete description of twist polymorphism in d(CpG) steps and a detailed picture of the molecular choreography associated with this conformational change. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Impact of DNA repair genes polymorphism (XPD and XRCC1) on the risk of breast cancer in Egyptian female patients.

PubMed

Hussien, Yousry Mostafa; Gharib, Amal F; Awad, Hanan A; Karam, Rehab A; Elsawy, Wael H

2012-02-01

The genes involved in DNA repair system play a crucial role in the protection against mutations. It has been hypothesized that functional deficiencies in highly conserved DNA repair processes resulting from polymorphic variation may increase genetic susceptibility to breast cancer (BC). The aim of the present study was to evaluate the association of genetic polymorphisms in 2 DNA repair genes, XPD (Asp312Asn) and XRCC1 (A399G), with BC susceptibility. We further investigated the potential combined effect of these DNA repair variants on BC risk. Both XPD (xeroderma pigmentosum group D) and XRCC1 (X-ray repair cross-complementing group 1) polymorphisms were characterized in 100 BC Egyptian females and 100 healthy women who had no history of any malignancy by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method and PCR with confronting two-pair primers (PCR-CTPP), using DNA from peripheral blood in a case control study. Our results revealed that the frequencies of AA genotype of XPD codon 312 polymorphism were significantly higher in the BC patients than in the normal individuals (P ≤ 0.003), and did not observe any association between the XRCC1 Arg399Gln polymorphism and risk of developing BC. Also, no association between both XPD Asp312Asn and XRCC1 A399G polymorphisms and the clinical characteristics of disease. Finally, the combination of AA(XPD) + AG(XRCC1) were significantly associated with BC risk. Our results suggested that, XPD gene is an important candidate gene for susceptibility to BC. Also, gene-gene interaction between XPD(AA) + XRCC1(AG) polymorphism may be associated with increased risk of BC in Egyptian women.

[The association between the DNA marker rs1842993 and risk for cardioembolic stroke in the Slavic population].

PubMed

Shetova, I M; Timofeev, D Iu; Shamalov, N A; Bondarenko, E A; Slominskiĭ, P A; Limborskaia, S A; Skvortsova, V I

2012-01-01

The analysis of association between DNA markers and total stroke risk was performed in 950 Slavonic patients. Patients with cardioembolic stroke were selected for a genome-wide association study. The HUMANCYTOSNP12 v.2 microchip was used to analyze all DNA samples on a panel of 301 000 single nucleotide polymorphisms. SNP rs1842993 on chromosome 7 was found to be associated with cardioembolic stroke risk.

Soil pretreatment and fast cell lysis for direct polymerase chain reaction from forest soils for terminal restriction fragment length polymorphism analysis of fungal communities

Treesearch

Fei Cheng; Lin Hou; Keith Woeste; Zhengchun Shang; Xiaobang Peng; Peng Zhao; Shuoxin Zhang

2016-01-01

Humic substances in soil DNA samples can influence the assessment of microbial diversity and community composition. Using multiple steps during or after cell lysis adds expenses, is time-consuming, and causes DNA loss. A pretreatment of soil samples and a single step DNA extraction may improve experimental results. In order to optimize a protocol for obtaining high...

Prenatal exclusion of Norrie disease with flanking DNA markers.

PubMed

Gal, A; Uhlhaas, S; Glaser, D; Grimm, T

1988-10-01

Three polymorphic DNA markers linked to the locus of Norrie disease were used for indirect genotype analysis in a ten-wk-old fetus at risk for the disease. When haplotypes of the family members and the estimated recombination frequency between Norrie gene and each of the DNA marker loci DXS7, DXS84, and DXS146 were taken into account, the risk that the fetus had inherited the mutation was about 1%.

DNA methylation and genome rearrangement characteristics of phase change in cultured shoots of Sequoia sempervirens.

PubMed

Huang, Li-Chun; Hsiao, Lin-June; Pu, Szu-Yuan; Kuo, Ching-I; Huang, Bau-Lian; Tseng, Tsung-Che; Huang, Hao-Jen; Chen, Yu-Ting

2012-06-01

Epigenetic machinery regulates the expression of individual genes and plays a crucial role in globally shaping and maintaining developmental patterning. We studied the extent of DNA methylation in the nucleus, mitochondrion and chloroplast in cultured Sequoia sempervirens (coast redwood) adult, juvenile and rejuvenated shoots by measuring the ratio of methylcytosine to total cytosine using high-performance liquid chromatography (HPLC). We also analyzed nuclear DNA (nuDNA) polymorphisms of different shoot types by methylation-sensitive amplified fragment length polymorphism (MSAP) and Southern blot analysis. The extent of nuDNA methylation was greater in the adult vegetative than juvenile and rejuvenated shoots (8% vs 6.5-7.5%). In contrast, the proportion of methylcytosine was higher in mitochondrial DNA (mDNA) of juvenile and rejuvenated shoots than adult shoots (6.6% vs 7.8-8.2%). MSAP and Southern blot analyses identified three MSAP fragments which could be applied as phase-specific molecular markers. We also found nuclear genome and mtDNA rearrangement may be as important as DNA methylation status during the phase change. Our findings strongly suggest that DNA methylation and genome rearrangement may affect the dynamic tissue- and cell type-specific changes that determine the developmental phase of S. sempervirens shoots. Copyright © Physiologia Plantarum 2012.

The Large Subunit rDNA Sequence of Plasmodiophora brassicae Does not Contain Intra-species Polymorphism

PubMed Central

Schwelm, Arne; Berney, Cédric; Dixelius, Christina; Bass, David; Neuhauser, Sigrid

2016-01-01

Clubroot disease caused by Plasmodiophora brassicae is one of the most important diseases of cultivated brassicas. P. brassicae occurs in pathotypes which differ in the aggressiveness towards their Brassica host plants. To date no DNA based method to distinguish these pathotypes has been described. In 2011 polymorphism within the 28S rDNA of P. brassicae was reported which potentially could allow to distinguish pathotypes without the need of time-consuming bioassays. However, isolates of P. brassicae from around the world analysed in this study do not show polymorphism in their LSU rDNA sequences. The previously described polymorphism most likely derived from soil inhabiting Cercozoa more specifically Neoheteromita-like glissomonads. Here we correct the LSU rDNA sequence of P. brassicae. By using FISH we demonstrate that our newly generated sequence belongs to the causal agent of clubroot disease. PMID:27750174

The effect of FokI vitamin D receptor polymorphism on bone mineral density in Jordanian perimenopausal women

PubMed Central

Kanan, Raed M.

2013-01-01

CONTEXT: Osteoporosis is a polygenic, multifactorial disease that is characterized by demineralization of bone, and thus presented with decreasing bone mineral mass. Vitamin D receptor (VDR) gene polymorphisms in the 3’-end region (as determined by the enzymes BsmI and ApaI) have been inconsistently associated with bone mineral mass. Another important VDR start codon polymorphism (as determined by the enzyme FokI) has been found to be related to adult bone mineral density (BMD) in pre-and post-menopausal American women. AIMS: This study aims to investigate the prevalence of the FokI VDR gene polymorphism in Jordanian perimenopausal women and study its relationship with bone mineral density. MATERIALS AND METHODS: DNA was isolated from 90 controls (Mean age = 50.41 ± 1.29 y), and 120 patients with symptomatic vertebral fractures (Mean age = 49.14 ± 3.19 y). Restriction Fragment Length Polymorphism (RFLP) analysis of FokI was performed on DNA samples. STATISTICAL ANALYSIS: Data was analyzed using SPSS v19 and Microsoft Excel 2007. RESULTS: The results showed that in controls, the FF (−0.70 ± 0.51) genotype is associated with high lumbar spine BMD Z-score as compared to Ff (−1.25 ± 0.26) and ff (−1.66 ± 0.47) genotypes (P = 0.0095). In patients, the ff genotype was associated with lower lumbar spine BMD in T-score (−2.31 ± 0.17) and Z-score (−1.56 ± 0.09) genotypes (P = 0.031). No significant association was seen in the femoral neck BMD. CONCLUSION: FokI polymorphism may be associated with low BMD in our studied population; however, further studies including other polymorphisms and large sample number are needed. PMID:24019627

Differentiation of Trichophyton rubrum clinical isolates from Japanese and Chinese patients by randomly amplified polymorphic DNA and DNA sequence analysis of the non-transcribed spacer region of the rRNA gene.

PubMed

Yang, Xiumin; Sugita, Takashi; Takashima, Masako; Hiruma, Masataro; Li, Ruoyu; Sudo, Hajime; Ogawa, Hideoki; Ikeda, Shigaku

2009-04-01

Trichophyton rubrum is the most common pathogen causing dermatophytosis worldwide. Recent genetic investigations showed that the microorganism originated in Africa and then spread to Europe and North America via Asia. We investigated the intraspecific diversity of T. rubrum isolated from two closely located Asian countries, Japan and China. A total of 150 clinical isolates of T. rubrum obtained from Japanese and Chinese patients were analyzed by randomly amplified polymorphic DNA (RAPD) and DNA sequence analysis of the non-transcribed spacer (NTS) region in the rRNA gene. RAPD analysis divided the 150 strains into two major clusters, A and B. Of the Japanese isolates, 30% belonged to cluster A and 70% belonged to cluster B, whereas 91% of the Chinese isolates were in cluster A. The NTS region of the rRNA gene was divided into four major groups (I-IV) based on DNA sequencing. The majority of Japanese isolates were type IV (51%), and the majority of Chinese isolates were type III (75%). These results suggest that although Japan and China are neighboring countries, the origins of T. rubrum isolates from these countries may not be identical. These findings provide information useful for tracing the global transmission routes of T. rubrum.

Assessing the germplasm of Laminaria (phaeophyceae) with random amplified polymorphic DNA (RAPD) method

NASA Astrophysics Data System (ADS)

He, Yingjun; Zou, Yuping; Wang, Xiaodong; Zheng, Zhiguo; Zhang, Daming; Duan, Delin

2003-06-01

Eighteen gametophytes including L. japonica, L. ochotensis and L. longissima, were verified with random amplified polymorphic DNA (RAPD) technique. Eighteen ten-base primers were chosen from 100 primers selected for final amplification test. Among the total of 205 bands amplified, 181 (88.3%) were polymorphic. The genetic distance among different strains ranged from 0.072 to 0.391. The dendrogram constructed by unweighted pair-group method with arithmetic (UPGMA) method showed that the female and male gametophytes of the same cell lines could be grouped in pairs respectively. It indicated that RAPD analysis could be used not only to distinguish different strains of Laminaria, but also to distinguish male and female gametophyte within the same cell lines. There is ambiguous systematic relationship if judged merely by the present data. It seems that the use of RAPD marker is limited to elucidation of the phylogenetic relationship among the species of Laminaria.

Genetic polymorphisms variants in interleukin-6 and interleukin-1beta patients with obstructive sleep apnea syndrome in East Northern Turkey.

PubMed

Gok, Ilhami; Huseyinoglu, Nergiz; Ilhan, Dogan

2015-08-01

To investigate the relationship of IL-1β and IL-6 cytokine gene polymorphisms with obstructive sleep apnea syndrome (OSAS) in 61 patients admitted to the neurology clinic in Kafkas University Hospital with insomnia problem who were diagnosed with OSAS in sleeping labs, and 80 healthy subjects not associated with the syndrome. METHODS :Blood samples were taken to isolate DNA from patients diagnosed with OSAS based on polysomnography results and healthy controls. DNA amplification of the genes was performed with PCR. Amplification products were cut with the restriction enzymes in order to determine IL-1 gene (TaqI) and IL-6 gene (Lwel) polymorphisms. The cut DNA fragments were carried out in agarose gel electrophoresis, and RFLP analysis was performed by utilizing the images with gel imaging system. PCR products were sequenced with an Applied Biosystems Automated Sequencer. Polymorphic changes were observed for IL-1β gene in 26 of 62 patients (41.9%), and 16 of the 80 (25.8%) in the control group. The incidence of polymorphic changes in IL-6 gene was in seen in seven (of the 62 patients) (11.3%), and in the 16 (20%) controls. The findings on the genomic level in OSAS may provide an important contribution to diagnosis of obstructive sleep apnea syndrome in clinical practice, as well as it helps to obtain the results easily about environmental and genetic interaction of OSAS patients. Copyright© by the Medical Assotiation of Zenica-Doboj Canton.

Prenatal diagnosis of glycogen storage disease type 1a by single stranded conformation polymorphism (SSCP).

PubMed

Parvari, R; Hershkovitz, E; Carmi, R; Moses, S

1996-09-01

Glycogen storage disease type 1a (GSD 1a), a severe metabolic disorder, is caused by the absence of glucose-6-phosphatase (G6Pase) activity. Diagnosis is currently established by demonstrating the lack of G6Pase activity in the patient's liver specimen. Enzymatic diagnosis cannot be performed in chorionic villi or amniocytes as G6Pase is active only in the liver, kidney, and intestinal mucosa. Recent cloning of the G6Pase gene and subsequent identification of the mutations causing GSD 1a have led to the possibility of performing DNA-based diagnosis in chorionic villus samples (CVS) or amniocytes. Here we report the first DNA-based prenatal diagnosis in two families in whom GSD 1a patients were diagnosed. In one Jewish family with a previously identified R83C mutation, single-stranded conformation polymorphism (SSCP) analysis of the DNA extracted from CVS showed a homozygous R83C mutant pattern. As a result, the pregnancy was terminated and the diagnosis was confirmed on DNA analysis of the aborted fetus. In another family of Arabic extraction in which a V166G mutation has been identified in one of the siblings, SSCP analysis performed on DNA extracted from CVS presented the pattern of a normal control. The pregnancy was carried to term and a healthy baby was born. Thus, once mutations causing the disease are identified, prenatal diagnosis of GSD 1a is possible. SSCP analysis of DNA prepared from CVS is reliable, simple and fast, making it a suitable method for prenatal diagnosis.

Differential DNA Methylation Patterns Are Related to Phellogen Origin and Quality of Quercus suber Cork.

PubMed

Inácio, Vera; Barros, Pedro M; Costa, Augusta; Roussado, Cristóvão; Gonçalves, Elsa; Costa, Rita; Graça, José; Oliveira, M Margarida; Morais-Cecílio, Leonor

2017-01-01

DNA methylation is thought to influence Quercus suber cork quality, which is the main constraint for its economic valorisation. However, a deep knowledge of the cytosine methylation patterns disclosing the epigenetic variability of trees with different cork quality types is totally missing. This study investigates the hypothesis that variations in DNA methylation contribute to differences in cork cellular characteristics directly related to original or traumatic phellogen activity. We used MSAPs (Methylation Sensitive Amplified Polymorphism) to assess DNA methylation patterns of cork and leaf tissues of Q. suber adult trees growing in three cork oak stands. The relationship between the detected polymorphisms and the diversity of cork quality traits was explored by a marker-trait analysis focusing on the most relevant quality characteristics. Populations differed widely in cork quality, but only slightly in degree of epigenetic differentiation. Four MSAP markers (1.3% of the total) were significantly associated with the most noteworthy quality traits: wood inclusions (nails) and porosity. This evidence supports the potential role of cytosine methylation in the modulation of differential phellogen activity either involved in localized cell death or in pore production, resulting in different cork qualities. Although, the underlying basis of the methylation polymorphism of loci affecting cork quality traits remain unclear, the disclosure of markers statistically associated with cork quality strengthens the potential role of DNA methylation in the regulation of these traits, namely at the phellogen level.

DNA methylation and methylation polymorphism in ecotypes of Jatropha curcas L. using methylation-sensitive AFLP markers.

PubMed

Mastan, Shaik G; Rathore, Mangal S; Bhatt, Vacha D; Chikara, J; Ghosh, A

2014-12-01

We investigated DNA methylation and polymorphism in the methylated DNA using AFLP based methylation-sensitive amplification polymorphism (MS-AFLP) markers in ecotypes of Jatropha curcas L. growing in similar and different geo-ecological conditions. Three ecotypes growing in different geo-ecological conditions with environmental heterogeneity (Group-1) and five ecotypes growing in similar environmental conditions (Group-2) were assessed. In ecotypes growing in group-1, 44.32 % DNA was methylated and of which 93.59 % DNA was polymorphic. While in group-2, 32.27 % DNA was methylated, of which 51.64 % DNA was polymorphic. In site 1 and site 2 of group-1, overall methylation was 18.94 and 22.44 % respectively with difference of 3.5 %, while overall polymorphism was 41.14 and 39.23 % with a difference of 1.91 %. In site 1 and site 2 of group-2, overall methylation was 24.68 and 24.18 % respectively with difference of 0.5 %, while overall polymorphism was 12.19 and 12.65 % with a difference of 0.46 %. The difference of methylation percentage and percentage of methylation polymorphism throughout the genome of J. curcas at site 1 and 2 of group-1 is higher than that of J. curcas at site 1 and 2 of group-2. These results correlated the physico-chemical properties of soil at these sites. The variations of physico-chemical properties of soil at Chorwadla (site 1 in group-1 and site 2 in group-2) compared to the soil at Brahmapur (site 2 in group-1) is higher than that of soil at Neswad (site 1 in group-2). The study suggests that these homologous nucleotide sequences probably play important role in ecotype adaptation to environmental heterogeneity by creating epiallelic variations hence in evolution of ecotypes/clines or forms of species showing phenotypic/genotypic differences in different geographical areas.

A Genetic Linkage Map of Longleaf Pine (Pinus palustris Mill.) Based on Random Amplified Polymorphic DNAs

Treesearch

C.D. Nelson; Thomas L. Kubisiak; M. Stine; W.L. Nance

1994-01-01

Eight megagametophyte DNA samples from a single longleaf pine (Pinus palustris Mill.) tree were used to screen 576 oligonucleotide primers for random amplified polymorphic DNA (RAPD) fragments. Primers amplifying repeatable polymorphic fragments were further characterized within a sample of 72 megagametophytes from the same tree. Fragments...

Lactobacillus hammesii sp. nov., isolated from French sourdough.

PubMed

Valcheva, Rosica; Korakli, Maher; Onno, Bernard; Prévost, Hervé; Ivanova, Iskra; Ehrmann, Matthias A; Dousset, Xavier; Gänzle, Michael G; Vogel, Rudi F

2005-03-01

Twenty morphologically different strains were chosen from French wheat sourdough isolates. Cells were Gram-positive, non-spore-forming, non-motile rods. The isolates were identified using amplified-fragment length polymorphism, randomly amplified polymorphic DNA and 16S rRNA gene sequence analysis. All isolates were members of the genus Lactobacillus. They were identified as representing Lactobacillus plantarum, Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus spicheri and Lactobacillus sakei. However, two isolates (LP38(T) and LP39) could be clearly discriminated from recognized Lactobacillus species on the basis of genotyping methods. 16S rRNA gene sequence similarity and DNA-DNA relatedness data indicate that the two strains belong to a novel Lactobacillus species, for which the name Lactobacillus hammesii is proposed. The type strain is LP38(T) (=DSM 16381(T)=CIP 108387(T)=TMW 1.1236(T)).

RAPD and SSR Polymorphisms in Mutant Lines of Transgenic Wheat Mediated by Low Energy Ion Beam

NASA Astrophysics Data System (ADS)

Wang, Tiegu; Huang, Qunce; Feng, Weisen

2007-10-01

Two types of markers-random amplified polymorphic DNA (RAPD) and simple sequence repeat DNA (SSR)-have been used to characterize the genetic diversity among nine mutant lines of transgenic wheat intermediated by low energy ion beam and their four receptor cultivars. The objectives of this study were to analyze RAPD-based and SSR-based genetic variance among transgenic wheat lines and with their receptors, and to find specific genetic markers of special traits of transgenic wheat lines. 170 RAPD primers were amplified to 733 fragments in all the experimental materials. There were 121 polymorphic fragments out of the 733 fragments with a ratio of polymorphic fragments of 16.5%. 29 SSR primer pairs were amplified to 83 fragments in all the experiment materials. There were 57 polymorphic fragments out of the 83 fragments with a ratio of polymorphic fragments of 68.7%. The dendrograms were prepared based on a genetic distance matrix using the UPGMA (Unweighted Pair-group Method with Arithmetic averaging) algorithm, which corresponded well to the results of the wheat pedigree analysis and separated the 13 genotypes into four groups. Association analysis between RAPD and SSR markers with the special traits of transgenic wheat mutant lines discovered that three RAPD markers, s1, opt-16, and f14, were significantly associated with the muticate trait, while three SSR markers, Rht8 (Xgwm261), Rht-B1b, and Rht-D1b, highly associated with the dwarf trait. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning.

Restriction fragment length polymorphism of the major histocompatibility complex of the dog.

PubMed

Sarmiento, U M; Storb, R F

1988-01-01

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D-homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (Bam HI, Eco RI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I, and Bgl II), separated by agarose gel electrophoresis, and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabeled HLA cDNA probes corresponding to DR, DQ, DP, and DO beta genes. The autoradiograms for all nine enzyme digests displayed multiple bands with the DRb, DQb, and DPb probes while the DOb probe hybridized with one to two bands. The RFLP patterns were highly polymorphic but consistent within each DLA-D type. Standard RFLP patterns were established for nine DLA-D types which could be discriminated from each other by using two enzymes (Rsa I and Pst I) and the HLA-DPb probe. Cluster analysis of the polymorphic restriction fragments detected by the DRb probe revealed four closely related supertypic groups or DLA-DR families: Dw3 + Dw4 + D1, Dw8 + D10, D7 + D16 + D9, and Dw1. This study provides the basis for DLA-D genotyping at a population level by RFLP analysis. These results also suggest that the genetic organization of the DLA-D region may closely resemble that of the HLA complex.

Chloroplast DNA Diversity among Trees, Populations and Species in the California Closed-Cone Pines (Pinus Radiata, Pinus Muricata and Pinus Attenuata)

PubMed Central

Hong, Y. P.; Hipkins, V. D.; Strauss, S. H.

1993-01-01

The amount, distribution and mutational nature of chloroplast DNA polymorphisms were studied via analysis of restriction fragment length polymorphisms in three closely related species of conifers, the California closed-cone pines-knobcone pine: Pinus attenuata Lemm.; bishop pine: Pinus muricata D. Don; and Monterey pine: Pinus radiata D. Don. Genomic DNA from 384 trees representing 19 populations were digested with 9-20 restriction enzymes and probed with cloned cpDNA fragments from Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] that comprise 82% of the chloroplast genome. Up to 313 restriction sites were surveyed, and 25 of these were observed to be polymorphic among or within species. Differences among species accounted for the majority of genetic (haplotypic) diversity observed [G(st) = 84(+/-13)%]; nucleotide diversity among species was estimated to be 0.3(+/-0.1)%. Knobcone pine and Monterey pine displayed almost no genetic variation within or among populations. Bishop pine also showed little variability within populations, but did display strong population differences [G(st) = 87(+/-8)%] that were a result of three distinct geographic groups. Mean nucleotide diversity within populations was 0.003(+/-0.002)%; intrapopulation polymorphisms were found in only five populations. This pattern of genetic variation contrasts strongly with findings from study of nuclear genes (allozymes) in the group, where most genetic diversity resides within populations rather than among populations or species. Regions of the genome subject to frequent length mutations were identified; estimates of subdivision based on length variant frequencies in one region differed strikingly from those based on site mutations or allozymes. Two trees were identified with a major chloroplast DNA inversion that closely resembled one documented between Pinus and Pseudotsuga. PMID:7905846

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans.

PubMed

Rand, D M; Kann, L M

1996-07-01

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.

Extensive genetic and DNA methylation variation contribute to heterosis in triploid loquat hybrids.

PubMed

Liu, Chao; Wang, Mingbo; Wang, Lingli; Guo, Qigao; Liang, Guolu

2018-04-24

We aim to overcome the unclear origin of the loquat and elucidate the heterosis mechanism of the triploid loquat. Here we investigated the genetic and epigenetic variations between the triploid plant and its parental lines using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified fragment length polymorphism (MSAP) analyses. We show that in addition to genetic variations, extensive DNA methylation variation occurred during the formation process of triploid loquat, with the triploid hybrid having increased DNA methylation compared to the parents. Furthermore, a correlation existed between genetic variation and DNA methylation remodeling, suggesting that genome instability may lead to DNA methylation variation or vice versa. Sequence analysis of the MSAP bands revealed that over 53% of them overlap with protein-coding genes, which may indicate a functional role of the differential DNA methylation in gene regulation and hence heterosis phenotypes. Consistent with this, the genetic and epigenetic alterations were associated closely to the heterosis phenotypes of triploid loquat, and this association varied for different traits. Our results suggested that the formation of triploid is accompanied by extensive genetic and DNA methylation variation, and these changes contribute to the heterosis phenotypes of the triploid loquats from the two cross lines.

Origin and evolution of Andigena potatoes revealed by chloroplast and nuclear DNA markers.

PubMed

Sukhotu, Thitaporn; Hosaka, Kazuyoshi

2006-06-01

Andigena potatoes (Solanum tuberosum L. subsp. andigena Hawkes) (2n = 4x = 48) are important, native-farmer-selected cultivars in the Andes, which form a primary gene pool for improving a worldwide grown potato (S. tuberosum subsp. tuberosum). To elucidate the origin of Andigena, 196 Andigena accessions were compared with 301 accessions of 33 closely related cultivated and wild species using several types of chloroplast DNA (ctDNA) markers and nuclear DNA (nDNA) restriction fragment length polymorphism (RFLP) markers. Fourteen ctDNA types (haplotypes) and 115 RFLP bands were detected in Andigena, of which the main haplotypes and frequent RFLP bands were mostly shared with a cultivated diploid species, S. stenotomum Juz. et Buk. Principal component analysis of nDNA polymorphisms revealed a progressive and continuous variation from Peruvian wild species with C-type ctDNA to a group of wild species having S-type ctDNA in its variation range (S. bukasovii, S. canasense, S. candolleanum, and S. multidissectum), to cultivated diploid potatoes (S. phureja and S. stenotomum), and to cultivated tetraploid potatoes (Andigena and Chilean S. tuberosum subsp. tuberosum). These results suggest that the initial Andigena population arose with multiple origins exclusively from S. stenotomum. The overall evolutionary process toward the present-day Andigena was discussed.

Characterization of class II alpha genes and DLA-D region allelic associations in the dog.

PubMed

Sarmiento, U M; Storb, R F

1988-10-01

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the alpha genes of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (BamHI, EcoRI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I and Bgl II), separated by agarose gel electrophoresis and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabelled HLA cDNA probes corresponding to DQ, DP, DZ and DR alpha genes. Clear evidence was obtained for the canine homologues of DQ and DR alpha genes with simple bi- or tri-allelic polymorphism respectively. Evidence for a single, nonpolymorphic DP alpha gene was also obtained. However, the presence of a DZ alpha gene could not be clearly demonstrated in canine genomic DNA. This report extends our previous RFLP analysis documenting polymorphism of DLA class II beta genes in the same panel of homozygous typing cell dogs, and provides the basis for DLA-D genotyping at a population level. This study also characterizes the RFLP-defined preferential allelic associations across the DLA-D region in nine different homozygous typing cell specificities.

Analysis of DNA Methylation of Gracilariopsis lemaneiformis Under Temperature Stress Using the Methylation Sensitive Amplification Polymorphism (MSAP) Technique

NASA Astrophysics Data System (ADS)

Peng, Chong; Sui, Zhenghong; Zhou, Wei; Hu, Yiyi; Mi, Ping; Jiang, Minjie; Li, Xiaodong; Ruan, Xudong

2018-06-01

Gracilariopsis lemaneiformis is an economically important agarophyte, which contains high quality gel and shows a high growth rate. Wild population of G. lemaneiformis displayed resident divergence, though with a low genetic diversity as was revealed by amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) analyses. In addition, different strains of G. lemaneiformis are diverse in morphology. The highly inconsistence between genetic background and physiological characteristics recommends strongly to the regulation at epigenetic level. In this study, the DNA methylation change in G. lemaneiformis among different generation branches and under different temperature stresses was assessed using methylation sensitive amplified polymorphism (MSAP) technique. It was shown that DNA methylation level among different generation branches was diverse. The full and total methylated DNA level was the lowest in the second generation branch and the highest in the third generation. The total methylation level was 61.11%, 60.88% and 64.12% at 15°C, 22°C and 26°C, respectively. Compared with the control group (22°C), the fully methylated and totally methylated ratios were increased in both experiment groups (15°C and 26°C). All of the cytosine methylation/demethylation transform (CMDT) was further analyzed. High temperature treatment could induce more CMDT than low temperature treatment did.

Methylenetetrahydrofolate reductase gene polymorphisms and risk of acute lymphoblastic leukemia in children.

PubMed

Sadananda Adiga, M N; Chandy, S; Ramachandra, N; Appaji, L; Aruna Kumari, B S; Ramaswamy, G; Savithri, H S; Krishnamoorthy, L

2010-01-01

Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism and is involved in DNA synthesis, DNA repair and DNA methylation. Genetic polymorphisms of this enzyme have been shown to impact several diseases, including cancer. Leukemias are malignancies arising from rapidly proliferating hematopoietic cells having great requirement of DNA synthesis. This case-control study was undertaken to analyze the association of the MTHFR gene polymorphisms 677 C"T and 1298 A"C and the risk of acute lymphoblastic leukemia in children. Eighty-six patients aged below 15 years with a confirmed diagnosis of acute lymphoblastic leukemia (ALL) and 99 matched controls were taken for this study. Analysis of the polymorphisms was done using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Frequency of MTHFR 677 CC and CT were 85.9% and 14.1% in the controls, and 84.9% and 15.1% in the cases. The 'T' allele frequency was 7% and 7.5% in cases and controls respectively. The frequency of MTHFR 1298 AA, AC, and CC were 28.3%, 55.6% and 16.1% for controls and 23.3%, 59.3% and 17.4% for cases respectively. The 'C' allele frequency for 1298 A-->C was 43.9% and 47% respectively for controls and cases. The odds ratio (OR) for C677T was 1.08 (95% CI 0.48-2.45, p = 0.851) and OR for A1298C was 1.29 (95% CI 0.65-2.29, p = 0.46) and OR for 1298 CC was 1.31 (95% CI 0.53-3.26, p = 0.56). The OR for the combined heterozygous status (677 CT and 1298 AC) was 1.94 (95% CI 0.58-6.52, p = 0.286). The prevalence of 'T' allele for 677 MTHFR polymorphism was low in the population studied. There was no association between MTHFR 677 C-->T and 1298 A-->C gene polymorphisms and risk of ALL, which may be due to the small sample size.

Production and characterization of interspecific somatic hybrids between Brassica oleracea var. botrytis and B. nigra and their progenies for the selection of advanced pre-breeding materials.

PubMed

Wang, Gui-xiang; Tang, Yu; Yan, Hong; Sheng, Xiao-guang; Hao, Wei-Wei; Zhang, Li; Lu, Kun; Liu, Fan

2011-10-01

Somatic hybridization is a potential method for gene transfer from wild relatives to cultivated crops that can overcome sexual incompatibilities of two distantly related species. In this study, interspecific asymmetric somatic hybrids of Brassica oleracea var. botrytis (cauliflower) and Brassica nigra (black mustard) were obtained by protoplast fusion and their backcrossed (BC(3)) and selfed (S(3)) offspring were analyzed. Cytological analysis showed that the B. nigra chromosomes were successively eliminated in the backcrosses with cauliflower. The fertility of the hybrid progenies was quite different due to the asynchronous and abnormal chromosome behavior of pollen mother cells (PMC) during meiosis. Analysis of sequence-related amplified polymorphism (SRAP) showed that all of these hybrids mainly had the DNA banding pattern from the two parents with some alterations. Genetically, the selfed generations were closer to B. nigra, while the backcrossed generations were closer to the cauliflower parent. Analysis of cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLP) showed that all somatic hybrids in this study contained chloroplast (cp) DNA of the donor parent black mustard, while mitochondrial (mt) DNA showed evidence of recombination and variations in the regions analyzed. Furthermore, three BC(3) plants (originated from somatic hybrids 3, 4, 10) with 2-8 B. nigra-derived chromosomes shown by genomic in situ hybridization (GISH) displayed a more cauliflower-like morphology and high resistance to black-rot. These plants were obtained as bridge materials for further analysis and breeding.

[Using IRAP markers for analysis of genetic variability in populations of resource and rare species of plants].

PubMed

Boronnikova, S V; Kalendar', R N

2010-01-01

Species-specific LTR retrotransposons were first cloned in five rare relic species of drug plants located in the Perm' region. Sequences of LTR retrotransposons were used for PCR analysis based on amplification of repeated sequences from LTR or other sites of retrotransposons (IRAP). Genetic diversity was studied in six populations of rare relic species of plants Adonis vernalis L. by means of the IRAP method; 125 polymorphic IRAP-markers were analyzed. Parameters for DNA polymorphism and genetic diversity of A. vernalis populations were determined.

Microsatellite marker development by partial sequencing of the sour passion fruit genome (Passiflora edulis Sims).

PubMed

Araya, Susan; Martins, Alexandre M; Junqueira, Nilton T V; Costa, Ana Maria; Faleiro, Fábio G; Ferreira, Márcio E

2017-07-21

The Passiflora genus comprises hundreds of wild and cultivated species of passion fruit used for food, industrial, ornamental and medicinal purposes. Efforts to develop genomic tools for genetic analysis of P. edulis, the most important commercial Passiflora species, are still incipient. In spite of many recognized applications of microsatellite markers in genetics and breeding, their availability for passion fruit research remains restricted. Microsatellite markers in P. edulis are usually limited in number, show reduced polymorphism, and are mostly based on compound or imperfect repeats. Furthermore, they are confined to only a few Passiflora species. We describe the use of NGS technology to partially assemble the P. edulis genome in order to develop hundreds of new microsatellite markers. A total of 14.11 Gbp of Illumina paired-end sequence reads were analyzed to detect simple sequence repeat sites in the sour passion fruit genome. A sample of 1300 contigs containing perfect repeat microsatellite sequences was selected for PCR primer development. Panels of di- and tri-nucleotide repeat markers were then tested in P. edulis germplasm accessions for validation. DNA polymorphism was detected in 74% of the markers (PIC = 0.16 to 0.77; number of alleles/locus = 2 to 7). A core panel of highly polymorphic markers (PIC = 0.46 to 0.77) was used to cross-amplify PCR products in 79 species of Passiflora (including P. edulis), belonging to four subgenera (Astrophea, Decaloba, Distephana and Passiflora). Approximately 71% of the marker/species combinations resulted in positive amplicons in all species tested. DNA polymorphism was detected in germplasm accessions of six closely related Passiflora species (P. edulis, P. alata, P. maliformis, P. nitida, P. quadrangularis and P. setacea) and the data used for accession discrimination and species assignment. A database of P. edulis DNA sequences obtained by NGS technology was examined to identify microsatellite repeats in the sour passion fruit genome. Markers were submitted to evaluation using accessions of cultivated and wild Passiflora species. The new microsatellite markers detected high levels of DNA polymorphism in sour passion fruit and can potentially be used in genetic analysis of P. edulis and other Passiflora species.

Optimization of subculture and DNA extraction steps within the whole genome sequencing workflow for source tracking of Salmonella enterica and Listeria monocytogenes.

PubMed

Gimonet, Johan; Portmann, Anne-Catherine; Fournier, Coralie; Baert, Leen

2018-06-16

This work shows that an incubation time reduced to 4-5 h to prepare a culture for DNA extraction followed by an automated DNA extraction can shorten the hands-on time, the turnaround time by 30% and increase the throughput while maintaining the WGS quality assessed by high quality Single Nucleotide Polymorphism analysis. Copyright © 2018. Published by Elsevier B.V.

16S-23S rDNA intergenic spacer region polymorphism of Lactococcus garvieae, Lactococcus raffinolactis and Lactococcus lactis as revealed by PCR and nucleotide sequence analysis.

PubMed

Blaiotta, Giuseppe; Pepe, Olimpia; Mauriello, Gianluigi; Villani, Francesco; Andolfi, Rosamaria; Moschetti, Giancarlo

2002-12-01

The intergenic spacer region (ISR) between the 16S and 23S rRNA genes was tested as a tool for differentiating lactococci commonly isolated in a dairy environment. 17 reference strains, representing 11 different species belonging to the genera Lactococcus, Streptococcus, Lactobacillus, Enterococcus and Leuconostoc, and 127 wild streptococcal strains isolated during the whole fermentation process of "Fior di Latte" cheese were analyzed. After 16S-23S rDNA ISR amplification by PCR, species or genus-specific patterns were obtained for most of the reference strains tested. Moreover, results obtained after nucleotide analysis show that the 16S-23S rDNA ISR sequences vary greatly, in size and sequence, among Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis as well as other streptococci from dairy environments. Because of the high degree of inter-specific polymorphism observed, 16S-23S rDNA ISR can be considered a good potential target for selecting species-specific molecular assays, such as PCR primer or probes, for a rapid and extremely reliable differentiation of dairy lactococcal isolates.

Polymorphism of the ACE gene and the risk of obstructive sleep apnoea.

PubMed

Chmielewska, Izabela; Mlak, Radosław; Krawczyk, Paweł; Czukiewska, Ewa; Milanowski, Janusz

2013-01-01

Obstructive sleep apnoea/hypopnea syndrome (OSA) is characterized by obstruction of the upper airway during sleep, resulting in repetitive breathing pauses accompanied by oxygen desaturation and arousal from sleep. Among the candidate genes affecting the risk of OSA, genes whose polymorphisms influence the development of diseases with similar pathogenesis such as OSA could be listed: APOE, genes for leptin and leptin receptor, TNFA1, ADRB2 and ACE (gene for angiotensin-converting enzyme). Until now there has been a confirmed relationship between ACE gene polymorphism and cardiovascular diseases, but its effect on the incidence of OSA is debatable. The aim of this study was to investigate the effect of ACE gene insertion/deletion (I/D) polymorphism on the risk of OSA. Fifty-five patients with confirmed diagnose of OSA and qualified to CPAP therapy entered the study. The control group included 50 subjects who did not complain of any sleep related symptoms. Diagnose of OSA was set on the basis of full overnight polysomnography together with Epworth Sleepiness Scale according to American Academy of Sleep Medicine guidelines. DNA was isolated from peripheral blood leukocytes with Qiagen DNA mini Kit. ACE gene polymorphism was determined in genomic DNA using allele specific polymerase chain reaction. Different sizes of PCR products were observed on agarose gel electrophoresis. There were non-significant differences in the frequency of ACE genotypes. However, allele D had significantly lower prevalence in the study group than in the control group. (χ(2) = 4.25 p = 0.04). Moreover, I allele carriers had a threefold greater risk of developing OSA (HR = 2.748, 95% CI = 1.029-7.340, p < 0.05). Analysis of ACE gene polymorphism might be useful to determine the risk of developing OSA in clinically predisposed patients.

Characterization of Erwinia chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR-amplified fragments of pel genes.

PubMed Central

Nassar, A; Darrasse, A; Lemattre, M; Kotoujansky, A; Dervin, C; Vedel, R; Bertheau, Y

1996-01-01

Conserved regions about 420 bp long of the pelADE cluster specific to Erwinia chrysanthemi were amplified by PCR and used to differentiate 78 strains of E. chrysanthemi that were obtained from different hosts and geographical areas. No PCR products were obtained from DNA samples extracted from other pectinolytic and nonpectinolytic species and genera. The pel fragments amplified from the E. chrysanthemi strains studied were compared by performing a restriction fragment length polymorphism (RFLP) analysis. On the basis of similarity coefficients derived from the RFLP analysis, the strains were separated into 16 PCR RFLP patterns grouped in six clusters, These clusters appeared to be correlated with other infraspecific levels of E. chrysanthemi classification, such as pathovar and biovar, and occasionally with geographical origin. Moreover, the clusters correlated well with the polymorphism of pectate lyase and pectin methylesterase isoenzymes. While the pectin methylesterase profiles correlated with host monocot-dicot classification, the pectate lyase polymorphism might reflect the cell wall microdomains of the plants belonging to these classes. PMID:8779560

Single nucleotide polymorphisms in common bean: their discovery and genotyping using a multiplex detection system

USDA-ARS?s Scientific Manuscript database

Single-nucleotide Polymorphism (SNP) markers are by far the most common form of DNA polymorphism in a genome. The objectives of this study were to discover SNPs in common bean comparing sequences from coding and non-coding regions obtained from Genbank and genomic DNA and to compare sequencing resu...

Were inefficient mitochondrial haplogroups selected during migrations of modern humans? A test using modular kinetic analysis of coupling in mitochondria from cybrid cell lines.

PubMed

Amo, Taku; Brand, Martin D

2007-06-01

We introduce a general test of the bioenergetic importance of mtDNA (mitochondrial DNA) variants: modular kinetic analysis of oxidative phosphorylation in mitochondria from cybrid cells with constant nuclear DNA but different mtDNA. We have applied this test to the hypothesis [Ruiz-Pesini, Mishmar, Brandon, Procaccio and Wallace (2004) Science 303, 223-226] that particular mtDNA haplogroups (specific combinations of polymorphisms) that cause lowered coupling efficiency, leading to generation of less ATP and more heat, were positively selected during radiations of modern humans into colder climates. Contrary to the predictions of this hypothesis, mitochondria from Arctic haplogroups had similar or even greater coupling efficiency than mitochondria from tropical haplogroups.

Analysis of DNA Cytosine Methylation Patterns Using Methylation-Sensitive Amplification Polymorphism (MSAP).

PubMed

Guevara, María Ángeles; de María, Nuria; Sáez-Laguna, Enrique; Vélez, María Dolores; Cervera, María Teresa; Cabezas, José Antonio

2017-01-01

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is the methylation-sensitive amplified polymorphism technique (MSAP) which is a modification of amplified fragment length polymorphism (AFLP). It has been used to study methylation of anonymous CCGG sequences in different fungi, plants, and animal species. The main variation of this technique resides on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent-cutter restriction enzyme. For each sample, MSAP analysis is performed using both EcoRI/HpaII- and EcoRI/MspI-digested samples. A comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) methylation-insensitive polymorphisms that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples and (2) methylation-sensitive polymorphisms which are associated with the amplified fragments that differ in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses the modifications that can be applied to adjust the technology to different species of interest.

Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation-sensitive amplified polymorphism.

PubMed

Kim, Nam-Soo; Im, Min-Ji; Nkongolo, Kabwe

2016-08-01

Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal-contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation-sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal-contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal-contaminated site compared to uncontaminated populations. Other genotypes from a different metal-contaminated site within the same region appear to be recalcitrant to metal-induced DNA alterations even ≥30 years of tree life exposure to nickel and copper. MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal-contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed.

A Meta-Analysis of the Association between DNMT1 Polymorphisms and Cancer Risk.

PubMed

Li, Hao; Liu, Jing-Wei; Sun, Li-Ping; Yuan, Yuan

2017-01-01

Previous studies have examined the associations of DNA methyltransferase 1 ( DNMT1 ) polymorphisms, including single nucleotide polymorphisms rs16999593 (T/C), rs2228611 (G/A), and rs2228612 (A/G), with cancer risk. However, the results are inconclusive. The aim of this meta-analysis is to elucidate the associations between DNMT1 polymorphisms and cancer susceptibility. The PubMed, Embase, Web of Science, and Chinese National Knowledge Infrastructure databases were searched systematically to identify potentially eligible reports. Odd ratios and 95% confidence intervals were used to evaluate the strength of association between three DNMT1 polymorphisms and cancer risk. A total of 16 studies were finally included in the meta-analysis, namely, nine studies of 3378 cases and 4244 controls for rs16999593, 11 studies of 3643 cases and 3866 controls for rs2228611, and three studies of 1343 cases and 1309 controls for rs2228612. The DNMT1 rs2228612 (A/G) polymorphism was significantly related to cancer risk in the recessive model. The meta-analysis also suggested that DNMT1 rs16999593 (T/C) may be associated with gastric cancer, while rs2228611 (G/A) may be associated with breast cancer. In future research, large-scale and well-designed studies are required to verify these findings.

PCR-based study of conserved and variable DNA sequences of Tritrichomonas foetus isolates from Saskatchewan, Canada.

PubMed Central

Riley, D E; Wagner, B; Polley, L; Krieger, J N

1995-01-01

The protozoan parasite Tritrichomonas foetus causes infertility and spontaneous abortion in cattle. In Saskatchewan, Canada, the culture prevalence of trichomonads was 65 of 1,048 (6%) among 1,048 bulls tested within a 1-year period ending in April 1994. Saskatchewan was previously thought to be free of the parasite. To confirm the culture results, possible T. foetus DNA presence was determined by the PCR. All of the 16 culture-positive isolates tested were PCR positive by a single-band test, but one PCR product was weak. DNA fingerprinting by both T17 PCR and randomly amplified polymorphic DNA PCR revealed genetic variation or polymorphism among the T. foetus isolates. T17 PCR also revealed conserved loci that distinguished these T. foetus isolates from Trichomonas vaginalis, from a variety of other protozoa, and from prokaryotes. TCO-1 PCR, a PCR test designed to sample DNA sequence homologous to the 5' flank of a highly conserved cell division control gene, detected genetic polymorphism at low stringency and a conserved, single locus at higher stringency. These findings suggested that T. foetus isolates exhibit both conserved genetic loci and polymorphic loci detectable by independent PCR methods. Both conserved and polymorphic genetic loci may prove useful for improved clinical diagnosis of T. foetus. The polymorphic loci detected by PCR suggested either a long history of infection or multiple lines of T. foetus infection in Saskatchewan. Polymorphic loci detected by PCR may provide data for epidemiologic studies of T. foetus. PMID:7615746

[Association analysis between SNPs of the growth hormone receptor gene and growth traits in arctic fox].

PubMed

DU, Zhi-Heng; Liu, Zong-Yue; Bai, Xiu-Juan

2010-06-01

Using single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing, single nucleotide polymorphisms (SNPs) of growth hormone receptor (GHR) gene were detected in an arctic fox population. Correlation analysis between GHR polymorphisms and growth traits were carried out using the appropriate model. Four SNPs, G3A in the 5'UTR, C99T in the first exon, T59C and G65A in the fifth exon were identified on the arctic fox GHR gene. The G3A and C99T polymorphisms of GHR were associated with female fox body weight (Pamp;0.05) and the T59C and G65A polymorphisms of GHR were associated with male fox body weight (Pamp;0.05) and the skin length of the female fox (Pamp;0.01). Therefore, marker assistant selection on body weight and skin length of arctic foxes using these SNPs can be applied to get big and high quality arctic foxes.

Typing single polymorphic nucleotides in mitochondrial DNA as a way to access Middle Pleistocene DNA

PubMed Central

Valdiosera, Cristina; García, Nuria; Dalén, Love; Smith, Colin; Kahlke, Ralf-Dietrich; Lidén, Kerstin; Angerbjörn, Anders; Arsuaga, Juan Luis; Götherström, Anders

2006-01-01

In this study, we have used a technique designed to target short fragments containing informative mitochondrial substitutions to extend the temporal limits of DNA recovery and study the molecular phylogeny of Ursus deningeri. We present a cladistic analysis using DNA recovered from 400 kyr old U. deningeri remains, which demonstrates U. deningeri's relation to Ursus spelaeus. This study extends the limits of recovery from skeletal remains by almost 300 kyr. Plant material from permafrost environments has yielded DNA of this age in earlier studies, and our data suggest that DNA in teeth from cave environments may be equally well preserved. PMID:17148299

Translational genomics for analysis of complex traits in peanut and sorghum

USDA-ARS?s Scientific Manuscript database

The integration of sequencing and genotype data from natural variation studies (by whole genome resequencing [wgs] or genotype by sequencing [gbs]), transcriptome (RNA-seq) and mutant analysis (also by wgs) facilitated the development of DNA markers in the form of single nucleotide polymorphic (SNP)...

Maternal cell traffic bounds for immune modulation: tracking maternal H-2 alleles in spleens of baby mice by DNA fingerprinting.

PubMed

Wan, Wenhan; Shimizu, Shoji; Ikawa, Hiromichi; Sugiyama, Kiyosh; Yamaguchi, Nobuo

2002-10-01

We have previously reported that the immunization of pregnant mice with T-dependent antigens successfully induced suppression of the antigen-specific plaque-forming cell (PFC) response to the relevant antigens in the offspring. This suppression was not caused by the administered antigens, the antibodies produced by the pregnant mother, or lactational transfer, but was dependent on the presence of the intact maternal T cells. It was major histocompatibility complex (MHC)-restricted manner tolerance, which continued for at least one-sixth of the murine life. Traditionally, the placenta acts as a natural barrier, not allowing the cells to pass through. However, the results presented strongly suggested that maternal T cells pass through the placenta and subsequently induce tolerance. In this present study, we attempted to substantiate the presence of maternal cells in the fetal circulation through the use of molecular techniques. We found that a highly polymorphic microsatellite sequence within the class II Eb gene of the H-2 complex is useful for the molecular detection of various H-2 alleles. DNA polymorphic analysis was used for tracking maternal H-2 alleles in the spleens of baby mice. The main procedure involved polymerase chain reaction amplification and restriction fragment length polymorphism analysis of the DNA sequence encompassing the H-2-specific microsatellite from the genomic DNA of baby mice. The results indicated that maternal T cells of immunized pregnant mice cross the placenta into the fetus, eventually inducing antigen-specific immunological tolerance in the offspring.

Cigarette smoking and hOGG1 Ser326Cys polymorphism are associated with 8-OHdG accumulation on mitochondrial DNA in thoracic esophageal squamous cell carcinoma.

PubMed

Lin, Chen-Sung; Wang, Liang-Shun; Chou, Teh-Ying; Hsu, Wen-Hu; Lin, Hui-Chen; Lee, Shu-Yu; Lee, Mau-Hua; Chang, Shi-Chuan; Wei, Yau-Huei

2013-12-01

We examined whether cigarette smoking affects the degrees of oxidative damage (8-hydroxyl-2'-deoxyguanosine [8-OHdG]) on mitochondrial DNA (mtDNA), whether the degree of 8-OHdG accumulation on mtDNA is related to the increased total mtDNA copy number, and whether human 8-oxoguanine DNA glycosylase 1 (hOGG1) Ser326Cys polymorphisms affect the degrees of 8-OHdG accumulation on mtDNA in thoracic esophageal squamous cell carcinoma (TESCC). DNA extracted from microdissected tissues of paired noncancerous esophageal muscles, noncancerous esophageal mucosa, and cancerous TESCC nests (n = 74) along with metastatic lymph nodes (n = 38) of 74 TESCC patients was analyzed. Both the mtDNA copy number and mtDNA integrity were analyzed by quantitative real-time polymerase chain reaction (PCR). The hOGG1 Ser326Cys polymorphisms were identified by restriction fragment length polymorphism PCR and PCR-based direct sequencing. Among noncancerous esophageal mucosa, cancerous TESCC nests, and metastatic lymph nodes, the mtDNA integrity decreased (95.2 to 47.9 to 18.6 %; P < 0.001) and the mtDNA copy number disproportionally increased (0.163 to 0.204 to 0.207; P = 0.026). In TESCC, higher indexes of cigarette smoking (0, 0-20, 20-40, and >40 pack-years) were related to an advanced pathologic N category (P = 0.038), elevated mtDNA copy number (P = 0.013), higher mtDNA copy ratio (P = 0.028), and increased mtDNA integrity (P = 0.069). The TESCC mtDNA integrity in patients with Ser/Ser, Ser/Cys, and Cys/Cys hOGG1 variants decreased stepwise from 65.2 to 52.1 to 41.3 % (P = 0.051). Elevated 8-OHdG accumulations on mtDNA in TESCC were observed. Such accumulations were associated with a compensatory increase in total mtDNA copy number, indexes of cigarette smoking, and hOGG1 Ser326Cys polymorphisms.

The Large Subunit rDNA Sequence of Plasmodiophora brassicae Does not Contain Intra-species Polymorphism.

PubMed

Schwelm, Arne; Berney, Cédric; Dixelius, Christina; Bass, David; Neuhauser, Sigrid

2016-12-01

Clubroot disease caused by Plasmodiophora brassicae is one of the most important diseases of cultivated brassicas. P. brassicae occurs in pathotypes which differ in the aggressiveness towards their Brassica host plants. To date no DNA based method to distinguish these pathotypes has been described. In 2011 polymorphism within the 28S rDNA of P. brassicae was reported which potentially could allow to distinguish pathotypes without the need of time-consuming bioassays. However, isolates of P. brassicae from around the world analysed in this study do not show polymorphism in their LSU rDNA sequences. The previously described polymorphism most likely derived from soil inhabiting Cercozoa more specifically Neoheteromita-like glissomonads. Here we correct the LSU rDNA sequence of P. brassicae. By using FISH we demonstrate that our newly generated sequence belongs to the causal agent of clubroot disease. Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.

Mitochondrial Haplogroup T Is Associated with Obesity in Austrian Juveniles and Adults

PubMed Central

Ebner, Sabine; Mangge, Harald; Langhof, Helmut; Halle, Martin; Siegrist, Monika; Aigner, Elmar; Paulmichl, Katharina; Paulweber, Bernhard; Datz, Christian; Sperl, Wolfgang; Kofler, Barbara; Weghuber, Daniel

2015-01-01

Background Recent publications have reported contradictory data regarding mitochondrial DNA (mtDNA) variation and its association with body mass index. The aim of the present study was to compare the frequencies of mtDNA haplogroups as well as control region (CR) polymorphisms of obese juveniles (n = 248) and obese adults (n = 1003) versus normal weight controls (njuvenile = 266, nadults = 595) in a well-defined, ethnically homogenous, age-matched comparative cohort of Austrian Caucasians. Methodology and Principal Findings Using SNP analysis and DNA sequencing, we identified the nine major European mitochondrial haplogroups and CR polymorphisms. Of these, only the T haplogroup frequency was increased in the juvenile obese cohort versus the control subjects [11.7% in obese vs. 6.4% in controls], although statistical significance was lost after adjustment for sex and age. Similar data were observed in a local adult cohort, in which haplogroup T was found at a significantly higher frequency in the overweight and obese subjects than in the normal weight group [9.7% vs. 6.2%, p = 0.012, adjusted for sex and age]. When all obese subjects were considered together, the difference in the frequency of haplogroup T was even more clearly seen [10.1% vs. 6.3%, p = 0.002, OR (95% CI) 1.71 (1.2–2.4), adjusted for sex and age]. The frequencies of the T haplogroup-linked CR polymorphisms C16294T and the C16296T were found to be elevated in both the juvenile and the adult obese cohort compared to the controls. Nevertheless, no mtDNA haplogroup or CR polymorphism was robustly associated with any of several investigated metabolic and cardiovascular parameters (e.g., blood pressure, blood glucose concentration, triglycerides, cholesterol) in all obese subjects. Conclusions and Significance By investigation of this large ethnically and geographically homogenous cohort of Middle European Caucasians, only mtDNA haplogroup T was identified as an obesity risk factor. PMID:26322975

Primary DNA damage and genetic polymorphisms for CYP1A1, EPHX and GSTM1 in workers at a graphite electrode manufacturing plant

PubMed Central

Moretti, Massimo; Dell'Omo, Marco; Villarini, Milena; Pastorelli, Roberta; Muzi, Giacomo; Airoldi, Luisa; Pasquini, Rossana

2007-01-01

Background The results of a cross-sectional study aimed to evaluate whether genetic polymorphisms (biomarkers of susceptibility) for CYP1A1, EPHX and GSTM1 genes that affect polycyclic aromatic hydrocarbons (PAH) activation and detoxification might influence the extent of primary DNA damage (biomarker of biologically effective dose) in PAH exposed workers are presented. PAH-exposure of the study populations was assessed by determining the concentration of 1-hydroxypyrene (1OHP) in urine samples (biomarker of exposure dose). Methods The exposed group consisted of workers (n = 109) at a graphite electrode manufacturing plant, occupationally exposed to PAH. Urinary 1OHP was measured by HPLC. Primary DNA damage was evaluated by the alkaline comet assay in peripheral blood leukocytes. Genetic polymorphisms for CYP1A1, EPHX and GSTM1 were determined by PCR or PCR/RFLP analysis. Results 1OHP and primary DNA damage were significantly higher in electrode workers compared to reference subjects. Moreover, categorization of subjects as normal or outlier highlighted an increased genotoxic risk OR = 2.59 (CI95% 1.32–5.05) associated to exposure to PAH. Polymorphisms in EPHX exons 3 and 4 was associated to higher urinary concentrations of 1OHP, whereas none of the genotypes analyzed (CYP1A1, EPHX, and GSTM1) had any significant influence on primary DNA damage as evaluated by the comet assay. Conclusion The outcomes of the present study show that molecular epidemiology approaches (i.e. cross-sectional studies of genotoxicity biomarkers) can play a role in identifying common genetic risk factors, also attempting to associate the effects with measured exposure data. Moreover, categorization of subjects as normal or outlier allowed the evaluation of the association between occupational exposure to PAH and DNA damage highlighting an increased genotoxic risk. PMID:17908297

Assessment of genetic relationship in Persea spp by traditional molecular markers.

PubMed

Reyes-Alemán, J C; Valadez-Moctezuma, E; Barrientos-Priego, A F

2016-04-04

Currently, the reclassification of the genus Persea is under discussion with molecular techniques for DNA analysis representing an alternative for inter- and intra-specific differentiation. In the present study, the traditional random-amplified polymorphic DNA (RAPD) and the inter simple sequence repeat (ISSR) markers were used to determine the genomic relationship of different species and hybrids representative of the subgenera Eriodaphne and Persea in a population conserved in a germplasm bank. The data were analyzed statistically using multivariate methods. In the RAPD analysis, a total of 190 polymorphic bands were produced, with an average of 23.7 bands per primer, the percentage contribution of each primer was from 7.66 to 19.63; the polymorphic information content (PIC) ranged from 0.23 to 0.45, with an average of 0.35. In the ISSR analysis, a total of 111 polymorphic bands were considered, with an average of 18.5 bands per primer, the percentage contribution of each was from 11.83 to 19.57; the PIC ranged from 0.35 to 0.48, with an average of 0.42. The phenograms obtained in each technique showed the relationship among the accessions through the clusters formed. In general, both the techniques grouped representatives of the Persea americana races (P. americana var. drymifolia, P. americana var. guatemalensis, and P. americana var. americana). However, it was not possible to separate the species of Persea used as reference into independent clades. In addition, they tended to separate the representatives of subgenera Eriodaphne and Persea.

Lactobacillus nantensis sp. nov., isolated from French wheat sourdough.

PubMed

Valcheva, Rosica; Ferchichi, Mounir F; Korakli, Maher; Ivanova, Iskra; Gänzle, Michael G; Vogel, Rudi F; Prévost, Hervé; Onno, Bernard; Dousset, Xavier

2006-03-01

A polyphasic taxonomic study of the bacterial flora isolated from traditional French wheat sourdough, using phenotypic characterization and phylogenetic as well as genetic methods, revealed a consistent group of isolates that could not be assigned to any recognized species. These results were confirmed by randomly amplified polymorphic DNA and amplified fragment length polymorphism fingerprinting analyses. Cells were Gram-positive, homofermentative rods. Comparative 16S rRNA gene sequence analysis of the representative strain LP33T indicated that these strains belong to the genus Lactobacillus and that they formed a branch distinct from their closest relatives Lactobacillus farciminis, Lactobacillus alimentarius, Lactobacillus paralimentarius and Lactobacillus mindensis. DNA-DNA reassociation experiments with the three phylogenetically closest Lactobacillus species confirmed that LP33T (= DSM 16982T = CIP 108546T = TMW 1.1265T) represents the type strain of a novel species, for which the name Lactobacillus nantensis sp. nov. is proposed.

Laser mass spectrometry for DNA fingerprinting for forensic applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, C.H.; Tang, K.; Taranenko, N.I.

The application of DNA fingerprinting has become very broad in forensic analysis, patient identification, diagnostic medicine, and wildlife poaching, since every individual`s DNA structure is identical within all tissues of their body. DNA fingerprinting was initiated by the use of restriction fragment length polymorphisms (RFLP). In 1987, Nakamura et al. found that a variable number of tandem repeats (VNTR) often occurred in the alleles. The probability of different individuals having the same number of tandem repeats in several different alleles is very low. Thus, the identification of VNTR from genomic DNA became a very reliable method for identification of individuals.more » DNA fingerprinting is a reliable tool for forensic analysis. In DNA fingerprinting, knowledge of the sequence of tandem repeats and restriction endonuclease sites can provide the basis for identification. The major steps for conventional DNA fingerprinting include (1) specimen processing (2) amplification of selected DNA segments by PCR, and (3) gel electrophoresis to do the final DNA analysis. In this work we propose to use laser desorption mass spectrometry for fast DNA fingerprinting. The process and advantages are discussed.« less

Evaluation of DNA extraction methods for the analysis of microbial community in biological activated carbon.

PubMed

Zheng, Lu; Gao, Naiyun; Deng, Yang

2012-01-01

It is difficult to isolate DNA from biological activated carbon (BAC) samples used in water treatment plants, owing to the scarcity of microorganisms in BAC samples. The aim of this study was to identify DNA extraction methods suitable for a long-term, comprehensive ecological analysis of BAC microbial communities. To identify a procedure that can produce high molecular weight DNA, maximizes detectable diversity and is relatively free from contaminants, the microwave extraction method, the cetyltrimethylammonium bromide (CTAB) extraction method, a commercial DNA extraction kit, and the ultrasonic extraction method were used for the extraction of DNA from BAC samples. Spectrophotometry, agarose gel electrophoresis and polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) analysis were conducted to compare the yield and quality of DNA obtained using these methods. The results showed that the CTAB method produce the highest yield and genetic diversity of DNA from BAC samples, but DNA purity was slightly less than that obtained with the DNA extraction-kit method. This study provides a theoretical basis for establishing and selecting DNA extraction methods for BAC samples.

Investigation of paternity establishing without the putative father using hypervariable DNA probes.

PubMed

Yokoi, T; Odaira, T; Nata, M; Sagisaka, K

1990-09-01

Seven kinds of DNA probes which recognize hypervariable loci were applied for paternity test. The putative father was decreased and unavailable for the test. The two legitimate children and their mother (the deceased's wife) and the four illegitimate children and their mother (the deceased's kept mistress) were available for analysis. Paternity index of four illegitimate child was investigated. Allelic frequencies and their confidence intervals among unrelated Japanese individuals were previously reported from our laboratory, and co-dominant segregation of the polymorphism was confirmed in family studies. Cumulative paternity indices of four illegitimate children from 16 kinds of standard blood group markers were 165, 42, 0.09, and 36, respectively. On the other hand, cumulative paternity indices from 7 kinds of DNA probes are 2,363, 4,685, 57,678, and 54,994, respectively, which are 14, 113, 640, 864, and 1,509 times higher than that from standard blood group markers. The DNA analyses gave nearly conclusive evidence that the putative father was the biological father of the children. Especially, the paternity relation of the third illegitimate child could not be established without the DNA analyses. Accordingly, DNA polymorphism is considered to be informative enough for paternity test.

LPA gene: interaction between the apolipoprotein(a) size ('kringle IV' repeat) polymorphism and a pentanucleotide repeat polymorphism influences Lp(a) lipoprotein level.

PubMed

Røsby, O; Berg, K

2000-01-01

In order to search for factors influencing the Lp(a) lipoprotein level, we have examined the apolipoprotein(a) (apo(a)) size polymorphism as well as a pentanucleotide (TTTTA) repeat polymorphism in the 5' control region of the LPA gene. Lp(a) lipoprotein levels were compared between individuals with different genotypes as defined by pulsed field gel electrophoresis of DNA plugs, and PCR of DNA samples followed by polyacrylamide gel electrophoresis. DNA plugs and DNA were prepared from blood samples collected from blood donors. Twenty-seven different K IV repeat alleles were observed in the 71 women and 92 men from which apo(a) size polymorphism results were obtained. Alleles encoding 26-32 Kringle IV repeats were the most frequent. Alleles encoding seven to 11 TTTTA repeats were detected in the 84 women and 122 men included in the pentanucleotide polymorphism study, and homozygosity for eight TTTTA repeats was the most common genotype. The eight TTTTA repeat allele occurred with almost any apo(a) allele. An inverse relationship between number of K IV repeats and Lp(a) concentration was confirmed. The contributions of the apo(a) size polymorphism and the pentanucleotide repeat polymorphism to the interindividual variance of Lp(a) lipoprotein concentrations were 9.7 and 3.5%, respectively (type IV sum of squares). Nineteen per cent of the variance in Lp(a) lipoprotein level appeared to be the result of the multiplication product (interaction) between the apo(a) size polymorphism and the pentanucleotide repeat polymorphism. The contribution of the apo(a) size polymorphism alone to the variation in Lp(a) lipoprotein level was lower than previously reported. However, the multiplicative interaction effect between the K IV repeat polymorphism and the pentanucleotide repeat polymorphism may be an important factor explaining the variation in Lp(a) lipoprotein levels among the populations.

High-sensitivity stable-isotope probing by a quantitative terminal restriction fragment length polymorphism protocol.

PubMed

Andeer, Peter; Strand, Stuart E; Stahl, David A

2012-01-01

Stable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)-TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli, Rhodococcus, Variovorax, and Microbacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.

Differential DNA Methylation Patterns Are Related to Phellogen Origin and Quality of Quercus suber Cork

PubMed Central

Costa, Augusta; Roussado, Cristóvão; Gonçalves, Elsa; Costa, Rita; Graça, José; Oliveira, M. Margarida

2017-01-01

DNA methylation is thought to influence Quercus suber cork quality, which is the main constraint for its economic valorisation. However, a deep knowledge of the cytosine methylation patterns disclosing the epigenetic variability of trees with different cork quality types is totally missing. This study investigates the hypothesis that variations in DNA methylation contribute to differences in cork cellular characteristics directly related to original or traumatic phellogen activity. We used MSAPs (Methylation Sensitive Amplified Polymorphism) to assess DNA methylation patterns of cork and leaf tissues of Q. suber adult trees growing in three cork oak stands. The relationship between the detected polymorphisms and the diversity of cork quality traits was explored by a marker-trait analysis focusing on the most relevant quality characteristics. Populations differed widely in cork quality, but only slightly in degree of epigenetic differentiation. Four MSAP markers (1.3% of the total) were significantly associated with the most noteworthy quality traits: wood inclusions (nails) and porosity. This evidence supports the potential role of cytosine methylation in the modulation of differential phellogen activity either involved in localized cell death or in pore production, resulting in different cork qualities. Although, the underlying basis of the methylation polymorphism of loci affecting cork quality traits remain unclear, the disclosure of markers statistically associated with cork quality strengthens the potential role of DNA methylation in the regulation of these traits, namely at the phellogen level. PMID:28045988

Single-strand conformation polymorphism (SSCP)-based mutation scanning approaches to fingerprint sequence variation in ribosomal DNA of ascaridoid nematodes.

PubMed

Zhu, X Q; Gasser, R B

1998-06-01

In this study, we assessed single-strand conformation polymorphism (SSCP)-based approaches for their capacity to fingerprint sequence variation in ribosomal DNA (rDNA) of ascaridoid nematodes of veterinary and/or human health significance. The second internal transcribed spacer region (ITS-2) of rDNA was utilised as the target region because it is known to provide species-specific markers for this group of parasites. ITS-2 was amplified by PCR from genomic DNA derived from individual parasites and subjected to analysis. Direct SSCP analysis of amplicons from seven taxa (Toxocara vitulorum, Toxocara cati, Toxocara canis, Toxascaris leonina, Baylisascaris procyonis, Ascaris suum and Parascaris equorum) showed that the single-strand (ss) ITS-2 patterns produced allowed their unequivocal identification to species. While no variation in SSCP patterns was detected in the ITS-2 within four species for which multiple samples were available, the method allowed the direct display of four distinct sequence types of ITS-2 among individual worms of T. cati. Comparison of SSCP/sequencing with the methods of dideoxy fingerprinting (ddF) and restriction endonuclease fingerprinting (REF) revealed that also ddF allowed the definition of the four sequence types, whereas REF displayed three of four. The findings indicate the usefulness of the SSCP-based approaches for the identification of ascaridoid nematodes to species, the direct display of sequence variation in rDNA and the detection of population variation. The ability to fingerprint microheterogeneity in ITS-2 rDNA using such approaches also has implications for studying fundamental aspects relating to mutational change in rDNA.

SNPs in DNA repair or oxidative stress genes and late subcutaneous fibrosis in patients following single shot partial breast irradiation

PubMed Central

2012-01-01

Background The aim of this study was to evaluate the potential association between single nucleotide polymorphisms related response to radiotherapy injury, such as genes related to DNA repair or enzymes involved in anti-oxidative activities. The paper aims to identify marker genes able to predict an increased risk of late toxicity studying our group of patients who underwent a Single Shot 3D-CRT PBI (SSPBI) after BCS (breast conserving surgery). Methods A total of 57 breast cancer patients who underwent SSPBI were genotyped for SNPs (single nucleotide polymorphisms) in XRCC1, XRCC3, GST and RAD51 by Pyrosequencing technology. Univariate analysis (ORs and 95% CI) was performed to correlate SNPs with the risk of developing ≥ G2 fibrosis or fat necrosis. Results A higher significant risk of developing ≥ G2 fibrosis or fat necrosis in patients with: polymorphic variant GSTP1 (Ile105Val) (OR = 2.9; 95%CI, 0.88-10.14, p = 0.047). Conclusions The presence of some SNPs involved in DNA repair or response to oxidative stress seem to be able to predict late toxicity. Trial Registration ClinicalTrials.gov: NCT01316328 PMID:22272830

ERCC2, ERCC1 polymorphisms and haplotypes, cooking oil fume and lung adenocarcinoma risk in Chinese non-smoking females.

PubMed

Yin, Zhihua; Su, Meng; Li, Xuelian; Li, Mingchuan; Ma, Rui; He, Qincheng; Zhou, Baosen

2009-12-14

Excision repair cross-complementing group 1 (ERCC1) and group 2 (ERCC2) proteins play important roles in the repair of DNA damage and adducts. Single nucleotide polymorphisms (SNPs) of DNA repair genes are suspected to influence the risk of lung cancer. This study aimed to investigate the association between the ERCC2 751, 312 and ERCC1 118 polymorphisms and the risk of lung adenocarcinoma in Chinese non-smoking females. A hospital-based case-control study of 285 patients and 285 matched controls was conducted. Information concerning demographic and risk factors was obtained for each case and control by a trained interviewer. After informed consent was obtained, each person donated 10 ml blood for biomarker testing. Three polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. This study showed that the individuals with the combined ERCC2 751AC/CC genotypes were at an increased risk for lung adenocarcinoma compared with those carrying the AA genotype [adjusted odds ratios (OR) 1.64, 95% confidence interval (CI) 1.06-2.52]. The stratified analysis suggested that increased risk associated with ERCC2 751 variant genotypes (AC/CC) was more pronounced in individuals without exposure to cooking oil fume (OR 1.98, 95%CI 1.18-3.32) and those without exposure to fuel smoke (OR 2.47, 95%CI 1.46-4.18). Haplotype analysis showed that the A-G-T and C-G-C haplotypes were associated with increased risk of lung adenocarcinoma among non-smoking females (ORs were 1.43 and 2.28, 95%CIs were 1.07-1.91 and 1.34-3.89, respectively). ERCC2 751 polymorphism may be a genetic risk modifier for lung adenocarcinoma in non-smoking females in China.

A tale of aborigines, conquerors and slaves: Alu insertion polymorphisms and the peopling of Canary Islands.

PubMed

Maca-Meyer, N; Villar, J; Pérez-Méndez, L; Cabrera de León, A; Flores, C

2004-11-01

Classical, mitochondrial DNA (mtDNA) and Y chromosome markers have been used to examine the genetic admixture in present day inhabitants of the Canary Islands. In this study, we report the analysis of ten autosomal Alu insertion polymorphisms in 364 samples from the seven main islands of the Archipelago, and their comparison to continental samples. The detection of population-specific alleles from the Iberian Peninsula and Northwest Africa, as well as their affinities on the basis of genetic distances and principal component analysis, support a clear link between these populations. Coincident with previous results, the Canarian gene pool can be distinguished as being halfway between those of its putative parents, although with a major Iberian contribution (62-78%). Both the substantial Northwest African contribution (23-38%), and the minor sub-Saharan African input (3%), suggest that the genetic legacy from the aborigines and slaves still persists in the Canary Islanders.

Rapid discrimination between Anopheles gambiae s.s. and Anopheles arabiensis by High-Resolution Melt (HRM) analysis.

PubMed

Zianni, Michael R; Nikbakhtzadeh, Mahmood R; Jackson, Bryan T; Panescu, Jenny; Foster, Woodbridge A

2013-04-01

There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software.

Rapid Discrimination between Anopheles gambiae s.s. and Anopheles arabiensis by High-Resolution Melt (HRM) Analysis

PubMed Central

Zianni, Michael R.; Nikbakhtzadeh, Mahmood R.; Jackson, Bryan T.; Panescu, Jenny; Foster, Woodbridge A.

2013-01-01

There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software. PMID:23543777

A standard photomap of ovarian nurse cell chromosomes and inversion polymorphism in Anopheles beklemishevi.

PubMed

Artemov, Gleb N; Gordeev, Mikhail I; Kokhanenko, Alina A; Moskaev, Anton V; Velichevskaya, Alena I; Stegniy, Vladimir N; Sharakhov, Igor V; Sharakhova, Maria V

2018-03-27

Anopheles beklemishevi is a member of the Maculipennis group of malaria mosquitoes that has the most northern distribution among other members of the group. Although a cytogenetic map for the larval salivary gland chromosomes of this species has been developed, a high-quality standard cytogenetic photomap that enables genomics and population genetics studies of this mosquito at the adult stage is still lacking. In this study, a cytogenetic map for the polytene chromosomes of An. beklemishevi from ovarian nurse cells was developed using high-resolution digital imaging from field collected mosquitoes. PCR-amplified DNA probes for fluorescence in situ hybridization (FISH) were designed based on the genome of An. atroparvus. The DNA probe obtained by microdissection procedures from the breakpoint region was labelled in a DOP-PCR reaction. Population analysis was performed on 371 specimens collected in 18 locations. We report the development of a high-quality standard photomap for the polytene chromosomes from ovarian nurse cells of An. beklemishevi. To confirm the suitability of the map for physical mapping, several PCR-amplified probes were mapped to the chromosomes of An. beklemishevi using FISH. In addition, we identified and mapped DNA probes to flanking regions of the breakpoints of two inversions on chromosome X of this species. Inversion polymorphism was determined in 13 geographically distant populations of An. beklemishevi. Four polymorphic inversions were detected. The positions of common chromosomal inversions were indicated on the map. The study constructed a standard photomap for ovarian nurse cell chromosomes of An. beklemishevi and tested its suitability for physical genome mapping and population studies. Cytogenetic analysis determined inversion polymorphism in natural populations of An. beklemishevi related to this species' adaptation.

High-dose folic acid supplementation alters the human sperm methylome and is influenced by the MTHFR C677T polymorphism

PubMed Central

Aarabi, Mahmoud; San Gabriel, Maria C.; Chan, Donovan; Behan, Nathalie A.; Caron, Maxime; Pastinen, Tomi; Bourque, Guillaume; MacFarlane, Amanda J.; Zini, Armand; Trasler, Jacquetta

2015-01-01

Dietary folate is a major source of methyl groups required for DNA methylation, an epigenetic modification that is actively maintained and remodeled during spermatogenesis. While high-dose folic acid supplementation (up to 10 times the daily recommended dose) has been shown to improve sperm parameters in infertile men, the effects of supplementation on the sperm epigenome are unknown. To assess the impact of 6 months of high-dose folic acid supplementation on the sperm epigenome, we studied 30 men with idiopathic infertility. Blood folate concentrations increased significantly after supplementation with no significant improvements in sperm parameters. Methylation levels of the differentially methylated regions of several imprinted loci (H19, DLK1/GTL2, MEST, SNRPN, PLAGL1, KCNQ1OT1) were normal both before and after supplementation. Reduced representation bisulfite sequencing (RRBS) revealed a significant global loss of methylation across different regions of the sperm genome. The most marked loss of DNA methylation was found in sperm from patients homozygous for the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, a common polymorphism in a key enzyme required for folate metabolism. RRBS analysis also showed that most of the differentially methylated tiles were located in DNA repeats, low CpG-density and intergenic regions. Ingenuity Pathway Analysis revealed that methylation of promoter regions was altered in several genes involved in cancer and neurobehavioral disorders including CBFA2T3, PTPN6, COL18A1, ALDH2, UBE4B, ERBB2, GABRB3, CNTNAP4 and NIPA1. Our data reveal alterations of the human sperm epigenome associated with high-dose folic acid supplementation, effects that were exacerbated by a common polymorphism in MTHFR. PMID:26307085

Expression and mutational analysis of Cip/Kip family in early glottic cancer.

PubMed

Kim, D-K; Lee, J H; Lee, O J; Park, C H

2015-02-01

Genetic alteration of cyclin-dependent kinase inhibitors has been associated with carcinogenesis mechanisms in various organs. This study aimed to evaluate the expression and mutational analysis of Cip/Kip family cyclin-dependent kinase inhibitors (p21CIP1/WAF1, p27KIP1 and p57KIP2) in early glottic cancer. Expressions of Cip/Kip family and p53 were determined by quantitative reverse transcription polymerase chain reaction and densitometry. For the analysis of p21 inactivation, sequence alteration was assessed using single-strand conformational polymorphism polymerase chain reaction. Additionally, the inactivation mechanism of p27 and p57 were investigated using DNA methylation analysis. Reduced expression of p27 and p57 were detected in all samples, whereas the expression of p21 was incompletely down-regulated in 6 of 11 samples. Additionally, single-strand conformational polymorphism polymerase chain reaction analysis showed the p53 mutation at exon 6. Methylation of p27 and p57 was detected by DNA methylation assay. Our results suggest that the Cip/Kip family may have a role as a molecular mechanism of carcinogenesis in early glottic cancer.

Precise Estimation of Allele Frequencies of Single-Nucleotide Polymorphisms by a Quantitative SSCP Analysis of Pooled DNA

PubMed Central

Sasaki, Tomonari; Tahira, Tomoko; Suzuki, Akari; Higasa, Koichiro; Kukita, Yoji; Baba, Shingo; Hayashi, Kenshi

2001-01-01

We show that single-nucleotide polymorphisms (SNPs) of moderate to high heterozygosity (minor allele frequencies >10%) can be efficiently detected, and their allele frequencies accurately estimated, by pooling the DNA samples and applying a capillary-based SSCP analysis. In this method, alleles are separated into peaks, and their frequencies can be reliably and accurately quantified from their peak heights (SD <1.8%). We found that as many as 40% of publicly available SNPs that were analyzed by this method have widely differing allele frequency distributions among groups of different ethnicity (parents of Centre d'Etude Polymorphisme Humaine families vs. Japanese individuals). These results demonstrate the effectiveness of the present pooling method in the reevaluation of candidate SNPs that have been collected by examination of limited numbers of individuals. The method should also serve as a robust quantitative technique for studies in which a precise estimate of SNP allele frequencies is essential—for example, in linkage disequilibrium analysis. PMID:11083945

[Quantitative characterization of nuclear polymorphism of the epithelial cells in fibroadenomatosis and cancer of the breast].

PubMed

Ganina, K P; Petunin, Iu I; Timoshenko, Ia G

1989-01-01

A method for quantitative analysis of epithelial cell nuclear polymorphism was suggested, viz. identification of general statistical population using Petunin's criterion. This criterion was employed to assess heterogeneity of visible surface of interphase epithelial cell nuclei and to assay nuclear DNA level in fibroadenomatous hyperplasia and cancer of the breast. Heterogeneity index (h), alongside with other parameters, appeared useful for quantitative assessment of the disease: heterogeneity index values ranging 0.1-0.4 point to pronounced heterogeneity of epithelial cell nucleus surface and DNA level, and are suggestive of malignant transformation of tissue, whereas benign proliferation of the epithelium is usually characterized by 0.4 less than h less than or equal to 0.9.

Analysis of mutational changes at the HLA locus in single human sperm.

PubMed

Huang, M M; Erlich, H A; Goodman, M F; Arnheim, N

1995-01-01

Using a simple and efficient single sperm PCR and direct sequencing method, we screened for HLA-DPB1 gene mutations that may give rise to new alleles at this highly polymorphic locus. More than 800 single sperm were studied from a heterozygous individual whose two alleles carried 16 nucleotide sequence differences clustered in six polymorphic regions. A potential microgene conversion event was detected. Unrepaired heteroduplex DNA similar to that which gives rise to postmeiotic segregation events in yeast was observed in three cases. Control experiments also revealed unusual sperm from DPB1 homozygous individuals. The data may help explain allelic diversity in the MHC and suggest that a possible source of human mosaicism may be incomplete DNA mismatch repair during gametogenesis.

DNA damage and genetic methylation changes caused by Cd in Arabidopsis thaliana seedlings.

PubMed

Li, Zhaoling; Liu, Zhihong; Chen, Ruijuan; Li, Xiaojun; Tai, Peidong; Gong, Zongqiang; Jia, Chunyun; Liu, Wan

2015-09-01

Amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MASP) techniques are sensitive to deoxyribonucleic acid (DNA) damage and genetic methylation, respectively. Using these 2 techniques, Arabidopsis thaliana cultured with 0 mg/L (control), 0.5 mg/L, 1.5 mg/L, and 5.0 mg/L Cd(2+) for 16 d was used to analyze the DNA damage and methylation changes as a result of cadmium (Cd). The DNA was amplified by 14 AFLP primer pairs and 13 MSAP primer combinations. In the AFLP experiment, 62 polymorphic sites were found in the patterns of 11 primer combinations and a total of 1116 fragments were obtained in these patterns. There were no polymorphic bands in the remaining 3 pairs. The proportions of polymorphic sites in the 0.5-mg/L Cd(2+) and 5.0-mg/L Cd(2+) treatments were significantly different. Seven polymorphic fragments were then separated and successfully sequenced, yielding 6 nucleobase substitutions and 1 nucleobase deletion. Similarly, in the MSAP experiment, the MSAP% and number of demethylated-type bands were unchanged after Cd treatment, but the number of methylated-type bands was increased significantly in the 5.0-mg/L Cd(2+) treatment group, a finding that may be associated with the AFLP results. The polymorphic bands were also sequenced and the functions of their homologous genes were determined. The DNA damage and methylation changes may be the primary cause of certain pathology changes as a result of Cd uptake in plants. © 2015 SETAC.

Compositions and methods for detecting single nucleotide polymorphisms

DOEpatents

Yeh, Hsin-Chih; Werner, James; Martinez, Jennifer S.

2016-11-22

Described herein are nucleic acid based probes and methods for discriminating and detecting single nucleotide variants in nucleic acid molecules (e.g., DNA). The methods include use of a pair of probes can be used to detect and identify polymorphisms, for example single nucleotide polymorphism in DNA. The pair of probes emit a different fluorescent wavelength of light depending on the association and alignment of the probes when hybridized to a target nucleic acid molecule. Each pair of probes is capable of discriminating at least two different nucleic acid molecules that differ by at least a single nucleotide difference. The methods can probes can be used, for example, for detection of DNA polymorphisms that are indicative of a particular disease or condition.

Isolation and characterization of new highly polymorphic DNA markers from the Huntington disease region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weber, B.; Hedrick, A.; Andrew, S.

1992-02-01

The defect causing Huntington disease (HD) has been mapped to 4p16.3, distal to the DNA marker D4S10. Subsequently, additional polymorphic markers closer to the HD gene have been isolated, which has led to the establishment of predictive testing programs for individuals at risk for HD. Approximately 17% of persons presenting to the Canadian collaborative study for predictive testing for HD have not received any modification of risk, in part because of limited informativeness of currently available DNA markers. Therefore, more highly polymorphic DNA markers are needed, which well further increase the accuracy and availability of predictive testing, specifically for familiesmore » with complex or incomplete pedigree structures. In addition, new markers are urgently needed in order to refine the breakpoints in the few known recombinant HD chromosomes, which could allow a more accurate localization of the HD gene within 4p16.3 and, therefore, accelerate the cloning of the disease gene. In this study, the authors present the identification and characterization of nine new polymorphic DNA markers, including three markers which detect highly informative multiallelic VNTR-like polymorphisms with PIC values of up to .84. These markers have been isolated from a cloned region of DNA which has been previously mapped approximately 1,000 kb from the 4p telomere.« less

[Study on once sampling quantitation based on information entropy of ISSR amplified bands of Houttuynia cordata].

PubMed

Wang, Haiqin; Liu, Wenlong; He, Fuyuan; Chen, Zuohong; Zhang, Xili; Xie, Xianggui; Zeng, Jiaoli; Duan, Xiaopeng

2012-02-01

To explore the once sampling quantitation of Houttuynia cordata through its DNA polymorphic bands that carried information entropy, from other form that the expression of traditional Chinese medicine polymorphism, genetic polymorphism, of traditional Chinese medicine. The technique of inter simple sequence repeat (ISSR) was applied to analyze genetic polymorphism of H. cordata samples from the same GAP producing area, the DNA genetic bands were transformed its into the information entropy, and the minimum once sampling quantitation with the mathematical mode was measured. One hundred and thirty-four DNA bands were obtained by using 9 screened ISSR primers to amplify from 46 strains DNA samples of H. cordata from the same GAP, the information entropy was H=0.365 6-0.978 6, and RSD was 14.75%. The once sampling quantitation was W=11.22 kg (863 strains). The "once minimum sampling quantitation" were calculated from the angle of the genetic polymorphism of H. cordata, and a great differences between this volume and the amount from the angle of fingerprint were found.

Were inefficient mitochondrial haplogroups selected during migrations of modern humans? A test using modular kinetic analysis of coupling in mitochondria from cybrid cell lines

PubMed Central

Amo, Taku; Brand, Martin D.

2007-01-01

We introduce a general test of the bioenergetic importance of mtDNA (mitochondrial DNA) variants: modular kinetic analysis of oxidative phosphorylation in mitochondria from cybrid cells with constant nuclear DNA but different mtDNA. We have applied this test to the hypothesis [Ruiz-Pesini, Mishmar, Brandon, Procaccio and Wallace (2004) Science 303, 223–226] that particular mtDNA haplogroups (specific combinations of polymorphisms) that cause lowered coupling efficiency, leading to generation of less ATP and more heat, were positively selected during radiations of modern humans into colder climates. Contrary to the predictions of this hypothesis, mitochondria from Arctic haplogroups had similar or even greater coupling efficiency than mitochondria from tropical haplogroups. PMID:17355224

Genome-wide analysis of intraspecific DNA polymorphism in 'Micro-Tom', a model cultivar of tomato (Solanum lycopersicum).

PubMed

Kobayashi, Masaaki; Nagasaki, Hideki; Garcia, Virginie; Just, Daniel; Bres, Cécile; Mauxion, Jean-Philippe; Le Paslier, Marie-Christine; Brunel, Dominique; Suda, Kunihiro; Minakuchi, Yohei; Toyoda, Atsushi; Fujiyama, Asao; Toyoshima, Hiromi; Suzuki, Takayuki; Igarashi, Kaori; Rothan, Christophe; Kaminuma, Eli; Nakamura, Yasukazu; Yano, Kentaro; Aoki, Koh

2014-02-01

Tomato (Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. The genome sequencing of the tomato cultivar 'Heinz 1706' was recently completed. To accelerate the progress of tomato genomics studies, systematic bioresources, such as mutagenized lines and full-length cDNA libraries, have been established for the cultivar 'Micro-Tom'. However, these resources cannot be utilized to their full potential without the completion of the genome sequencing of 'Micro-Tom'. We undertook the genome sequencing of 'Micro-Tom' and here report the identification of single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) between 'Micro-Tom' and 'Heinz 1706'. The analysis demonstrated the presence of 1.23 million SNPs and 0.19 million indels between the two cultivars. The density of SNPs and indels was high in chromosomes 2, 5 and 11, but was low in chromosomes 6, 8 and 10. Three known mutations of 'Micro-Tom' were localized on chromosomal regions where the density of SNPs and indels was low, which was consistent with the fact that these mutations were relatively new and introgressed into 'Micro-Tom' during the breeding of this cultivar. We also report SNP analysis for two 'Micro-Tom' varieties that have been maintained independently in Japan and France, both of which have served as standard lines for 'Micro-Tom' mutant collections. Approximately 28,000 SNPs were identified between these two 'Micro-Tom' lines. These results provide high-resolution DNA polymorphic information on 'Micro-Tom' and represent a valuable contribution to the 'Micro-Tom'-based genomics resources.

[Tissue-specific Changes in the Polymorphism of Simple Repeats in DNA of the Offspring of Different Sex Born from Irradiated Male or Female Mice].

PubMed

Lomaeva, M G; Fomenko, L A; Vasil'eva, G V; Bezlepkin, V G

2016-01-01

Evidence is presented indicating the differences in the polymorphism of microsatellite (MCS) repeats in DNA of somatic tissues in the offspring of BALB/c mice of different sex born from preconceptionally irradiated males or females. Brother-sister groups of the offspring born by non-irradiated parental pairs were compared with the offspring obtained after the irradiation of one parent in the same pairs. The number of MCS repeats in DNA of somatic tissues of the offspring from irradiated males or females was compared by a polymerase chain reaction using an arbitrary primer. It was found that changes in the polymorphism of the number of MCS repeats in the offspring from the males irradiated at a dose of 2 Gy was insignificant as compared with the offspring from control animals. In the offspring born by the females irradiated at a dose of 2 Gy (which does not impair the reproductive capacity), a statistically significant increase in the polymorphism was observed. Changes in the polymorphism were different in the offspring of different sex. A higher level of polymorphism was revealed in the female offspring born from the females of the F0 generation after their irradiation at a dose of 2 Gy. The increase in the polymorphism of the number of MCS repeats in DNA was more pronounced in postmitotic tissues compared with proliferating tissues.

Surveyor nuclease detection of mutations and polymorphisms of mtDNA in children.

PubMed

Pilch, Jacek; Asman, Marek; Jamroz, Ewa; Kajor, Maciej; Kotrys-Puchalska, Elżbieta; Goss, Małgorzata; Krzak, Maria; Witecka, Joanna; Gmiński, Jan; Sieroń, Aleksander L

2010-11-01

Mitochondrial encephalomyopathies are complex disorders with wide range of clinical manifestations. Particularly time-consuming is the identification of mutations in mitochondrial DNA. A group of 20 children with clinical manifestations of mitochondrial encephalomyopathies was selected for molecular studies. The aims were (a) to identify mutations in mtDNA isolated from muscle and (b) to verify detected mutations in DNA isolated from blood, in order to assess the utility of a Surveyor nuclease assay kit for patient screening. The most common changes found were polymorphisms, including a few missense mutations altering the amino acid sequence of mitochondrial proteins. In two boys with MELAS (i.e., mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), a mutation A→G3243 was detected in the tRNALeu gene of mtDNA isolated from muscle and blood. In one boy, the carrier status of his mother was confirmed, based on molecular analysis of DNA isolated from blood. A method using Surveyor nuclease allows systematic screening for small mutations in mtDNA, using as its source blood of the patients and asymptomatic carriers. The method still requires confirmation studying a larger group. In some patients, the use of this method should precede and might limit indications for traumatic muscle and skin biopsy. Copyright © 2010 Elsevier Inc. All rights reserved.

MTHFR, TS and XRCC1 genetic variants may affect survival in patients with myelodysplastic syndromes treated with supportive care or azacitidine.

PubMed

Visani, G; Loscocco, F; Ruzzo, A; Galimberti, S; Graziano, F; Voso, M T; Giacomini, E; Finelli, C; Ciabatti, E; Fabiani, E; Barulli, S; Volpe, A; Magro, D; Piccaluga, P; Fuligni, F; Vignetti, M; Fazi, P; Piciocchi, A; Gabucci, E; Rocchi, M; Magnani, M; Isidori, A

2017-12-05

We evaluated the impact of genomic polymorphisms in folate-metabolizing, DNA synthesis and DNA repair enzymes on the clinical outcome of 108 patients with myelodysplastic syndromes (MDS) receiving best supportive care (BSC) or azacitidine. A statistically significant association between methylenetetrahydrofolate reductase (MTHFR) 677T/T, thymidylate synthase (TS) 5'-untranslated region (UTR) 3RG, TS 3'-UTR -6 bp/-6 bp, XRCC1 399G/G genotypes and short survival was found in patients receiving BSC by multivariate analysis (P<0.001; P=0.026; P=0.058; P=0.024). MTHFR 677T/T, TS 3'-UTR -6 bp/-6 bp and XRCC1 399G/G genotypes were associated with short survival in patients receiving azacitidine by multivariate analysis (P<0.001; P=0.004; P=0.002). We then performed an exploratory analysis to evaluate the effect of the simultaneous presence of multiple adverse variant genotypes. Interestingly, patients with ⩾1 adverse genetic variants had a short survival, independently from their International Prognostic Scoring System (IPSS) and therapy received. To our knowledge, this is the first study showing that polymorphisms in folate-metabolizing pathway, DNA synthesis and DNA repair genes could influence survival of MDS patients.The Pharmacogenomics Journal advance online publication, 5 December 2017; doi:10.1038/tpj.2017.48.

Melting analysis on microbeads in rapid temperature-gradient inside microchannels for single nucleotide polymorphisms detectiona)

PubMed Central

Li, Kan-Chien; Ding, Shih-Torng; Lin, En-Chung; Wang, Lon (Alex); Lu, Yen-Wen

2014-01-01

A continuous-flow microchip with a temperature gradient in microchannels was utilized to demonstrate spatial melting analysis on microbeads for clinical Single Nucleotide Polymorphisms (SNPs) genotyping on animal genomic DNA. The chip had embedded heaters and thermometers, which created a rapid and yet stable temperature gradient between 60 °C and 85 °C in a short distance as the detection region. The microbeads, which served as mobile supports carrying the target DNA and fluorescent dye, were transported across the temperature gradient. As the surrounding temperature increased, the fluorescence signals of the microbeads decayed with this relationship being acquired as the melting curve. Fast DNA denaturation, as a result of the improved heat transfer and thermal stability due to scaling, was also confirmed. Further, each individual microbead could potentially bear different sequences and pass through the detection region, one by one, for a series of melting analysis, with multiplex, high-throughput capability being possible. A prototype was tested with target DNA samples in different genotypes (i.e., wild and mutant types) with a SNP location from Landrace sows. The melting temperatures were obtained and compared to the ones using a traditional tube-based approach. The results showed similar levels of SNP discrimination, validating our proposed technique for scanning homozygotes and heterozygotes to distinguish single base changes for disease research, drug development, medical diagnostics, agriculture, and animal production. PMID:25553186

Useful DNA polymorphisms are identified by snapback, a midrepetitive element in Tribolium castaneum.

PubMed

Stuart, J J; De Gortari, M J; Hall, P S; Maxwell, M E; Mocelin, G; Brown, S J; Muir, W M

1996-06-01

The red flour bettle, Tribolium castaneum, is both a pest of stored grain products and an important experimental organism. To improve its facility as a genetic model, we are developing DNA fingerprinting methods for this insect. A Tribolium DNA fragment, snapback-1 (SBI), identified among sequences that reassociate before a Cot of 0.03 mol.s/L, was found to produce a banding pattern in restriction endonuclease digested genomic DNA that is characteristic of a midrepetitive element. DNA fingerprints of individual beetles demonstrated that unvarying inherited DNA polymorphism is revealed, and that polymorphism is inherited in a dominant Mendelian fashion. Linkage between bands was minimal. The sequence of SBI was determined, and hybridization experiments indicated that SBI is a fragment of a larger midrepetitive element. Fingerprinting individuals with known inbreeding coefficients indicated that SBI loci have relatively high mutation rates. The possibility that SBI is a fragment of a transposable element is discussed.

New genetic variants associated with prostate cancer

Cancer.gov

Researchers have newly identified 23 common genetic variants -- one-letter changes in DNA known as single-nucleotide polymorphisms or SNPs -- that are associated with risk of prostate cancer. These results come from an analysis of more than 10 million SNP

Association of transforming growth-factor alpha gene polymorphisms with nonsyndromic cleft palate only (CPO)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shiang, R.; Lidral, A.C.; Ardinger, H.H.

1993-10-01

Genetic analysis and tissue-specific expression studies support a role for transforming growth-factor alpha (TGFA) in craniofacial development. Previous studies have confirmed an association of alleles for TGFA with nonsyndromic cleft lip with or without cleft palate (CL/P) in humans. The authors carried out a retrospective association study to determine whether specific allelic variants of the TGFA gene are also associated with cleft palate only (CPO). The PCR products from 12 overlapping sets of primers to the TGFA cDNA were examined by using single-strand conformational polymorphism analysis. Four DNA polymorphic sites for TGFA were identified in the 3[prime] untranslated region ofmore » the TGFA gene. These variants, as well as previously identified RFLPs for TGFA, were characterized in case and control populations for CPO by using X[sup 2] analysis. A significant association between alleles of TGFA and CPO was identified which further supports a role for this gene as one of the genetic determinants of craniofacial development. Sequence analysis of the variants disclosed a cluster of three variable sites within 30 bp of each other in the 3[prime] untranslated region previously associated with an antisense transcript. These studies extend the role for TGFA in craniofacial morphogenesis and support an interrelated mechanism underlying nonsyndromic forms of CL/P. 46 refs., 3 figs., 3 tabs.« less

Nuclear Ribosomal DNA Variation and Pathogenic Specialization in Alternaria Fungi Known To Produce Host-Specific Toxins †

PubMed Central

Kusaba, Motoaki; Tsuge, Takashi

1994-01-01

A total of 99 strains of 11 Alternaria species, including 68 strains of seven fungi known to produce host-specific toxins, were subjected to analysis of restriction fragment length polymorphism (RFLP) in nuclear ribosomal DNA (rDNA). Total DNA was digested with XbaI, and the Southern blots were probed with a nuclear rDNA clone of Alternaria kikuchiana. The hybridization gave 17 different RFLPs from the 99 strains. On the basis of these RFLPs, populations of host-specific toxin-producing fungi could not be differentiated from one another nor from nonpathogenic A. alternata. Each population of the toxin-producing fungi carried rDNA variants. Nine different types, named A1 to A6 and B1 to B3, were detected among the toxin-producing fungi and nonpathogenic A. alternata. All of the populations contained the type A4 variant, and the other rDNA types were also shared by different toxin-producing fungi and A. alternata. In contrast, Alternaria species that are morphologically distinguishable from A. alternata could be differentiated from A. alternata on the basis of the rDNA RFLPs. Polymorphisms in rDNA digested with HaeIII and MspI were also evaluated in 61 Alternaria strains. These restriction enzymes produced 31 variations among all of the samples. The seven toxin-producing fungi and nonpathogenic A. alternata could not be resolved by phylogenetic analysis based on the RFLPs, although they could be differentiated from the other Alternaria species studied. These results provide support for the hypothesis that Alternaria fungi known to produce host-specific toxins are intraspecific variants of A. alternata specialized in pathogenicity. Images PMID:16349367

Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups.

PubMed

Herrnstadt, Corinna; Elson, Joanna L; Fahy, Eoin; Preston, Gwen; Turnbull, Douglass M; Anderson, Christen; Ghosh, Soumitra S; Olefsky, Jerrold M; Beal, M Flint; Davis, Robert E; Howell, Neil

2002-05-01

The evolution of the human mitochondrial genome is characterized by the emergence of ethnically distinct lineages or haplogroups. Nine European, seven Asian (including Native American), and three African mitochondrial DNA (mtDNA) haplogroups have been identified previously on the basis of the presence or absence of a relatively small number of restriction-enzyme recognition sites or on the basis of nucleotide sequences of the D-loop region. We have used reduced-median-network approaches to analyze 560 complete European, Asian, and African mtDNA coding-region sequences from unrelated individuals to develop a more complete understanding of sequence diversity both within and between haplogroups. A total of 497 haplogroup-associated polymorphisms were identified, 323 (65%) of which were associated with one haplogroup and 174 (35%) of which were associated with two or more haplogroups. Approximately one-half of these polymorphisms are reported for the first time here. Our results confirm and substantially extend the phylogenetic relationships among mitochondrial genomes described elsewhere from the major human ethnic groups. Another important result is that there were numerous instances both of parallel mutations at the same site and of reversion (i.e., homoplasy). It is likely that homoplasy in the coding region will confound evolutionary analysis of small sequence sets. By a linkage-disequilibrium approach, additional evidence for the absence of human mtDNA recombination is presented here.

Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.)

PubMed Central

Urasaki, Naoya; Goeku, Satoko; Kaneshima, Risa; Takamine, Tomonori; Tarora, Kazuhiko; Takeuchi, Makoto; Moromizato, Chie; Yonamine, Kaname; Hosaka, Fumiko; Terakami, Shingo; Matsumura, Hideo; Yamamoto, Toshiya; Shoda, Moriyuki

2015-01-01

To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a ‘piping-leaf-type’ cultivar, ‘Yugafu’, and a ‘spiny-tip-leaf-type’ variety, ‘Yonekura’. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the ‘spiny-leaf type’ as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding. PMID:26175625

Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.).

PubMed

Urasaki, Naoya; Goeku, Satoko; Kaneshima, Risa; Takamine, Tomonori; Tarora, Kazuhiko; Takeuchi, Makoto; Moromizato, Chie; Yonamine, Kaname; Hosaka, Fumiko; Terakami, Shingo; Matsumura, Hideo; Yamamoto, Toshiya; Shoda, Moriyuki

2015-06-01

To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a 'piping-leaf-type' cultivar, 'Yugafu', and a 'spiny-tip-leaf-type' variety, 'Yonekura'. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the 'spiny-leaf type' as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding.

Restriction fragment length polymorphism among Israeli Holstein-Friesian dairy bulls.

PubMed

Beckmann, J S; Kashi, Y; Hallerman, E M; Nave, A; Soller, M

1986-01-01

Israeli Holstein-Friesian dairy bulls were screened for restriction fragment length polymorphisms by hybridizing cloned DNA probes for bovine growth hormone, for chymosin, and for rat muscle beta-actin to restriction endonuclease-digested DNA immobilized on nitrocellulose filters. The population proved to be polymorphic at the growth hormone locus, with evidence consistent with the phenotypes being inherited in allelic fashion. A low level of polymorphism was also observed at one of the beta-actin gene family loci. The chymosin locus was monomorphic with the restriction enzymes utilized. The results illustrate the power of restriction fragment length polymorphism methodology in visualizing genetic variability in dairy cattle populations.

Polymorphic Imprinting of SLC38A4 Gene in Bovine Placenta.

PubMed

Xu, Da; Zhang, Cui; Li, Junliang; Wang, Guannan; Chen, Weina; Li, Dongjie; Li, Shijie

2018-05-21

Imprinted genes are characterized by monoallelic expression that is dependent on parental origin. Comparative analysis of imprinted genes between species is a powerful tool for understanding the biological significance of genomic imprinting. The slc38a4 gene encodes a neutral amino acid transporter and is identified as imprinted in mice. In this study, the imprinting status of SLC38A4 was assessed in bovine adult tissues and placenta using a polymorphism-based approach. Results indicate that SLC38A4 is not imprinted in eight adult bovine tissues including heart, liver, spleen, lung, kidney, muscle, fat, and brain. It was interesting to note that SLC38A4 showed polymorphic status in five heterogeneous placentas, with three exhibiting paternal monoallelic expression and two exhibiting biallelic expression. Monoallelic expression of imprinted genes is generally associated with allele-specific differentially methylation regions (DMRs) of CpG islands (CGIs)-encompassed promoter; therefore, the DNA methylation statuses of three CGIs in the SLC38A4 promoter and exon 1 region were tested in three placentas (two exhibiting paternal monoallelic and one showing biallelic expression of SLC38A4) and their corresponding paternal sperms. Unexpectedly, extreme hypomethylation (< 3%) of the DNA was observed in all the three detected placentas and their corresponding paternal sperms. The absence of DMR in bovine SLC38A4 promoter region implied that DNA methylation of these three CGIs does not directly or indirectly affect the polymorphic imprinting of SLC38A4 in bovine placenta. This suggested other epigenetic features other than DNA methylation are needed in regulating the imprinting of bovine SLC38A4, which is different from that of mouse with respect to a DMR existence at the mouse's slc38a4 promoter region. Although further work is needed, this first characterization of polymorphic imprinting status of SLC38A4 in cattle placenta provides valuable information on investigating the genomic imprinting phenomenon itself.

Association between polymorphisms at promoters of XRCC5 and XRCC6 genes and risk of breast cancer.

PubMed

Rajaei, Mehrdad; Saadat, Iraj; Omidvari, Shahpour; Saadat, Mostafa

2014-04-01

Variation in DNA repair genes is one of the mechanisms that may lead to variation in DNA repair capacity. Ku, a heterodimeric DNA-binding complex, is directly involved in repair of DNA double-strand breaks. Ku consists of two subunits, Ku70 and Ku80, which are encoded by the XRCC6 and XRCC5 genes, respectively. In the present study, we investigated whether common genetic variant in variable number of tandem repeats (VNTR) XRCC5 and T-991C XRCC6 was associated with an altered risk of breast cancer. The present study included 407 females with breast cancer and 395 age frequency-matched controls which were randomly selected from the healthy female blood donors. The XRCC5 and XRCC6 polymorphisms were determined using PCR-based methods. For XRCC5 polymorphism, in comparison with the 1R/1R genotype, the 0R/0R genotype increased breast cancer risk (OR 9.55, 95%CI 1.19-76.64, P = 0.034). The 1R/3R genotype compared with 1R/1R genotype decreased the risk of breast cancer (Fisher's exact test P = 0.015). There was no association between T-991C polymorphism of XRCC6 and breast cancer risk. Mean of age at diagnosis of breast cancer for 0, 1, 2, 3, and >4 repeat in XRCC5 were 39.2, 41.9, 44.3, 45.8, and 47.3 years, respectively. The Kaplan-Meier survival analysis revealed that the number of repeat was associated with age at diagnosis of breast cancer (log rank statistic = 13.90, df = 4, P = 0.008). The findings of the present study revealed that either breast cancer risk or age at diagnosis of breast cancer was associated with the VNTR polymorphism at promoter region of XRCC5.

The hOGG1 Ser326Cys polymorphism contributes to digestive system cancer susceptibility: evidence from 48 case-control studies.

PubMed

Wang, Yang; Gao, Xujie; Wei, Feng; Zhang, Xinwei; Yu, Jinpu; Zhao, Hua; Sun, Qian; Yan, Fan; Yan, Cihui; Li, Hui; Ren, Xiubao

2015-02-01

The Ser326Cys polymorphism in the human 8-oxogunaine DNA glycosylase (hOGG1) gene had been implicated in cancer susceptibility. Studies investigating the associations between the Ser326Cys polymorphism and digestion cancer susceptibility showed conflicting results. Therefore, a meta-analysis was performed to derive a more precise estimation of the relationship. We conducted a meta-analysis of 48 studies that included 12,073 cancer cases and 19,557 case-free controls. We assessed the strength of the association using odds ratios (ORs) with 95% confidence intervals (CIs). In our analysis, the hOGG1 Ser326Cys polymorphism was significantly associated with the risk of digestive system cancers (Cys/Cys vs. Ser/Ser: OR = 1.17, 95% CI = 1.00-1.35, P < 0.001; Cys/Cys vs. Cys/Ser + Ser/Ser: OR = 1.14, 95% CI = 1.00-1.29, P < 0.001). In subgroup analyses by cancer types, we found that the hOGG1 Ser326Cys polymorphism may increase hepatocellular cancer and colorectal cancer risks, but decrease the risk of oral cancer. These findings supported that hOGG1 Ser326Cys polymorphism may contribute to the susceptibility of digestive cancers.

DNA repair genes polymorphisms and genetic susceptibility to Philadelphia-negative myeloproliferative neoplasms in a Portuguese population: The role of base excision repair genes polymorphisms.

PubMed

Azevedo, Ana P; Silva, Susana N; De Lima, João P; Reichert, Alice; Lima, Fernando; Júnior, Esmeraldina; Rueff, José

2017-06-01

The role of base excision repair (BER) genes in Philadelphia-negative (PN)-myeloproliferative neoplasms (MPNs) susceptibility was evaluated by genotyping eight polymorphisms [apurinic/apyrimidinic endodeoxyribonuclease 1, mutY DNA glycosylase, earlier mutY homolog ( E. coli ) (MUTYH), 8-oxoguanine DNA glycosylase 1, poly (ADP-ribose) polymerase (PARP) 1, PARP4 and X-ray repair cross-complementing 1 (XRCC1)] in a case-control study involving 133 Caucasian Portuguese patients. The results did not reveal a correlation between individual BER polymorphisms and PN-MPNs when considered as a whole. However, stratification for essential thrombocythaemia revealed i) borderline effect/tendency to increased risk when carrying at least one variant allele for XRCC1_399 single-nucleotide polymorphism (SNP); ii) decreased risk for Janus kinase 2-positive patients carrying at least one variant allele for XRCC1_399 SNP; and iii) decreased risk in females carrying at least one variant allele for MUTYH SNP. Combination of alleles demonstrated an increased risk to PN-MPNs for one specific haplogroup. These findings may provide evidence for gene variants in susceptibility to MPNs. Indeed, common variants in DNA repair genes may hamper the capacity to repair DNA, thus increasing cancer susceptibility.

DNA repair genes polymorphisms and genetic susceptibility to Philadelphia-negative myeloproliferative neoplasms in a Portuguese population: The role of base excision repair genes polymorphisms

PubMed Central

Azevedo, Ana P.; Silva, Susana N.; De Lima, João P.; Reichert, Alice; Lima, Fernando; Júnior, Esmeraldina; Rueff, José

2017-01-01

The role of base excision repair (BER) genes in Philadelphia-negative (PN)-myeloproliferative neoplasms (MPNs) susceptibility was evaluated by genotyping eight polymorphisms [apurinic/apyrimidinic endodeoxyribonuclease 1, mutY DNA glycosylase, earlier mutY homolog (E. coli) (MUTYH), 8-oxoguanine DNA glycosylase 1, poly (ADP-ribose) polymerase (PARP) 1, PARP4 and X-ray repair cross-complementing 1 (XRCC1)] in a case-control study involving 133 Caucasian Portuguese patients. The results did not reveal a correlation between individual BER polymorphisms and PN-MPNs when considered as a whole. However, stratification for essential thrombocythaemia revealed i) borderline effect/tendency to increased risk when carrying at least one variant allele for XRCC1_399 single-nucleotide polymorphism (SNP); ii) decreased risk for Janus kinase 2-positive patients carrying at least one variant allele for XRCC1_399 SNP; and iii) decreased risk in females carrying at least one variant allele for MUTYH SNP. Combination of alleles demonstrated an increased risk to PN-MPNs for one specific haplogroup. These findings may provide evidence for gene variants in susceptibility to MPNs. Indeed, common variants in DNA repair genes may hamper the capacity to repair DNA, thus increasing cancer susceptibility. PMID:28599464

Simple sequence repeats in Escherichia coli: abundance, distribution, composition, and polymorphism.

PubMed

Gur-Arie, R; Cohen, C J; Eitan, Y; Shelef, L; Hallerman, E M; Kashi, Y

2000-01-01

Computer-based genome-wide screening of the DNA sequence of Escherichia coli strain K12 revealed tens of thousands of tandem simple sequence repeat (SSR) tracts, with motifs ranging from 1 to 6 nucleotides. SSRs were well distributed throughout the genome. Mononucleotide SSRs were over-represented in noncoding regions and under-represented in open reading frames (ORFs). Nucleotide composition of mono- and dinucleotide SSRs, both in ORFs and in noncoding regions, differed from that of the genomic region in which they occurred, with 93% of all mononucleotide SSRs proving to be of A or T. Computer-based analysis of the fine position of every SSR locus in the noncoding portion of the genome relative to downstream ORFs showed SSRs located in areas that could affect gene regulation. DNA sequences at 14 arbitrarily chosen SSR tracts were compared among E. coli strains. Polymorphisms of SSR copy number were observed at four of seven mononucleotide SSR tracts screened, with all polymorphisms occurring in noncoding regions. SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identification, and detection of loci affecting key phenotypes) and for evolutionary adaptation of microbes.

Molecular analysis of RAPD DNA based markers: their potential use for the detection of genetic variability in jojoba (Simmondsia chinensis L Schneider).

PubMed

Amarger, V; Mercier, L

1995-01-01

We have applied the recently developed technique of random amplified polymorphic DNA (RAPD) for the discrimination between two jojoba clones at the genomic level. Among a set of 30 primers tested, a simple reproducible pattern with three distinct fragments for clone D and two distinct fragments for clone E was obtained with primer OPB08. Since RAPD products are the results of arbitrarily priming events and because a given primer can amplify a number of non-homologous sequences, we wondered whether or not RAPD bands, even those of similar size, were derived from different loci in the two clones. To answer this question, two complementary approaches were used: i) cloning and sequencing of the amplification products from clone E; and ii) complementary Southern analysis of RAPD gels using cloned or amplified fragments (directly recovered from agarose gels) as RFLP probes. The data reported here show that the RAPD reaction generates multiple amplified fragments. Some fragments, although resolved as a single band on agarose gels, contain different DNA species of the same size. Furthermore, it appears that the cloned RAPD products of known sequence that do not target repetitive DNA can be used as hybridization probes in RFLP to detect a polymorphism among individuals.

Human beta-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old.

PubMed

Béraud-Colomb, E; Roubin, R; Martin, J; Maroc, N; Gardeisen, A; Trabuchet, G; Goosséns, M

1995-12-01

Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the beta-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for beta-globin frameworks by sequencing through two variable positions and for a polymorphic (AT) chi (T) gamma microsatellite 500 bp upstream of the beta-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible.

Specific Polymorphic Variation in the Mitochondrial Genome and Increased In-Hospital Mortality After Severe Trauma

PubMed Central

Canter, Jeffrey A.; Norris, Patrick R.; Moore, Jason H.; Jenkins, Judith M.; Morris, John A.

2007-01-01

Objective: To determine whether specific genetic variations in the mtDNA that impact energy production and free-radical generation are potential new risk factors for in-hospital mortality after severe trauma. Summary Background Data: Each of the 3 mitochondrial DNA polymorphisms selected for this study (at positions 4216, 10398, 4917) alter the amino acid sequence of different key subunits of Complex I in the electron transport chain. They have been previously implicated in phenotypes involving tissues with high-energy demand, such as the brain and retina. Methods: Seven hundred forty-five consecutive patients admitted to the trauma intensive care unit at Vanderbilt University Medical Center between April 11, 2005, and February 27, 2006, were potentially eligible for this study. Under an Institutional Review Board-approved protocol (which excluded patients <18 years of age and prisoners), 666 patients had DNA extracted from a blood sample. Detailed demographic and clinical covariates were also obtained (including age, gender, ethnicity, lactate measurements, and injury severity score). A flurogenic 5′ nuclease allelic discrimination Taqman assay and the ABI 7900HT Sequence Detection System (v2.1) was used to genotype the T4216C, A10398G, and A4917G polymorphisms. The primary outcome was in-hospital mortality. Results: Multivariate logistic regression analysis revealed that the 4216T allele was a significant independent predictor of in-hospital mortality (OR = 2.63, 95% CI 1.14–6.07, P = 0.02) after adjustment for age, gender, injury severity score, highest lactate level, mechanism of injury, and the 10398 polymorphism. Conclusions: Variation in the mtDNA, specifically the 4216T allele, appears to increase the risk of in-hospital mortality after severe injury. PMID:17717444

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers.

PubMed

Cui, Cuiju; Li, Yan; Liu, Yanling; Li, Xiaojie; Luo, Shiju; Zhang, Zhuangzhi; Wu, Ruina; Liang, Guangjin; Sun, Juan; Peng, Jie; Tian, Pingping

2017-02-01

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r=0.78) was better than that of the RAPD marker system (r=0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding. Copyright © 2016 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

Genomic profiling of plastid DNA variation in the Mediterranean olive tree

PubMed Central

2011-01-01

Background Characterisation of plastid genome (or cpDNA) polymorphisms is commonly used for phylogeographic, population genetic and forensic analyses in plants, but detecting cpDNA variation is sometimes challenging, limiting the applications of such an approach. In the present study, we screened cpDNA polymorphism in the olive tree (Olea europaea L.) by sequencing the complete plastid genome of trees with a distinct cpDNA lineage. Our objective was to develop new markers for a rapid genomic profiling (by Multiplex PCRs) of cpDNA haplotypes in the Mediterranean olive tree. Results Eight complete cpDNA genomes of Olea were sequenced de novo. The nucleotide divergence between olive cpDNA lineages was low and not exceeding 0.07%. Based on these sequences, markers were developed for studying two single nucleotide substitutions and length polymorphism of 62 regions (with variable microsatellite motifs or other indels). They were then used to genotype the cpDNA variation in cultivated and wild Mediterranean olive trees (315 individuals). Forty polymorphic loci were detected on this sample, allowing the distinction of 22 haplotypes belonging to the three Mediterranean cpDNA lineages known as E1, E2 and E3. The discriminating power of cpDNA variation was particularly low for the cultivated olive tree with one predominating haplotype, but more diversity was detected in wild populations. Conclusions We propose a method for a rapid characterisation of the Mediterranean olive germplasm. The low variation in the cultivated olive tree indicated that the utility of cpDNA variation for forensic analyses is limited to rare haplotypes. In contrast, the high cpDNA variation in wild populations demonstrated that our markers may be useful for phylogeographic and populations genetic studies in O. europaea. PMID:21569271

Longevity-associated mitochondrial DNA 5178 C/A polymorphism modulates the effects of coffee consumption on erythrocytic parameters in Japanese men: an exploratory cross-sectional analysis.

PubMed

Kokaze, Akatsuki; Ishikawa, Mamoru; Matsunaga, Naomi; Karita, Kanae; Yoshida, Masao; Ohtsu, Tadahiro; Ochiai, Hirotaka; Shirasawa, Takako; Nanri, Hinako; Saga, Nobuyuki; Ohtsu, Iichiro; Hoshino, Hiromi; Takashima, Yutaka

2014-12-20

Mitochondrial DNA 5178 cytosine/adenine (Mt5178 C/A) polymorphism reportedly modulates the effects of coffee consumption on the risk of hypertension, dyslipidemia and abnormal glucose tolerance. The objective of this analysis was to investigate whether Mt5178 C/A polymorphism modifies the effects of coffee consumption on erythrocytic parameters in male Japanese health check-up examinees. A total of 436 men (mean age ± standard deviation, 54.1 ± 7.8 years) were selected from among individuals visiting the hospital for regular medical check-ups. After Mt5178 C/A genotyping, an exploratory cross-sectional analysis assessing the joint effects of Mt5178 C/A polymorphism and coffee consumption on red blood cell counts, hematocrit and hemoglobin was conducted. For Mt5178C genotypic men, after adjustment for age, body mass index, alcohol consumption, habitual smoking and green tea consumption, coffee consumption significantly decreased red blood cell counts (P for trend = 0.022) and hemoglobin (P for trend = 0.035). The risk of anemia, defined as hemoglobin of <14 g/dL, after the aforementioned adjustment, appeared to depend on coffee consumption (P for trend = 0.078), and the adjusted odds ratio for anemia was significantly higher in men who consumed ≥4 cups of coffee per day than in those who consumed <1 cup per day (odds ratio = 3.771, 95% confidence interval: 1.088 to 13.06, P = 0.036). For Mt5178A genotypic men, coffee consumption possibly reduced the risk of anemia (P for trend = 0.049). However, after the aforementioned adjustment, the statistical significance disappeared (P for trend = 0.137). This exploratory cross-sectional analysis suggests that Mt5178 C/A polymorphism modulates the effects of coffee consumption on erythrocytic parameters and the risk of anemia in male Japanese health check-up examinees.

Ex vivo study for the assessment of behavioral factor and gene polymorphisms in individual susceptibility to oxidative DNA damage metals-induced.

PubMed

Di Pietro, Angela; Baluce, Barbara; Visalli, Giuseppa; La Maestra, Sebastiano; Micale, Rosanna; Izzotti, Alberto

2011-06-01

Transition metals in fine particulate matter generated by combustion induce oxidative DNA damage and inflammation. However, there is remarkable inter-individual variability in susceptibility to these damages. To assess this variability, an ex vivo study was performed using lymphocytes of 47 Caucasian healthy subjects. Cell samples were exposed to a water solution of oil fly ash (OFA). This was formed by the distinctive transition metals vanadium, iron, and nickel. Oxidative DNA damage was evaluated by testing cell viability, intracellular ROS production and 8-oxo-dG. DNA fragmentation and DNA repair capacity were assessed by using the Alkaline-Halo assay. GSTM1, GSTT1, hOGG1, and C677T and A1298C MTHFR gene polymorphisms were tested. Demographic and behavioral factors, collected by questionnaire, were also considered. OFA induced damages showed: (a) a 20-fold variation in range among different subjects in ROS production, (b) a 7-fold variation in range of 8-oxo-dG, and (c) a 25-fold variation in range in DNA repair capacity. A significant increase in DNA damage was detected in GSTT1-deficent subjects compared with wild type genotype carriers. Increases in cytoplasmic ROS and decreases in DNA repair capacity (P<0.05) were observed in C677T and A1298C variants of MTHFR. A remarkable protective effect of high fruits and vegetable intake was observed for ROS production and DNA damage. Conversely, an adverse effect of meat intake was observed on ROS increase, DNA damage and repair capacity, probably due to the increased intake of bioavailable iron. Smoking decreased DNA repair capacity, while age increased OFA-induced DNA damage. The wide comparative analysis of the complex interactions network, between genetic and behavioral factors provides evidence of the remarkable role of several lifestyle factors. In comparison to genetic polymorphisms they seem to have a higher weight in determining individual susceptibility to the adverse effects of airborne pollutants as transition metals. Copyright © 2011 Elsevier GmbH. All rights reserved.

Mismatch repair gene MSH3 polymorphism is associated with the risk of sporadic prostate cancer.

PubMed

Hirata, Hiroshi; Hinoda, Yuji; Kawamoto, Ken; Kikuno, Nobuyuki; Suehiro, Yutaka; Okayama, Naoko; Tanaka, Yuichiro; Dahiya, Rajvir

2008-05-01

The mismatch repair system is a DNA repair mechanism that corrects mispaired bases during DNA replication errors. Cancer cells deficient in MMR proteins have a 10(2) to 10(3)-fold increase in the mutation rate. Single nucleotide polymorphisms of mismatch repair genes have been shown to cause a decrease in DNA repair activity. We hypothesized that mismatch repair gene polymorphism could be a risk factor for prostate cancer and p53 Pro/Pro genotype carriers could influence MSH3 and MSH6 polymorphisms. DNA samples from 110 patients with prostate cancer and 110 healthy controls were analyzed by single strand conformational polymorphism and polymerase chain reaction-restriction fragment length polymorphism to determine the genotypic frequency of 5 polymorphic loci on 2 MMR genes (MSH3 and MSH6) and p53 codon72. The chi-square test was applied to compare genotype frequency between patients and controls. A significant increase in the G/A+A/A genotype of MSH3 Pro222Pro was observed in patients compared to controls (OR 1.87, 95% CI 1.0-3.5). The frequency of A/G + G/G genotypes of MSH3 exon23 Thr1036Ala also tended to increase in patients (OR 1.57, 95% CI 0.92-2.72). In p53 codon72 Arg/Pro + Pro/Pro carriers the frequency of the AG + GG genotype of MSH3 exon23 was significantly increased in patients compared to controls (OR 2.1, 95% CI 1.05-4.34). To our knowledge this is the first report of the association of MSH3 gene polymorphisms in prostate cancer. These results suggest that the MSH3 polymorphism may be a risk factor for prostate cancer.

Varietal Tracing of Virgin Olive Oils Based on Plastid DNA Variation Profiling

PubMed Central

Pérez-Jiménez, Marga; Besnard, Guillaume; Dorado, Gabriel; Hernandez, Pilar

2013-01-01

Olive oil traceability remains a challenge nowadays. DNA analysis is the preferred approach to an effective varietal identification, without any environmental influence. Specifically, olive organelle genomics is the most promising approach for setting up a suitable set of markers as they would not interfere with the pollinator variety DNA traces. Unfortunately, plastid DNA (cpDNA) variation of the cultivated olive has been reported to be low. This feature could be a limitation for the use of cpDNA polymorphisms in forensic analyses or oil traceability, but rare cpDNA haplotypes may be useful as they can help to efficiently discriminate some varieties. Recently, the sequencing of olive plastid genomes has allowed the generation of novel markers. In this study, the performance of cpDNA markers on olive oil matrices, and their applicability on commercial Protected Designation of Origin (PDO) oils were assessed. By using a combination of nine plastid loci (including multi-state microsatellites and short indels), it is possible to fingerprint six haplotypes (in 17 Spanish olive varieties), which can discriminate high-value commercialized cultivars with PDO. In particular, a rare haplotype was detected in genotypes used to produce a regional high-value commercial oil. We conclude that plastid haplotypes can help oil traceability in commercial PDO oils and set up an experimental methodology suitable for organelle polymorphism detection in the complex olive oil matrices. PMID:23950947

Prenatal detection of fetal triploidy from cell-free DNA testing in maternal blood.

PubMed

Nicolaides, Kypros H; Syngelaki, Argyro; del Mar Gil, Maria; Quezada, Maria Soledad; Zinevich, Yana

2014-01-01

To investigate potential performance of cell-free DNA (cfDNA) testing in maternal blood in detecting fetal triploidy. Plasma and buffy coat samples obtained at 11-13 weeks' gestation from singleton pregnancies with diandric triploidy (n=4), digynic triploidy (n=4), euploid fetuses (n=48) were sent to Natera, Inc. (San Carlos, Calif., USA) for cfDNA testing. Multiplex polymerase chain reaction amplification of cfDNA followed by sequencing of single nucleotide polymorphic loci covering chromosomes 13, 18, 21, X, and Y was performed. Sequencing data were analyzed using the NATUS algorithm which identifies copy number for each of the five chromosomes. cfDNA testing provided a result in 44 (91.7%) of the 48 euploid cases and correctly predicted the fetal sex and the presence of two copies each of chromosome 21, 18 and 13. In diandric triploidy, cfDNA testing identified multiple paternal haplotypes (indicating fetal trisomy 21, trisomy 18 and trisomy 13) suggesting the presence of either triploidy or dizygotic twins. In digynic triploidy the fetal fraction corrected for maternal weight and gestational age was below the 0.5th percentile. cfDNA testing by targeted sequencing and allelic ratio analysis of single nucleotide polymorphisms covering chromosomes 21, 18, 13, X, and Y can detect diandric triploidy and raise the suspicion of digynic triploidy. © 2013 S. Karger AG, Basel.

Phylogenetic analysis of mtDNA lineages in South American mummies.

PubMed

Monsalve, M V; Cardenas, F; Guhl, F; Delaney, A D; Devine, D V

1996-07-01

Some studies of mtDNA propose that contemporary Amerindians have descended from four haplotype groups, each defined by specific sets of polymorphisms. One recent study also found evidence of other potential founder haplotypes. We wanted to determine whether the four haplotypes in modern populations were also present in ancient South American aboriginals. We subjected mtDNA from Colombian mummies (470 to 1849 AD) to PCR amplification and restriction endonuclease analysis. The mtDNA D-loop region was surveyed for sequence variation by restriction analysis and a segment of this region was sequenced for each mummy to characterize the haplotypes. Our mummies exhibited three of the four major characteristic haplotypes of Amerindian populations defined by four markers. With sequence data obtained in the ancient samples and published data on contemporary Amerindians it was possible to infer the origin of these six mummies.

Methylation-sensitive amplification polymorphism analysis of fat and muscle tissues in pigs.

PubMed

Ma, J D; Li, M Z; Zhou, S L; Zhou, C W; Li, X W

2012-09-26

DNA methylation may be involved in regulating the expression of protein-coding genes, resulting in different fat and muscle phenotypes. Using a methylation-sensitive amplified polymorphism approach, we obtained 7423 bands by selective amplification of genomic DNA from six different fat depots and two heterogeneous muscle types from Duroc/Landrace/Yorkshire cross-bred pigs. The degrees of DNA methylation, determined by the percentages of hemi- and fully methylated sites relative to the total number of CCGG sites, were similar in male and female pigs for each specific tissue [χ(2) test; P (two-tailed) > 0.05]. Gender bias was therefore ignored. There were significant differences in the degree of DNA methylation among the eight tissue types [χ(2) test; P(total) (two-tailed) = 0.009]. However, similar degrees of methylation were observed among the six fat depots [χ(2) test; P(fat) (two-tailed) = 0.24 > 0.05]and between the two muscle types [χ(2) test; P(muscle) (two-tailed) = 0.76 > 0.05]. We conclude that the degree of DNA methylation differs between porcine fat and muscle tissue, but that the methylation status of a particular tissue type is similar, despite being deposited at different body sites.

Genetic variability in selected date palm (Phoenix dactylifera L.) cultivars of United Arab Emirates using ISSR and DAMD markers.

PubMed

Purayil, Fayas T; Robert, Gabriel A; Gothandam, Kodiveri M; Kurup, Shyam S; Subramaniam, Sreeramanan; Cheruth, Abdul Jaleel

2018-02-01

Nine (9) different date palm ( Phoenix dactylifera L.) cultivars from UAE, which differ in their flower timings were selected to determine the polymorphism and genetic relationship between these cultivars. Hereditary differences and interrelationships were assessed utilizing inter-simple sequence repeat (ISSR) and directed amplification of minisatellite DNA region (DAMD) primers. Analysis on eight DAMD and five ISSR markers produced total of 113 amplicon including 99 polymorphic and 14 monomorphic alleles with a polymorphic percentage of 85.45. The average polymorphic information content for the two-marker system was almost similar (DAMD, 0.445 and ISSR, 0.459). UPGMA based clustering of DAMD and ISSR revealed that mid-season cultivars, Mkh (Khlas) and MB (Barhee) grouped together to form a subcluster in both the marker systems. The genetic similarity analysis followed by clustering of the cumulative data from the DAMD and ISSR resulted in two major clusters with two early-season cultivars (ENg and Ekn), two mid-season cultivars (MKh and MB) and one late-season cultivar (Lkhs) in cluster 1, cluster 2 includes two late-season cultivars, one early-season cultivar and one mid-season cultivar. The cluster analysis of both DAMD and ISSR marker revealed that, the patterns of variation between some of the tested cultivars were similar in both DNA marker systems. Hence, the present study signifies the applicability of DAMD and ISSR marker system in detecting genetic diversity of date palm cultivars flowering at different seasons. This may facilitate the conservation and improvement of date palm cultivars in the future.

Analysis of isolates within species of anuran trypanosomes using random amplified polymorphic DNA.

PubMed

Lun, Z R; Desser, S S

1996-01-01

A total of 20 decamer primers were used to generate random applied polymorphic DNA (RAPD) markers from 5 isolates of Trypanosoma fallisi, 3 isolates of T. ranarum, 2 isolates of T. rotatorium, and 2 isolates of T. rotatorium-like trypanosomes in addition to 2 species from the American Type Culture Collection, T. chattoni (ATCC 50294) and Trypanosoma sp. (ATCC 50295). A slight polymorphism was observed among the four isolates of T. fallisi obtained form American toads, Bufo americanus, collected in Algonquin Park, Ontario, Canada, and an isolate obtained from the same species of host collected in Marquette, Michigan, United States, and produced similarity coefficients ranging from 80.7% to 96.9%. Pronounced polymorphism was recorded among the three isolates of T. ranarum from bullfrogs, Rana catesbeiana, collected in Ontario, Canada, and in Maryland, United States, and from a Northern leopard frog, R. pipiens, collected in Minnesota (USA). The similarity coefficients ranged from 54.7% to 59.5%, suggesting that alleles of these isolates were conserved over a wide geographic range. The high degree of polymorphism observed in two isolates of T. rotatorium from a bullfrog collected in Ontario and two isolates of a T. rotatorium-like parasite from the green frog R. clamitans, collected in Louisiana (USA) suggests that they are different species. These results reflect the high similarity among isolates from the same geographic location and the pronounced polymorphism apparent among isolates from distant geographic locations.

Polymorphisms of apolipoprotein E and angiotensin-converting enzyme genes and carotid atherosclerosis in heavy drinkers.

PubMed

Bednarska-Makaruk, Małgorzata; Rodo, Maria; Markuszewski, Cezary; Rozenfeld, Anna; Swiderska, Malgorzata; Habrat, Bogusław; Wehr, Hanna

2005-01-01

To investigate the influence of apolipoprotein E (APOE) and angiotensin-converting enzyme (ACE) gene polymorphisms on carotid artery atherosclerosis in alcoholism. Polymorphism of both genes was identified by DNA analysis in 130 male alcohol-dependent patients. Intima-media thickness (IMT) was measured ultrasonographically. Multivariate regression analysis showed that of all the known risk factors the greatest impact on carotid atherosclerosis in alcoholics was exerted by age, hypertension, LDL cholesterol and fasting plasma glucose levels. Subjects carrying the APO E epsilon4 allele were more liable to develop atherosclerotic changes in carotid arteries compared with subjects with the epsilon3/3 genotype, which showed statistical significance in patients under 50 years of age. No association was shown between ACE I/D polymorphism and carotid atherosclerosis. APO E polymorphism can increase the risk of carotid atherosclerosis development in an alcoholic subject. The association of the APO E epsilon4 allele with carotid atherosclerosis was significant in younger patients. Since the elevated carotid IMT is considered to be a good marker of increased risk of generalized atherosclerosis the consequences could involve both cardiac and cerebrovascular events.

Mapped DNA probes from Ioblolly pine can be used for restriction fragment length polymorphism mapping in other conifers

Treesearch

M.R. Ahuja; M.E. Devey; A.T. Groover; K.D. Jermstad; D.B Neale

1994-01-01

A high-density genetic map based on restriction fragment length polymorphisms (RFLPs) is being constructed for loblolly pine (Pinus taeda L.). Consequently, a large number of DNA probes from loblolly pine are potentially available for use in other species. We have used some of these DNA probes to detect RFLPs in 12 conifers and an angiosperm....

Characterization of a highly polymorphic region 5′ to JH in the human immunoglobulin heavy chain

PubMed Central

Silva, Alcino J.; Johnson, John P.; White, Raymond L.

1987-01-01

A cloned DNA segment 1.25 kilobases (kb) upstream from the joining segments of the human heavy chain immunoglobulin gene revealed extensive polymorphic variation at this locus, and the polymorphic pattern was stably transmitted to the next generation. Genomic restriction analysis showed that the polymorphism was caused by insertions/deletions within an MspI/BamHI fragment. Sequencing of one allele, 848 base pairs (bp) long, revealed eleven 50-base-pair tandem repeats. A second allele, 648 bp long, was cloned from a human genomic cosmid library, sequenced, and found to contain four fewer repeats than the first allele. A survey of 186 chromosomes from unrelated individuals of primarily northern European descent revealed at least six alleles. Images PMID:2884636

Association analysis of a polymorphism of the monoamine oxidase B gene with Parkinson`s disease in a Japanese population

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morimoto, Yuji; Murayama, Nobuhiro; Kuwano, Akira

1995-12-18

The polymorphic allele of the monoamine oxidase B (MAO-B) gene detected by polymerase chain reaction (PCR) and single-stranded conformation polymorphism (SSCP) was associated with Parkinson`s disease (PD) in Caucasians. We characterized this polymorphic allele, allele 1, of the MAO-B gene using direct sequencing of PCR products. A single DNA substitution (G-A), resulting gain of Mae III restriction site was detected in intron 13 of the MAO-B gene. The allele associated with PD in Caucasians was twice as frequent as in healthy Japanese, but the association of the allele of the MAO-B gene was not observed in Japanese patients with PD.more » 7 refs., 2 figs., 1 tab.« less

Single nucleotide polymorphism detection in aldehyde dehydrogenase 2 (ALDH2) gene using bacterial magnetic particles based on dissociation curve analysis.

PubMed

Maruyama, Kohei; Takeyama, Haruko; Nemoto, Etsuo; Tanaka, Tsuyoshi; Yoda, Kiyoshi; Matsunaga, Tadashi

2004-09-20

Single nucleotide polymorphism (SNP) detection for aldehyde dehydrogenase 2 (ALDH2) gene based on DNA thermal dissociation curve analysis was successfully demonstrated using an automated system with bacterial magnetic particles (BMPs) by developing a new method for avoiding light scattering caused by nanometer-size particles when using commercially available fluorescent dyes such as FITC, Cy3, and Cy5 as labeling chromophores. Biotin-labeled PCR products in ALDH2, two allele-specific probes (Cy3-labeled detection probe for ALDH2*1 and Cy5-labeled detection probe for ALDH2*2), streptavidin-immobilized BMPs (SA-BMPs) were simultaneously mixed. The mixture was denatured at 70 degrees C for 3 min, cooled slowly to 25 degrees C, and incubated for 10 min, allowing the DNA duplex to form between Cy3- or Cy5-labeled detection probes and biotin-labeled PCR products on SA-BMPs. Then duplex DNA-BMP complex was heated to 58 degrees C, a temperature determined by dissociation curve analysis and a dissociated single-base mismatched detection probe was removed at the same temperature under precise control. Furthermore, fluorescence signal from the detection probe was liberated into the supernatant from completely matched duplex DNA-BMP complex by heating to 80 degrees C and measured. In the homozygote target DNA (ALDH2*1/*1 and ALDH2*2/*2), the fluorescence signals from single-base mismatched were decreased to background level, indicating that mismatched hybridization was efficiently removed by the washing process. In the heterozygote target DNA (ALDH2*1/*2), each fluorescence signals was at a similar level. Therefore, three genotypes of SNP in ALDH2 gene were detected using the automated detection system with BMPs. Copyright 2004 Wiley Periodicals, Inc.

The Use and Effectiveness of Triple Multiplex System for Coding Region Single Nucleotide Polymorphism in Mitochondrial DNA Typing of Archaeologically Obtained Human Skeletons from Premodern Joseon Tombs of Korea

PubMed Central

Oh, Chang Seok; Lee, Soong Deok; Kim, Yi-Suk; Shin, Dong Hoon

2015-01-01

Previous study showed that East Asian mtDNA haplogroups, especially those of Koreans, could be successfully assigned by the coupled use of analyses on coding region SNP markers and control region mutation motifs. In this study, we tried to see if the same triple multiplex analysis for coding regions SNPs could be also applicable to ancient samples from East Asia as the complementation for sequence analysis of mtDNA control region. By the study on Joseon skeleton samples, we know that mtDNA haplogroup determined by coding region SNP markers successfully falls within the same haplogroup that sequence analysis on control region can assign. Considering that ancient samples in previous studies make no small number of errors in control region mtDNA sequencing, coding region SNP analysis can be used as good complimentary to the conventional haplogroup determination, especially of archaeological human bone samples buried underground over long periods. PMID:26345190

Variation in the number of nucleoli and incomplete homogenization of 18S ribosomal DNA sequences in leaf cells of the cultivated Oriental ginseng (Panax ginseng Meyer).

PubMed

Chelomina, Galina N; Rozhkovan, Konstantin V; Voronova, Anastasia N; Burundukova, Olga L; Muzarok, Tamara I; Zhuravlev, Yuri N

2016-04-01

Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440-640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine.

Variation in the number of nucleoli and incomplete homogenization of 18S ribosomal DNA sequences in leaf cells of the cultivated Oriental ginseng (Panax ginseng Meyer)

PubMed Central

Chelomina, Galina N.; Rozhkovan, Konstantin V.; Voronova, Anastasia N.; Burundukova, Olga L.; Muzarok, Tamara I.; Zhuravlev, Yuri N.

2015-01-01

Background Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. Methods The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. Results In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440–640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. Conclusion This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine. PMID:27158239

A Polymorphism in Mitochondrial DNA Associated with IQ?

ERIC Educational Resources Information Center

Skuder, Patricia; And Others

1995-01-01

Of 100 DNA markers examined in an allelic association study, only 1 showed a replicated association with IQ in samples totaling 107 children. How the gene marked by the particular restriction fragment length polymorphism was tracked and its mitochondrial origin identified is described. (SLD)

A Neutrality Test for Detecting Selection on DNA Methylation Using Single Methylation Polymorphism Frequency Spectrum

PubMed Central

Wang, Jun; Fan, Chuanzhu

2015-01-01

Inheritable epigenetic mutations (epimutations) can contribute to transmittable phenotypic variation. Thus, epimutations can be subject to natural selection and impact the fitness and evolution of organisms. Based on the framework of the modified Tajima’s D test for DNA mutations, we developed a neutrality test with the statistic “Dm” to detect selection forces on DNA methylation mutations using single methylation polymorphisms. With computer simulation and empirical data analysis, we compared the Dm test with the original and modified Tajima’s D tests and demonstrated that the Dm test is suitable for detecting selection on epimutations and outperforms original/modified Tajima’s D tests. Due to the higher resetting rate of epimutations, the interpretation of Dm on epimutations and Tajima’s D test on DNA mutations could be different in inferring natural selection. Analyses using simulated and empirical genome-wide polymorphism data suggested that genes under genetic and epigenetic selections behaved differently. We applied the Dm test to recently originated Arabidopsis and human genes, and showed that newly evolved genes contain higher level of rare epialleles, suggesting that epimutation may play a role in origination and evolution of genes and genomes. Overall, we demonstrate the utility of the Dm test to detect whether the loci are under selection regarding DNA methylation. Our analytical metrics and methodology could contribute to our understanding of evolutionary processes of genes and genomes in the field of epigenetics. The Perl script for the “Dm” test is available at http://fanlab.wayne.edu/ (last accessed December 18, 2014). PMID:25539727

Identification of Legionella Species by Random Amplified Polymorphic DNA Profiles

PubMed Central

Lo Presti, François; Riffard, Serge; Vandenesch, François; Etienne, Jerome

1998-01-01

Random amplified polymorphic DNA (RAPD) was used for the identification of Legionella species. Primer SK2 (5′-CGGCGGCGGCGG-3′) and standardized RAPD conditions gave the technique a reproducibility of 93 to 100%, depending on the species tested. Species-specific patterns corresponding to the 42 Legionella species were consequently defined by this method; the patterns were dependent on the recognition of a core of common bands for each species. This specificity was demonstrated by testing 65 type strains and 265 environmental and clinical isolates. No serogroup-specific profiles were obtained. A number of unidentified Legionella isolates potentially corresponding to new species were clustered in four groups. RAPD analysis appears to be a rapid and reproducible technique for identification of Legionella isolates to the species level without further restriction or hybridization. PMID:9774564

Polymorphism in xeroderma pigmentosum complementation group C codon 939 and aflatoxin B1-related hepatocellular carcinoma in the Guangxi population.

PubMed

Long, Xi-Dai; Ma, Yun; Zhou, Yuan-Feng; Ma, Ai-Min; Fu, Guo-Hui

2010-10-01

Genetic polymorphisms in DNA repair genes may influence individual variations in DNA repair capacity, and this may be associated with the risk and outcome of hepatocellular carcinoma (HCC) related to aflatoxin B1 (AFB1) exposure. In this study, we focused on the polymorphism of xeroderma pigmentosum complementation group C (XPC) codon 939 (rs#2228001), which is involved in nucleotide excision repair. We conducted a case-control study including 1156 HCC cases and 1402 controls without any evidence of hepatic disease to evaluate the associations between this polymorphism and HCC risk and prognosis in the Guangxi population. AFB1 DNA adduct levels, XPC genotypes, and XPC protein levels were tested with a comparative enzyme-linked immunosorbent assay, TaqMan polymerase chain reaction for XPC genotypes, and immunohistochemistry, respectively. Higher AFB1 exposure was observed among HCC patients versus the control group [odds ratio (OR) = 9.88 for AFB1 exposure years and OR = 6.58 for AFB1 exposure levels]. The XPC codon 939 Gln alleles significantly increased HCC risk [OR = 1.25 (95% confidence interval = 1.03-1.52) for heterozygotes of the XPC codon 939 Lys and Gln alleles (XPC-LG) and OR = 1.81 (95% confidence interval = 1.36-2.40) for homozygotes of the XPC codon 939 Gln alleles (XPC-GG)]. Significant interactive effects between genotypes and AFB1 exposure status were also observed in the joint-effects analysis. This polymorphism, moreover, was correlated with XPC expression levels in cancerous tissues (r = -0.369, P < 0.001) and with the overall survival of HCC patients (the median survival times were 30, 25, and 19 months for patients with homozygotes of the XPC codon 939 Lys alleles, XPC-LG, and XPC-GG, respectively), especially under high AFB1 exposure conditions. Like AFB1 exposure, the XPC codon 939 polymorphism was an independent prognostic factor influencing the survival of HCC. Additionally, this polymorphism multiplicatively interacted with the xeroderma pigmentosum complementation group D codon 751 polymorphism with respect to HCC risk (OR(interaction) = 1.71). These results suggest that the XPC codon 939 polymorphism may be associated with the risk and outcome of AFB1-related HCC in the Guangxi population and may interact with AFB1 exposure in the process of HCC induction by AFB1.

A novel MALDI–TOF based methodology for genotyping single nucleotide polymorphisms

PubMed Central

Blondal, Thorarinn; Waage, Benedikt G.; Smarason, Sigurdur V.; Jonsson, Frosti; Fjalldal, Sigridur B.; Stefansson, Kari; Gulcher, Jeffery; Smith, Albert V.

2003-01-01

A new MALDI–TOF based detection assay was developed for analysis of single nucleotide polymorphisms (SNPs). It is a significant modification on the classic three-step minisequencing method, which includes a polymerase chain reaction (PCR), removal of excess nucleotides and primers, followed by primer extension in the presence of dideoxynucleotides using modified thermostable DNA polymerase. The key feature of this novel assay is reliance upon deoxynucleotide mixes, lacking one of the nucleotides at the polymorphic position. During primer extension in the presence of depleted nucleotide mixes, standard thermostable DNA polymerases dissociate from the template at positions requiring a depleted nucleotide; this principal was harnessed to create a genotyping assay. The assay design requires a primer- extension primer having its 3′-end one nucleotide upstream from the interrogated site. The assay further utilizes the same DNA polymerase in both PCR and the primer extension step. This not only simplifies the assay but also greatly reduces the cost per genotype compared to minisequencing methodology. We demonstrate accurate genotyping using this methodology for two SNPs run in both singleplex and duplex reactions. We term this assay nucleotide depletion genotyping (NUDGE). Nucleotide depletion genotyping could be extended to other genotyping assays based on primer extension such as detection by gel or capillary electrophoresis. PMID:14654708

Human 8-oxoguanine DNA glycosylase gene polymorphism (Ser326Cys) and cancer risk: updated meta-analysis

PubMed Central

Park, Hae Jeong; Chung, Joo-Ho; Ban, Ju Yeon

2017-01-01

Genetic polymorphism of human 8-oxoguanine glycosylase 1 (hOGG1) has been reported to have a relationship with the risk of the development of various cancers. Many studies have described the influence of Ser326Cys polymorphism of the hOGG1 gene on cancer susceptibility. However, the results have remained inconclusive and controversial. Therefore, we performed a meta-analysis to more precisely determine the relationship between the hOGG1 polymorphism and the development of cancer. Electronic databases including PubMed, Embase, Google Scholar, and the Korean Studies Information Service System (KISS) were searched. The odds ratio (OR), 95% confidence interval (CI), and p value were calculated to assess the strength of the association with the risk of cancer using Comprehensive Meta-analysis software (Corporation, NJ, USA). The 127 studies including 38,757 cancer patients and 50,177 control subjects were analyzed for the meta-analysis. Our meta-analysis revealed that G allele of Ser326Cys polymorphism of the hOGG1 gene statistically increased the susceptibility of cancer (all population, OR = 1.092, 95% CI = 1.051-1.134, p < 0.001; in Asian, OR = 1.095, 95% CI = 1.048-1.145, p < 0.001; in Caucasian, OR = 1.097, 95% CI = 1.033-1.179, p = 0.002). Also, other genotype models showed significant association with cancer (p < 0.05, respectively). The present meta-analysis concluded that the G allele was associated with an increased risk of cancer. It suggested that the hOGG1 polymorphism may be a candidate marker of cancer. PMID:28415770

Genome-wide single-nucleotide polymorphism arrays demonstrate high fidelity of multiple displacement-based whole-genome amplification.

PubMed

Tzvetkov, Mladen V; Becker, Christian; Kulle, Bettina; Nürnberg, Peter; Brockmöller, Jürgen; Wojnowski, Leszek

2005-02-01

Whole-genome DNA amplification by multiple displacement (MD-WGA) is a promising tool to obtain sufficient DNA amounts from samples of limited quantity. Using Affymetrix' GeneChip Human Mapping 10K Arrays, we investigated the accuracy and allele amplification bias in DNA samples subjected to MD-WGA. We observed an excellent concordance (99.95%) between single-nucleotide polymorphisms (SNPs) called both in the nonamplified and the corresponding amplified DNA. This concordance was only 0.01% lower than the intra-assay reproducibility of the genotyping technique used. However, MD-WGA failed to amplify an estimated 7% of polymorphic loci. Due to the algorithm used to call genotypes, this was detected only for heterozygous loci. We achieved a 4.3-fold reduction of noncalled SNPs by combining the results from two independent MD-WGA reactions. This indicated that inter-reaction variations rather than specific chromosomal loci reduced the efficiency of MD-WGA. Consistently, we detected no regions of reduced amplification, with the exception of several SNPs located near chromosomal ends. Altogether, despite a substantial loss of polymorphic sites, MD-WGA appears to be the current method of choice to amplify genomic DNA for array-based SNP analyses. The number of nonamplified loci can be substantially reduced by amplifying each DNA sample in duplicate.

A Natural Polymorphism in rDNA Replication Origins Links Origin Activation with Calorie Restriction and Lifespan

PubMed Central

Kwan, Elizabeth X.; Foss, Eric J.; Tsuchiyama, Scott; Alvino, Gina M.; Kruglyak, Leonid; Kaeberlein, Matt; Raghuraman, M. K.; Brewer, Bonita J.; Kennedy, Brian K.; Bedalov, Antonio

2013-01-01

Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics. PMID:23505383

A natural polymorphism in rDNA replication origins links origin activation with calorie restriction and lifespan.

PubMed

Kwan, Elizabeth X; Foss, Eric J; Tsuchiyama, Scott; Alvino, Gina M; Kruglyak, Leonid; Kaeberlein, Matt; Raghuraman, M K; Brewer, Bonita J; Kennedy, Brian K; Bedalov, Antonio

2013-01-01

Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics.

[Analysis of genomic DNA methylation level in radish under cadmium stress by methylation-sensitive amplified polymorphism technique].

PubMed

Yang, Jin-Lan; Liu, Li-Wang; Gong, Yi-Qin; Huang, Dan-Qiong; Wang, Feng; He, Ling-Li

2007-06-01

The level of cytosine methylation induced by cadmium in radish (Raphanus sativus L.) genome was analysed using the technique of methylation-sensitive amplified polymorphism (MSAP). The MSAP ratios in radish seedling exposed to cadmium chloride at the concentration of 50, 250 and 500 mg/L were 37%, 43% and 51%, respectively, and the control was 34%; the full methylation levels (C(m)CGG in double strands) were at 23%, 25% and 27%, respectively, while the control was 22%. The level of increase in MSAP and full methylation indicated that de novo methylation occurred in some 5'-CCGG sites under Cd stress. There was significant positive correlation between increase of total DNA methylation level and CdCl(2) concentration. Four types of MSAP patterns: de novo methylation, de-methylation, atypical pattern and no changes of methylation pattern were identified among CdCl(2) treatments and the control. DNA methylation alteration in plants treated with CdCl(2) was mainly through de novo methylation.

MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform

PubMed Central

Suyama, Yoshihisa; Matsuki, Yu

2015-01-01

Restriction-enzyme (RE)-based next-generation sequencing methods have revolutionized marker-assisted genetic studies; however, the use of REs has limited their widespread adoption, especially in field samples with low-quality DNA and/or small quantities of DNA. Here, we developed a PCR-based procedure to construct reduced representation libraries without RE digestion steps, representing de novo single-nucleotide polymorphism discovery, and its genotyping using next-generation sequencing. Using multiplexed inter-simple sequence repeat (ISSR) primers, thousands of genome-wide regions were amplified effectively from a wide variety of genomes, without prior genetic information. We demonstrated: 1) Mendelian gametic segregation of the discovered variants; 2) reproducibility of genotyping by checking its applicability for individual identification; and 3) applicability in a wide variety of species by checking standard population genetic analysis. This approach, called multiplexed ISSR genotyping by sequencing, should be applicable to many marker-assisted genetic studies with a wide range of DNA qualities and quantities. PMID:26593239

Cohort analysis of a single nucleotide polymorphism on DNA chips.

PubMed

Schwonbeck, Susanne; Krause-Griep, Andrea; Gajovic-Eichelmann, Nenad; Ehrentreich-Förster, Eva; Meinl, Walter; Glatt, Hansrüdi; Bier, Frank F

2004-11-15

A method has been developed to determine SNPs on DNA chips by applying a flow-through bioscanner. As a practical application we demonstrated the fast and simple SNP analysis of 24 genotypes in an array of 96 spots with a single hybridisation and dissociation experiment. The main advantage of this methodical concept is the parallel and fast analysis without any need of enzymatic digestion. Additionally, the DNA chip format used is appropriate for parallel analysis up to 400 spots. The polymorphism in the gene of the human phenol sulfotransferase SULT1A1 was studied as a model SNP. Biotinylated PCR products containing the SNP (The SNP summary web site: ) (mutant) and those containing no mutation (wild-type) were brought onto the chips coated with NeutrAvidin using non-contact spotting. This was followed by an analysis which was carried out in a flow-through biochip scanner while constantly rinsing with buffer. After removing the non-biotinylated strand a fluorescent probe was hybridised, which is complementary to the wild-type sequence. If this probe binds to a mutant sequence, then one single base is not fully matching. Thereby, the mismatched hybrid (mutant) is less stable than the full-matched hybrid (wild-type). The final step after hybridisation on the chip involves rinsing with a buffer to start dissociation of the fluorescent probe from the immobilised DNA strand. The online measurement of the fluorescence intensity by the biochip scanner provides the possibility to follow the kinetics of the hybridisation and dissociation processes. According to the different stability of the full-match and the mismatch, either visual discrimination or kinetic analysis is possible to distinguish SNP-containing sequence from the wild-type sequence.

Molecular identification and genetic variation of varieties of Styphnolobium japonicum (Fabaceae) using SRAP markers.

PubMed

Sun, R X; Zhang, C H; Zheng, Y Q; Zong, Y C; Yu, X D; Huang, P

2016-05-06

Thirty-four Styphnolobium japonicum varieties were analyzed using sequence-related amplified polymorphism (SRAP) markers, to investigate genetic variation and test the effectiveness of SRAP markers in DNA fingerprint establishment. Twelve primer pairs were selected from 120 primer combinations for their reproducibility and high polymorphism. We found a total of 430 amplified fragments, of which 415 fragments were considered polymorphic with an average of 34.58 polymorphic fragments for each primer combination. The percentage of polymorphic fragments was 96.60%, and four primer pairs showed 100% polymorphism. Moreover, simple matched coefficients ranged between 0.68 and 0.89, with an average of 0.785, indicating that the genetic variation among varieties was relatively low. This could be because of the narrow genetic basis of the selected breeding material. Based on the similarity coefficient value of 0.76, the varieties were divided into four major groups. In addition, abundant and clear SRAP fingerprints were obtained and could be used to establish DNA fingerprints. In the DNA fingerprints, each variety had its unique pattern that could be easily distinguished from others. The results demonstrated that 34 varieties of S. japonicum had a relatively narrow genetic variation. Hence, a broadening of the genetic basis of breeding material is necessary. We conclude that establishment of DNA fingerprint is feasible by means of SRAP markers.

Assessment of genetic diversity and relationships among Egyptian mango (Mangifera indica L.) cultivers grown in Suez Canal and Sinai region using RAPD markers.

PubMed

Mansour, Hassan; Mekki, Laila E; Hussein, Mohammed A

2014-01-01

DNA-based RAPD (Random Amplification of Polymorphic DNA) markers have been used extensively to study genetic diversity and relationships in a number of fruit crops. In this study, 10 (7 commercial mango cultivars and 3 accessions) mango genotypes traditionally grown in Suez Canal and Sinai region of Egypt, were selected to assess genetic diversity and relatedness. Total genomic DNA was extracted and subjected to RAPD analysis using 30 arbitrary 10-mer primers. Of these, eleven primers were selected which gave 92 clear and bright fragments. A total of 72 polymorphic RAPD bands were detected out of 92 bands, generating 78% polymorphisms. The mean PIC values scores for all loci were of 0.85. This reflects a high level of discriminatory power of a marker and most of these primers produced unique band pattern for each cultivar. A dendrogram based on Nei's Genetic distance co-efficient implied a moderate degree of genetic diversity among the cultivars used for experimentation, with some differences. The hybrid which had derived from cultivar as female parent was placed together. In the cluster, the cultivars and accessions formed separate groups according to bearing habit and type of embryo and the members in each group were very closely linked. Cluster analysis clearly showed two main groups, the first consisting of indigenous to the Delta of Egypt cultivars and the second consisting of indigenous to the Suez Canal and Sinai region. From the analysis of results, it appears the majority of mango cultivars originated from a local mango genepool and were domesticated later. The results indicated the potential of RAPD markers for the identification and management of mango germplasm for breeding purposes.

Whole-loop mitochondrial DNA D-loop sequence variability in Egyptian Arabian equine matrilines

PubMed Central

Hudson, William

2017-01-01

Background Egyptian Arabian horses have been maintained in a state of genetic isolation for over a hundred years. There is only limited genetic proof that the studbook records of female lines of Egyptian Arabian pedigrees are reliable. This study characterized the mitochondrial DNA (mtDNA) signatures of 126 horses representing 14 matrilines in the Egyptian Agricultural Organization (EAO) horse-breeding program. Findings Analysis of the whole D-loop sequence yielded additional information compared to hypervariable region-1 (HVR1) analysis alone, with 42 polymorphic sites representing ten haplotypes compared to 16 polymorphic sites representing nine haplotypes, respectively. Most EAO haplotypes belonged to ancient haplogroups, suggesting origin from a wide geographical area over many thousands of years, although one haplotype was novel. Conclusions Historical families share haplotypes and some individuals from different strains belonged to the same haplogroup: the classical EAO strain designation is not equivalent to modern monophyletic matrilineal groups. Phylogenetic inference showed that the foundation mares of the historical haplotypes were highly likely to have the same haplotypes as the animals studied (p > 0.998 in all cases), confirming the reliability of EAO studbook records and providing the opportunity for breeders to confirm the ancestry of their horses. PMID:28859174

Analysis of genetic diversity and genome relationships of four eggplant species (Solanum melongena L) using RAPD markers

NASA Astrophysics Data System (ADS)

Susilo; Setyaningsih, M.

2018-01-01

Solanum melongena (eggplant) is one of the diversity of the Solanum family which is grown and widely spread in Indonesia and widely used by the community. This research explored the genetic diversity of four local Indonesian eggplant species namely leuca, tekokak, gelatik and kopek by using RAPD (Random Amplified Polymorphic DNA). The samples were obtained from Agricultural Technology Assessment Institute (BPTP) Bogor, Indonesia. The result of data observation was in the form of Solanum melongena plant’s DNA profile analyzed descriptively and quantitatively. 30 DNA bands (28 polymorphic and 2 monomorphic) were successfully scored by using four primers (OPF-01, OPF-02, OPF-03, and OPF-04). The Primers were used able to amplify all of the four eggplant samples. The result of PCR-RAPD visualization produces bands of 300-1500 bp. The result of cluster analysis showed the existence of three clusters (A, B, and C). Cluster A (coefficient of equal to 49%) consisted of a gelatik, cluster B (coefficient of 65% equilibrium) consisted of TPU (Kopek) and TK (Tekokak), and cluster C (55% equilibrium coefficient) consisted of LC (Leunca). These results indicated that the closest proximity is found in samples of TK (Tekokak) and TPU (Kopek).

Analysis of DNA methylation related to rice adult plant resistance to bacterial blight based on methylation-sensitive AFLP (MSAP) analysis.

PubMed

Sha, A H; Lin, X H; Huang, J B; Zhang, D P

2005-07-01

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. The rice cultivar Wase Aikoku 3 becomes resistant to the blight pathogen Xanthomonas oryzae pv. oryzae at the adult stage. Using methylation-sensitive amplified polymorphism (MSAP) analysis, we compared the patterns of cytosine methylation in seedlings and adult plants of the rice cultivar Wase Aikoku 3 that had been inoculated with the pathogen Xanthomonas oryzae pv. oryzae, subjected to mock inoculation or left untreated. In all, 2000 DNA fragments, each representing a recognition site cleaved by either or both of two isoschizomers, were amplified using 60 pairs of selective primers. A total of 380 sites were found to be methylated. Of these, 45 showed differential cytosine methylation among the seedlings and adult plants subjected to different treatments, and overall levels of methylation were higher in adult plants than in seedlings. All polymorphic fragments were sequenced, and six showed homology to genes that code for products of known function. Northern analysis of three fragments indicated that their expression varied with methylation pattern, with hypermethylation being correlated with repression of transcription, as expected. The results suggest that significant differences in cytosine methylation exist between seedlings and adult plants, and that hypermethylation or hypomethylation of specific genes may be involved in the development of adult plant resistance (APR) in rice plants.

Cross-species amplification of mitochondrial DNA sequence-tagged-site markers in conifers: the nature of polymorphism and variation within and among species in Picea.

PubMed

Jaramillo-Correa, J P; Bousquet, J; Beaulieu, J; Isabel, N; Perron, M; Bouillé, M

2003-05-01

Primers previously developed to amplify specific non-coding regions of the mitochondrial genome in Angiosperms, and new primers for additional non-coding mtDNA regions, were tested for their ability to direct DNA amplification in 12 conifer taxa and to detect sequence-tagged-site (STS) polymorphisms within and among eight species in Picea. Out of 12 primer pairs, nine were successful at amplifying mtDNA in most of the taxa surveyed. In conifers, indels and substitutions were observed for several loci, allowing them to distinguish between families, genera and, in some cases, between species within genera. In Picea, interspecific polymorphism was detected for four loci, while intraspecific variation was observed for three of the mtDNA regions studied. One of these (SSU rRNA V1 region) exhibited indel polymorphisms, and the two others ( nad1 intron b/c and nad5 intron1) revealed restriction differences after digestion with Sau3AI (PCR-RFLP). A fourth locus, the nad4L- orf25 intergenic region, showed a multibanding pattern for most of the spruce species, suggesting a possible gene duplication. Maternal inheritance, expected for mtDNA in conifers, was observed for all polymorphic markers except the intergenic region nad4L- orf25. Pooling of the variation observed with the remaining three markers resulted in two to six different mtDNA haplotypes within the different species of Picea. Evidence for intra-genomic recombination was observed in at least two taxa. Thus, these mitotypes are likely to be more informative than single-locus haplotypes. They should be particularly useful for the study of biogeography and the dynamics of hybrid zones.

Use of SSR markers for DNA fingerprinting and diversity analysis of Pakistani sugarcane (Saccharum spp. hybrids) cultivars

USDA-ARS?s Scientific Manuscript database

In recent years SSR markers have been used widely for genetic analysis. The objective of this study was to use an SSR-based marker system to develop the molecular fingerprints and analyze the genetic relationship of sugarcane cultivars grown in Pakistan. Twenty-one highly polymorphic SSR markers wer...

Inter- and Intraspecific Identification of the New World Screwworm Using Random Amplified Polymorphic DNA-Polymerase Chain Reaction

USDA-ARS?s Scientific Manuscript database

New World screwworms (NWS), Cochliomyia hominivorax (Coquerel), are one of the most important arthropod pests of livestock in the Western Hemisphere. Early instars are very difficult to distinguish morphologically from several closely related blow fly species. Random amplified polymorphic DNA polyme...

KDNA Genetic Signatures Obtained by LSSP-PCR Analysis of Leishmania (Leishmania) infantum Isolated from the New and the Old World

PubMed Central

Alvarenga, Janaína Sousa Campos; Ligeiro, Carla Maia; Gontijo, Célia Maria Ferreira; Cortes, Sofia; Campino, Lenea; Vago, Annamaria Ravara; Melo, Maria Norma

2012-01-01

Background Visceral Leishmaniasis (VL) caused by species from the Leishmania donovani complex is the most severe form of the disease, lethal if untreated. VL caused by Leishmania infantum is a zoonosis with an increasing number of human cases and millions of dogs infected in the Old and the New World. In this study, L. infantum (syn. L.chagasi) strains were isolated from human and canine VL cases. The strains were obtained from endemic areas from Brazil and Portugal and their genetic polymorphism was ascertained using the LSSP-PCR (Low-Stringency Single Specific Primer PCR) technique for analyzing the kinetoplastid DNA (kDNA) minicircles hypervariable region. Principal Findings KDNA genetic signatures obtained by minicircle LSSP-PCR analysis of forty L. infantum strains allowed the grouping of strains in several clades. Furthermore, LSSP-PCR profiles of L. infantum subpopulations were closely related to the host origin (human or canine). To our knowledge this is the first study which used this technique to compare genetic polymorphisms among strains of L. infantum originated from both the Old and the New World. Conclusions LSSP-PCR profiles obtained by analysis of L. infantum kDNA hypervariable region of parasites isolated from human cases and infected dogs from Brazil and Portugal exhibited a genetic correlation among isolates originated from the same reservoir, human or canine. However, no association has been detected among the kDNA signatures and the geographical origin of L. infantum strains. PMID:22912862

Mitochondrial DNA G10398A variant is not associated with breast cancer in African-American women

PubMed Central

Setiawan, Veronica Wendy; Chu, Li-Hao; John, Esther M.; Ding, Yuan Chun; Ingles, Sue A.; Bernstein, Leslie; Press, Michael F.; Ursin, Giske; Haiman, Christopher A.; Neuhausen, Susan L

2009-01-01

Mitochondria play important roles in cellular energy production, free radical generation and apoptosis. In a previous report in Cancer Research, the mitochondrial DNA (mtDNA) G10398A (Thr → Ala) polymorphism was associated with breast cancer risk in African-American women. Here, we seek to replicate the association by genotyping the G10398A polymorphism in three established population-based case-control studies of breast cancer in African-American women. The 10398A allele was not significantly associated with risk in any of the studies [San Francisco study (542 cases, 282 controls, odds ratio (OR) = 1.73; 95% confidence interval (CI): 0.87, 3.47, P = 0.12); Multiethnic Cohort (391 cases, 460 controls, OR = 1.08; 95% CI: 0.62, 1.86, P = 0.79); CARE/LIFE study (524 cases, 236 controls, OR = 0.81; 95% CI: 0.43, 1.52, P = 0.50)]. When pooling the data across the three studies (1456 cases and 978 controls), no significant association was observed with the 10398A allele (OR = 1.14; 95% CI: 0.80, 1.62, P = 0.47, P heterogeneity=0.30). In analysis of advanced breast cancer cases (n=674), there also was no significant association (OR = 1.18; 95% CI: 0.76, 1.82, P = 0.46). Our results do not support the hypothesis that the mtDNA G10398A polymorphism is a marker of breast cancer risk in African Americans as previously reported. PMID:18262047

A genotyping system capable of simultaneously analyzing >1000 single nucleotide polymorphisms in a haploid genome.

PubMed

Wang, Hui-Yun; Luo, Minjie; Tereshchenko, Irina V; Frikker, Danielle M; Cui, Xiangfeng; Li, James Y; Hu, Guohong; Chu, Yi; Azaro, Marco A; Lin, Yong; Shen, Li; Yang, Qifeng; Kambouris, Manousos E; Gao, Richeng; Shih, Weichung; Li, Honghua

2005-02-01

A high-throughput genotyping system for scoring single nucleotide polymorphisms (SNPs) has been developed. With this system, >1000 SNPs can be analyzed in a single assay, with a sensitivity that allows the use of single haploid cells as starting material. In the multiplex polymorphic sequence amplification step, instead of attaching universal sequences to the amplicons, primers that are unlikely to have nonspecific and productive interactions are used. Genotypes of SNPs are then determined by using the widely accessible microarray technology and the simple single-base extension assay. Three SNP panels, each consisting of >1000 SNPs, were incorporated into this system. The system was used to analyze 24 human genomic DNA samples. With 5 ng of human genomic DNA, the average detection rate was 98.22% when single probes were used, and 96.71% could be detected by dual probes in different directions. When single sperm cells were used, 91.88% of the SNPs were detectable, which is comparable to the level that was reached when very few genetic markers were used. By using a dual-probe assay, the average genotyping accuracy was 99.96% for 5 ng of human genomic DNA and 99.95% for single sperm. This system may be used to significantly facilitate large-scale genetic analysis even if the amount of DNA template is very limited or even highly degraded as that obtained from paraffin-embedded cancer specimens, and to make many unpractical research projects highly realistic and affordable.

A meta-analysis of xeroderma pigmentosum gene D Ls751Gln polymorphism and susceptibility to hepatocellular carcinoma.

PubMed

Wang, Yu; Zhao, Yingren; Zhang, Aiyun; Ma, Juan; Wang, Zhenzhen; Zhang, Xu

2015-01-01

Hepatocellular carcinoma (HCC) is one of most common malignant tumors worldwide, but with unclear mechanisms. Xeroderma pigmentosum gene D (XPD) is one important DNA damage repair gene and can be involved in protein mutation. Currently little has been known about XPD polymorphism and HCC susceptibility in Chinese people. This study used a meta-analysis approach to comprehensively investigate the correlation between XPD polymorphism and HCC susceptibility in Chinese population, based on previously published literatures. A computer retrieval system was used to collect all case-control studies about XPD Lys751Gln polymorphism and HCC susceptibility. Data in literatures were extracted for meta-analysis. After the primary screening, four independent studies, which were published in 3 English articles and one Chinese article, were recruited in this study. There were 1,717 samples included in all studies. Using Gln/Gln + Lys/Gln, Lys/Lys + Lys/Gln and Lys allels as the reference, HCC disease alleles including Lys/Lys, Gln/Gln and Gln had OR values (95% CI, I(2)) of 1.007 (0.657~4.672, 91%), 3.516 (0.220~20.661, 48%) and 3.225 (0.278~12.326, 84%), respectively. The polymorphism of XPD751 loci is closely correlated with primary HCC. Lys751Gln polymorphism of XPD gene can be used as one susceptibility factor for HCC.

High-throughput multiplex HLA-typing by ligase detection reaction (LDR) and universal array (UA) approach.

PubMed

Consolandi, Clarissa

2009-01-01

One major goal of genetic research is to understand the role of genetic variation in living systems. In humans, by far the most common type of such variation involves differences in single DNA nucleotides, and is thus termed single nucleotide polymorphism (SNP). The need for improvement in throughput and reliability of traditional techniques makes it necessary to develop new technologies. Thus the past few years have witnessed an extraordinary surge of interest in DNA microarray technology. This new technology offers the first great hope for providing a systematic way to explore the genome. It permits a very rapid analysis of thousands genes for the purpose of gene discovery, sequencing, mapping, expression, and polymorphism detection. We generated a series of analytical tools to address the manufacturing, detection and data analysis components of a microarray experiment. In particular, we set up a universal array approach in combination with a PCR-LDR (polymerase chain reaction-ligation detection reaction) strategy for allele identification in the HLA gene.

Comparison of Ribotyping, Randomly Amplified Polymorphic DNA Analysis, and Pulsed-Field Gel Electrophoresis in Typing of Lactobacillus rhamnosus and L. casei Strains

PubMed Central

Tynkkynen, Soile; Satokari, Reetta; Saarela, Maria; Mattila-Sandholm, Tiina; Saxelin, Maija

1999-01-01

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 334, respectively, but did not give any signal with the recently revived species L. zeae, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as L. casei. Our results are in accordance with the suggested new classification of the L. casei group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively. PMID:10473394

Comparison of ribotyping, randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis in typing of Lactobacillus rhamnosus and L. casei strains.

PubMed

Tynkkynen, S; Satokari, R; Saarela, M; Mattila-Sandholm, T; Saxelin, M

1999-09-01

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 334, respectively, but did not give any signal with the recently revived species L. zeae, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as L. casei. Our results are in accordance with the suggested new classification of the L. casei group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively.

Lyme disease with facial nerve palsy: rapid diagnosis using a nested polymerase chain reaction-restriction fragment length polymorphism analysis.

PubMed

Hashimoto, Y; Takahashi, H; Kishiyama, K; Sato, Y; Nakao, M; Miyamoto, K; Iizuka, H

1998-02-01

A 64-year-old woman with Lyme disease and manifesting facial nerve palsy had been bitten by a tick on the left frontal scalp 4 weeks previously. Erythema migrans appeared on the left forehead, accompanied by left facial paralysis. Nested polymerase chain reaction-restriction fragment length polymorphism analysis (nested PCR-RFLP) was performed on DNA extracted from a skin biopsy of the erythema on the left forehead. Borrelia flagellin gene DNA was detected and its RFLP pattern indicated that the organism was B. garinii, Five weeks later, B. garinii was isolated by conventional culture from the erythematous skin lesion, but not from the cerebrospinal fluid. After treatment with ceftriaxone intravenously for 10 days and oral administration of minocycline for 7 days, both the erythema and facial nerve palsy improved significantly. Nested PCR and culture taken after the lesion subsided, using skin samples obtained from a site adjacent to the original biopsy, were both negative. We suggest that nested PCR-RFLP analysis might be useful for the rapid diagnosis of Lyme disease and for evaluating therapy.

Nuclear DNA analyses in genetic studies of populations: practice, problems and prospects.

PubMed

Zhang, De-Xing; Hewitt, Godfrey M

2003-03-01

Population-genetic studies have been remarkably productive and successful in the last decade following the invention of PCR technology and the introduction of mitochondrial and microsatellite DNA markers. While mitochondrial DNA has proven powerful for genealogical and evolutionary studies of animal populations, and microsatellite sequences are the most revealing DNA markers available so far for inferring population structure and dynamics, they both have important and unavoidable limitations. To obtain a fuller picture of the history and evolutionary potential of populations, genealogical data from nuclear loci are essential, and the inclusion of other nuclear markers, i.e. single copy nuclear polymorphic (scnp) sequences, is clearly needed. Four major uncertainties for nuclear DNA analyses of populations have been facing us, i.e. the availability of scnp markers for carrying out such analysis, technical laboratory hurdles for resolving haplotypes, difficulty in data analysis because of recombination, low divergence levels and intraspecific multifurcation evolution, and the utility of scnp markers for addressing population-genetic questions. In this review, we discuss the availability of highly polymorphic single copy DNA in the nuclear genome, describe patterns and rate of evolution of nuclear sequences, summarize past empirical and theoretical efforts to recover and analyse data from scnp markers, and examine the difficulties, challenges and opportunities faced in such studies. We show that although challenges still exist, the above-mentioned obstacles are now being removed. Recent advances in technology and increases in statistical power provide the prospect of nuclear DNA analyses becoming routine practice, allowing allele-discriminating characterization of scnp loci and microsatellite loci. This certainly will increase our ability to address more complex questions, and thereby the sophistication of genetic analyses of populations.

Reconciling patterns of inter-ocean molecular variance from four classes of molecular markers in blue marlin (Makaira nigricans).

PubMed

Buonaccorsi, V P; McDowell, J R; Graves, J E

2001-05-01

Different classes of molecular markers occasionally yield discordant views of population structure within a species. Here, we examine the distribution of molecular variance from 14 polymorphic loci comprising four classes of molecular markers within approximately 400 blue marlin individuals (Makaira nigricans). Samples were collected from the Atlantic and Pacific Oceans over 5 years. Data from five hypervariable tetranucleotide microsatellite loci and restriction fragment length polymorphism (RFLP) analysis of whole molecule mitochondrial DNA (mtDNA) were reported and compared with previous analyses of allozyme and single-copy nuclear DNA (scnDNA) loci. Temporal variance in allele frequencies was nonsignificant in nearly all cases. Mitochondrial and microsatellite loci revealed striking phylogeographic partitioning among Atlantic and Pacific Ocean samples. A large cluster of alleles was present almost exclusively in Atlantic individuals at one microsatellite locus and for mtDNA, suggesting that, if gene flow occurs, it is likely to be unidirectional from Pacific to Atlantic oceans. Mitochondrial DNA inter-ocean divergence (FST) was almost four times greater than microsatellite or combined nuclear divergences including allozyme and scnDNA markers. Estimates of Neu varied by five orders of magnitude among marker classes. Using mathematical and computer simulation approaches, we show that substantially different distributions of FST are expected from marker classes that differ in mode of inheritance and rate of mutation, without influence of natural selection or sex-biased dispersal. Furthermore, divergent FST values can be reconciled by quantifying the balance between genetic drift, mutation and migration. These results illustrate the usefulness of a mitochondrial analysis of population history, and relative precision of nuclear estimates of gene flow based on a mean of several loci.

Genome-wide DNA polymorphisms in Kavuni, a traditional rice cultivar with nutritional and therapeutic properties.

PubMed

Rathinasabapathi, Pasupathi; Purushothaman, Natarajan; Parani, Madasamy

2016-05-01

Although rice genome was sequenced in the year 2002, efforts in resequencing the large number of available accessions, landraces, traditional cultivars, and improved varieties of this important food crop are limited. We have initiated resequencing of the traditional cultivars from India. Kavuni is an important traditional rice cultivar from South India that attracts premium price for its nutritional and therapeutic properties. Whole-genome sequencing of Kavuni using Illumina platform and SNPs analysis using Nipponbare reference genome identified 1 150 711 SNPs of which 377 381 SNPs were located in the genic regions. Non-synonymous SNPs (62 708) were distributed in 19 251 genes, and their number varied between 1 and 115 per gene. Large-effect DNA polymorphisms (7769) were present in 3475 genes. Pathway mapping of these polymorphisms revealed the involvement of genes related to carbohydrate metabolism, translation, protein-folding, and cell death. Analysis of the starch biosynthesis related genes revealed that the granule-bound starch synthase I gene had T/G SNPs at the first intron/exon junction and a two-nucleotide combination, which were reported to favour high amylose content and low glycemic index. The present study provided a valuable genomics resource to study the rice varieties with nutritional and medicinal properties.

The OGG1 Ser326Cys polymorphism and the risk of esophageal cancer: a meta-analysis.

PubMed

Wang, Zhan; Gan, Lu; Nie, Wei; Geng, Yan

2013-10-01

The oxoguanine DNA glycosylase (OGG1) Ser326Cys polymorphism has been implicated in susceptibility to esophageal cancer. Several studies investigated the association of this polymorphism with esophageal cancer in different populations. However, the results were contradictory. A meta-analysis was conducted to assess the association between the OGG1 Ser326Cys polymorphism and esophageal cancer susceptibility. Databases, including PubMed, EMBASE, China National Knowledge Infrastructure (CNKI), and Weipu Database were searched to find relevant studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of associations. A random-effects model was used. Twelve studies involving 2363 cases and 3621 controls were included. Overall, a significant association between the OGG1 Ser326Cys polymorphism and esophageal cancer was observed for Cys/Cys versus Cys/Ser+Ser/Ser (OR=1.40; 95% CI 1.12-1.74; p=0.003; Pheterogeneity=0.18). In the subgroup analysis by ethnicity, a significant association was found among Asians (OR=1.51; 95% CI 1.15-1.96; p=0.002; Pheterogeneity=0.22), but not among Caucasians (OR=1.21; 95% CI 0.81-1.81; p=0.35; Pheterogeneity=0.21). In the subgroup analysis by pathologic type, we found that the Cys/Cys genotype was associated with increased esophageal squamous cell carcinoma risk (OR=1.86; 95% CI 1.36-2.53; p<0.0001; Pheterogeneity=0.73). This meta-analysis suggested that the OGG1 Ser326Cys polymorphism was a risk factor of esophageal cancer.

The role of the RTEL1 rs2297440 polymorphism in the risk of glioma development: a meta-analysis.

PubMed

Zhang, Cuiping; Lu, Yu; Zhang, Xiaolian; Yang, Dongmei; Shang, Shuxin; Liu, Denghe; Jiang, Kongmei; Huang, Weiqiang

2016-07-01

The regulator of the telomere elongation helicase1 (RTEL1) gene plays a crucial role in the DNA double-stand break-repair pathway by maintaining genomic stability. Recent epidemiological studies showed that the rs2297440 polymorphism in the RTEL1 gene was a potential risk locus for glioma development, but the results were inconclusive. To clarify the association between this polymorphism and the risk of glioma, we performed a comprehensive meta-analysis. The PubMed, EMBASE, Web of Science, and China National Knowledge Infrastructure databases were systematically searched to identify all relevant published studies up to 30 August 2015. Four eligible studies were finally included. The pooled results indicated that the RTEL1 rs2297440 polymorphism moderately increased the risk of glioma in all genetic models. A comparison of the dominant model CT + CC versus TT (OR 1.40; 95 % CI 1.24-1.60; p < 0.001) indicated that having the C allele conferred a 40 % increased risk of developing glioma. In a subgroup analysis based on geographic location (Europe, Asia, and America), there was an association between the rs2297440 polymorphism and the risk of glioma in all three areas. The results of the subgroup analysis based on source of control indicated an elevated risk of glioma in population-based control studies. This meta-analysis demonstrates that the RTEL1 rs2297440 polymorphism plays a moderate, but significant role in the risk of glioma. Further studies with larger sample sizes are necessary to confirm this finding.

Mapping of the Gynoecy in Bitter Gourd (Momordica charantia) Using RAD-Seq Analysis

PubMed Central

Matsumura, Hideo; Miyagi, Norimichi; Taniai, Naoki; Fukushima, Mai; Tarora, Kazuhiko; Shudo, Ayano; Urasaki, Naoya

2014-01-01

Momordica charantia is a monoecious plant of the Cucurbitaceae family that has both male and female unisexual flowers. Its unique gynoecious line, OHB61-5, is essential as a maternal parent in the production of F1 cultivars. To identify the DNA markers for this gynoecy, a RAD-seq (restriction-associated DNA tag sequencing) analysis was employed to reveal genome-wide DNA polymorphisms and to genotype the F2 progeny from a cross between OHB61-5 and a monoecious line. Based on a RAD-seq analysis of F2 individuals, a linkage map was constructed using 552 co-dominant markers. In addition, after analyzing the pooled genomic DNA from monoecious or gynoecious F2 plants, several SNP loci that are genetically linked to gynoecy were identified. GTFL-1, the closest SNP locus to the putative gynoecious locus, was converted to a conventional DNA marker using invader assay technology, which is applicable to the marker-assisted selection of gynoecy in M. charantia breeding. PMID:24498029

Novel primer specific false terminations during DNA sequencing reactions: danger of inaccuracy of mutation analysis in molecular diagnostics

PubMed Central

Anwar, R; Booth, A; Churchill, A J; Markham, A F

1996-01-01

The determination of nucleotide sequence is fundamental to the identification and molecular analysis of genes. Direct sequencing of PCR products is now becoming a commonplace procedure for haplotype analysis, and for defining mutations and polymorphism within genes, particularly for diagnostic purposes. A previously unrecognised phenomenon, primer related variability, observed in sequence data generated using Taq cycle sequencing and T7 Sequenase sequencing, is reported. This suggests that caution is necessary when interpreting DNA sequence data. This is particularly important in situations where treatment may be dependent on the accuracy of the molecular diagnosis. Images PMID:16696096

Evaluation of Prediction of Polymorphisms of DNA Repair Genes on the Efficacy of Platinum-Based Chemotherapy in Patients With Non-Small Cell Lung Cancer: A Network Meta-Analysis.

PubMed

Yu, Shao-Nan; Liu, Gui-Feng; Li, Xue-Feng; Fu, Bao-Hong; Dong, Li-Xin; Zhang, Shu-Hua

2017-12-01

This network meta-analysis (NMA) was conducted to compare the predictive value of 14 SNPs in eight DNA repair genes on the efficacy of platinum-based chemotherapy in patients with non-small cell lung cancer (NSCLC). These included ERCC1 (rs11615, rs3212986, rs3212948), XRCC1 (rs25487, rs25489, rs1799782), XPD (rs13181, rs1799793), XPG (rs1047768, rs17655), XPA (rs1800975), XRCC3 (rs861539), APE1 (rs3136820), and RRM1 (rs1042858). The PubMed and Cochrane library databases were reviewed from their inception to February 2017 and studies which met our inclusion criteria were included in our investigation. This network meta-analysis combines direct and indirect evidence to assess the predictive value of 14 SNPs in eight DNA repair genes on the efficacy of platinum-based chemotherapy in NSCLC. We evaluated the predictive value through the use of the odd ratios (OR) and drawing surface under the cumulative ranking curves (SUCRA). A total of 26 eligible cohort studies were enrolled in this NMA. The pairwise meta-analysis indicated that in terms of overall response ratio (ORR), ERCC1 (rs11615), XRCC1 (rs25487, rs1799782), and XPD (rs13181) polymorphisms are associated with the efficacy of platinum-based chemotherapy in NSCLC. The result of this NMA suggests that there is no significant difference in predictive value of 8 DNA repair genes on the efficacy of platinum-based chemotherapy in NSCLC patients. The rank of SUCRA values of the 14 SNPs in the eight DNA repair genes were: XPD (rs1799793)→ERCC1 (rs3212986)→XPA(rs1800975)→ERCC1(rs3212948)→XRCC1(rs25487)→XRCC3(rs861539)→APE1(rs3136820)→ERCC1(rs11615)→XRCC1(rs1799782)→RRM1(rs1042858)→XPD(rs13181)→XPG (rs1047768)→XPG(rs17655)→XRCC1(rs25489). ERCC1(rs11615), XRCC1(rs25487, rs1799782) and XPD(rs13181) polymorphisms were better predictors in evaluating the efficacy of platinum-based chemotherapy in NSCLC patients. J. Cell. Biochem. 118: 4782-4791, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Genotyping and Molecular Identification of Date Palm Cultivars Using Inter-Simple Sequence Repeat (ISSR) Markers.

PubMed

Ayesh, Basim M

2017-01-01

Molecular markers are credible for the discrimination of genotypes and estimation of the extent of genetic diversity and relatedness in a set of genotypes. Inter-simple sequence repeat (ISSR) markers rapidly reveal high polymorphic fingerprints and have been used frequently to determine the genetic diversity among date palm cultivars. This chapter describes the application of ISSR markers for genotyping of date palm cultivars. The application involves extraction of genomic DNA from the target cultivars with reliable quality and quantity. Subsequently the extracted DNA serves as a template for amplification of genomic regions flanked by inverted simple sequence repeats using a single primer. The similarity of each pair of samples is measured by calculating the number of mono- and polymorphic bands revealed by gel electrophoresis. Matrices constructed for similarity and genetic distance are used to build a phylogenetic tree and cluster analysis, to determine the molecular relatedness of cultivars. The protocol describes 3 out of 9 tested primers consistently amplified 31 loci in 6 date palm cultivars, with 28 polymorphic loci.

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

Rapid identification of red-flesh loquat cultivars using EST-SSR markers based on manual cultivar identification diagram strategy.

PubMed

Li, X Y; Xu, H X; Chen, J W

2014-04-29

Manual cultivar identification diagram is a new strategy for plant cultivar identification based on DNA markers, providing information to efficiently separate cultivars. We tested 25 pairs of apple EST-SSR primers for amplification of PCR products from loquat cultivars. These EST-SSR primers provided clear amplification products from the loquat cultivars, with a relatively high transferability rate of 84% to loquat; 11 pairs of primers amplified polymorphic products. After analysis of 24 red-fleshed loquat accessions, we found that only 7 pairs of primers could clearly separate all of them. A cultivar identification diagram of the 24 cultivars was constructed using polymorphic bands from the DNA fingerprints and EST-SSR primers. Any two of the 24 cultivars could be rapidly separated from each other, according to the polymorphic bands from the cultivars; the corresponding primers were marked in the correct position on the cultivar identification diagram. This red-flesh loquat cultivar identification diagram can separate the 24 red-flesh loquat cultivars, which is of benefit for loquat cultivar identification for germplasm management and breeding programs.

Chromosome evolution in the Thermotogales: large-scale inversions and strain diversification of CRISPR sequences.

PubMed

DeBoy, Robert T; Mongodin, Emmanuel F; Emerson, Joanne B; Nelson, Karen E

2006-04-01

In the present study, the chromosomes of two members of the Thermotogales were compared. A whole-genome alignment of Thermotoga maritima MSB8 and Thermotoga neapolitana NS-E has revealed numerous large-scale DNA rearrangements, most of which are associated with CRISPR DNA repeats and/or tRNA genes. These DNA rearrangements do not include the putative origin of DNA replication but move within the same replichore, i.e., the same replicating half of the chromosome (delimited by the replication origin and terminus). Based on cumulative GC skew analysis, both the T. maritima and T. neapolitana lineages contain one or two major inverted DNA segments. Also, based on PCR amplification and sequence analysis of the DNA joints that are associated with the major rearrangements, the overall chromosome architecture was found to be conserved at most DNA joints for other strains of T. neapolitana. Taken together, the results from this analysis suggest that the observed chromosomal rearrangements in the Thermotogales likely occurred by successive inversions after their divergence from a common ancestor and before strain diversification. Finally, sequence analysis shows that size polymorphisms in the DNA joints associated with CRISPRs can be explained by expansion and possibly contraction of the DNA repeat and spacer unit, providing a tool for discerning the relatedness of strains from different geographic locations.

Genetic variability in isolates of Chromobacterium violaceum from pulmonary secretion, water, and soil.

PubMed

Santini, A C; Magalhães, J T; Cascardo, J C M; Corrêa, R X

2016-04-28

Chromobacterium violaceum is a free-living Gram-negative bacillus usually found in the water and soil in tropical regions, which causes infections in humans. Chromobacteriosis is characterized by rapid dissemination and high mortality. The aim of this study was to detect the genetic variability among C. violaceum type strain ATCC 12472, and seven isolates from the environment and one from a pulmonary secretion from a chromobacteriosis patient from Ilhéus, Bahia. The molecular characterization of all samples was performed by polymerase chain reaction (PCR) sequencing and 16S rDNA analysis. Primers specific for two ATCC 12472 pathogenicity genes, hilA and yscD, as well as random amplified polymorphic DNA (RAPD), were used for PCR amplification and comparative sequencing of the products. For a more specific approach, the PCR products of 16S rDNA were digested with restriction enzymes. Seven of the samples, including type-strain ATCC 12472, were amplified by the hilA primers; these were subsequently sequenced. Gene yscD was amplified only in type-strain ATCC 12472. MspI and AluI digestion revealed 16S rDNA polymorphisms. This data allowed the generation of a dendogram for each analysis. The isolates of C. violaceum have variability in random genomic regions demonstrated by RAPD. Also, these isolates have variability in pathogenicity genes, as demonstrated by sequencing and restriction enzyme digestion.

Association of P53 gene polymorphism with gastric cancer in Northern Iran as a high-risk region.

PubMed

Hedayatizadeh-Omran, Akbar; Alizadeh-Navaei, Reza; Janbabaei, Ghasem; Omrani-Nava, Versa; Hasheminasab, Yahya; Amjadi, Omolbanin; Tehrani, Mohsen

2018-05-01

Gastric cancer has the fourth highest morbidity rate of all cancers worldwide. Genetic factors including alterations in oncogenes and tumor suppressor genes serve an important role in gastric cancer development and progression. The P53 gene acts as a tumor suppressor gene by regulating the cell cycle, DNA transcription and repair, apoptosis, senescence and genome stability. In addition to somatic P53 mutations in cancer development, germline polymorphisms are also involved in different malignancies. The polymorphism of P53 at codon 72 (Arg72Pro) is established as a common variant that increases susceptibility to various cancers. The present case-control study was conducted to evaluate the possible association between this P53 polymorphism and gastric cancer in the Iranian population. A total of 59 patients with gastric cancer and 59 healthy controls were enrolled in the present study. Genomic DNA was extracted from peripheral blood mononuclear cells and genotype analysis was performed using a polymerase chain reaction-based restriction fragment length polymorphism assay. Genotype frequencies did not differ significantly between the patients and controls (P=0.4); the frequencies of the three genotypes Arg/Arg, Arg/Pro and Pro/Pro in gastric cancer patients were 28.8, 49.2 and 22.0%, and in controls were 37.3, 49.2 and 13.6%. Additionally, there were no differences in genotype frequencies based on tumor location, histological differentiation or tumor stage. Based on these findings, it may be concluded that the P53 codon 72 polymorphism does not contribute to gastric cancer susceptibility in Northern Iran.

RFC-1 80G>A polymorphism in case-mother/control-mother dyads is associated with risk of nephroblastoma and neuroblastoma.

PubMed

Montalvão-de-Azevedo, Rafaela; Vasconcelos, Gisele M; Vargas, Fernando R; Thuler, Luiz Claudio; Pombo-de-Oliveira, Maria S; de Camargo, Beatriz

2015-02-01

Embryonic tumors are associated with an interruption during normal organ development; they may be related to disturbances in the folate pathway involved in DNA synthesis, methylation, and repair. Prenatal supplementation with folic acid is associated with a decreased risk of neuroblastoma, brain tumors, retinoblastoma, and nephroblastoma. The aim of this study was to investigate the association between MTHFR rs1801133 (C677T) and RFC-1 rs1051266 (G80A) genotypes with the risk of developing nephroblastoma and neuroblastoma. Case-mother/control-mother dyad study. Samples from Brazilian children with nephroblastoma (n=80), neuroblastoma (n=66), healthy controls (n=453), and their mothers (case n=93; control n=75) were analyzed. Genomic DNA was isolated from peripheral blood cells and/or buccal cells and genotyped to identify MTHFR C677T and RFC-1 G80A polymorphisms. Differences in genotype distribution between patients and controls were tested by multiple logistic regression analysis. Risk for nephroblastoma and neuroblastoma was two- to fourfold increased among children with RFC-1 polymorphisms. An increased four- to eightfold risk for neuroblastoma and nephroblastoma was seen when the child and maternal genotypes were combined. Our results suggest that mother and child RFC-1 G80A genotypes play a role on the risk of neuroblastoma and nephroblastoma since this polymorphism may impair the intracellular levels of folate, through carrying fewer folate molecules to the cell interior, and thus, the intracellular concentration is not enough to maintain regular DNA synthesis and methylation pathways.

Molecular marker-based genetic diversity analysis of scantly studied Brazilian accessions of a medicinal plant, Morinda citrifolia L. (noni).

PubMed

Bordallo, P N; Monteiro, A M R; Sousa, J A; Aragão, F A S

2017-02-23

Morinda citrifolia L., commonly known as noni, has been used for the treatment of various diseases for over two centuries. It was introduced and widely disseminated in Brazil because of its high market value and ease of adaptation to the soil and climatic conditions of the country. The aim of this study was to estimate the genetic variability of noni accessions from the collection of Embrapa Agroindústria Tropical in Brazil. We evaluated 36 plants of the 13 accessions of noni from the germplasm collection of M. citrifolia. Several methods of DNA extraction were tested. After definition of the method, the DNA of each sample was subjected to polymerase chain reactions using 20 random amplified polymorphic DNA primers. The band patterns on agarose gel were converted into a binary data matrix, which was used to estimate the genetic distances between the plants and to perform the cluster analyses. Of the total number of markers used in this study, 125 (81.1%) were polymorphic. The genetic distances between the genotypes ranged from 0.04 to 0.49. Regardless of the high number of polymorphic bands, the genetic variability of the noni plants evaluated was low since most of the genotypes belonged to the same cluster as shown by the dendrogram and Tocher's cluster analysis. The low genetic diversity among the studied noni individuals indicates that additional variability should be introduced in the germplasm collection of noni by gathering new individuals and/or by hybridizing contrasting individuals.

The murine Cd48 gene: allelic polymorphism in the IgV-like region.

PubMed

Cabrero, J G; Freeman, G J; Reiser, H

1998-12-01

The murine CD48 molecule is a member of the immunoglobulin superfamily which regulates the activation of T lymphocytes. prior cloning experiments using mRNA from two different mouse strains had yielded discrepant sequences within the IgV-like domain of murine CD48. To resolve this issue, we have directly sequenced genomic DNA of 10 laboratory strains and two inbred strains of wild origin. The results of our analysis reveal an allelic polymorphism within the IgV-like domain of murine CD48.

Extensive mitochondrial heteroplasmy in the neotropical ants of the Ectatomma ruidum complex (Formicidae: Ectatomminae)

USDA-ARS?s Scientific Manuscript database

Recent advances in DNA sequencing and polymorphism detection technology have made it possible to test for the existence of more than one type of organellar genome within a cell or individual (heteroplasmy). Here we investigated whether the origin of mitochondrial (mt) DNA polymorphism in members of ...

Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Lactococcus lactis ssp. lactis and Lactococcus garvieae isolated from bovine intramammary infections.

PubMed

Plumed-Ferrer, C; Barberio, A; Franklin-Guild, R; Werner, B; McDonough, P; Bennett, J; Gioia, G; Rota, N; Welcome, F; Nydam, D V; Moroni, P

2015-09-01

In total, 181 streptococci-like bacteria isolated from intramammary infections (IMI) were submitted by a veterinary clinic to Quality Milk Production Services (QMPS, Cornell University, Ithaca, NY). The isolates were characterized by sequence analysis, and 46 Lactococcus lactis ssp. lactis and 47 Lactococcus garvieae were tested for susceptibility to 17 antibiotics. No resistant strains were found for β-lactam antibiotics widely used in clinical practice (penicillin, ampicillin, and amoxicillin), and all minimum inhibitory concentrations (MIC) were far from the resistance breakpoints. Eight strains had MIC intermediate to cefazolin. The random amplification of polymorphic DNA (RAPD)-PCR fingerprint patterns showed a slightly higher heterogeneity for Lc. lactis ssp. lactis isolates than for Lc. garvieae isolates. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Variability of ribosomal RNA genes in Rauwolfia species: parallelism between tissue culture-induced rearrangements and interspecies polymorphism.

PubMed

Andreev, I O; Spiridonova, K V; Solovyan, V T; Kunakh, V A

2005-01-01

An analysis of 18S-25S and 5S rRNA genes in intact plants and cultured tissues of some Rauwolfia species was performed to compare these sequences variability occurred as a result of the species evolution in nature and that induced by tissue culture. The restriction fragment length polymorphism of 18S-25S and 5S rDNA was found both in intact plants of various Rauwolfia species and in long-term Rauwolfia serpentina tissue cultures. In addition, changes in the amount of 18S-25S rRNA genes were observed in long-term R. serpentina tissue cultures. The results demonstrate that rDNA variability observed in intact plants as well as in long-term cultures is attributed to differences in the same regions of ribosomal RNA genes.

Association of ACE Gene I/D polymorphism with migraine in Kashmiri population.

PubMed

Wani, Irfan Yousuf; Sheikh, Saleem; Shah, Zafar Amin; Pandith, Arshid A; Wani, Mushtaq; Asimi, Ravouf; Wani, Maqbool; Sheikh, Shahnawaz; Mehraj, Iqra

2016-01-01

Migraine is a complex, recurrent headache disorder that is one of the most common complaints in neurology practice. The role of various genes in its pathogenesis is being studied. We did this study to see whether an association exists between ACE gene I/D polymorphism and migraine in our region. The study included 100 patients diagnosed with migraine and 121 healthy controls. The study subject were age and gender matched. The analysis was based on Polymerase Chain Reaction (PCR) and included following steps: DNA extraction from blood, PCR and Restriction Fragment Length Polymorphism (RFLP). Out of 100 cases, 69 were females and 31 were males. Fifty-seven were having migraine without aura and 43 had migraine with aura. 45 of the cases had II polymorphism, 40 had ID polymorphism and 15 had DD polymorphism in ACE gene. We were not able to find a statistically significant association between ACE gene I/D polymorphism with migraine. The reason for difference in results between our study and other studies could be because of different ethnicity in study populations. So a continuous research is needed in this regard in order to find the genes and different polymorphism that increase the susceptibility of Kashmiri population to migraine.

Analysis of genetic diversity using SNP markers in oat

USDA-ARS?s Scientific Manuscript database

A large-scale single nucleotide polymorphism (SNP) discovery was carried out in cultivated oat using Roche 454 sequencing methods. DNA sequences were generated from cDNAs originating from a panel of 20 diverse oat cultivars, and from Diversity Array Technology (DArT) genomic complexity reductions fr...

High-dose folic acid supplementation alters the human sperm methylome and is influenced by the MTHFR C677T polymorphism.

PubMed

Aarabi, Mahmoud; San Gabriel, Maria C; Chan, Donovan; Behan, Nathalie A; Caron, Maxime; Pastinen, Tomi; Bourque, Guillaume; MacFarlane, Amanda J; Zini, Armand; Trasler, Jacquetta

2015-11-15

Dietary folate is a major source of methyl groups required for DNA methylation, an epigenetic modification that is actively maintained and remodeled during spermatogenesis. While high-dose folic acid supplementation (up to 10 times the daily recommended dose) has been shown to improve sperm parameters in infertile men, the effects of supplementation on the sperm epigenome are unknown. To assess the impact of 6 months of high-dose folic acid supplementation on the sperm epigenome, we studied 30 men with idiopathic infertility. Blood folate concentrations increased significantly after supplementation with no significant improvements in sperm parameters. Methylation levels of the differentially methylated regions of several imprinted loci (H19, DLK1/GTL2, MEST, SNRPN, PLAGL1, KCNQ1OT1) were normal both before and after supplementation. Reduced representation bisulfite sequencing (RRBS) revealed a significant global loss of methylation across different regions of the sperm genome. The most marked loss of DNA methylation was found in sperm from patients homozygous for the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, a common polymorphism in a key enzyme required for folate metabolism. RRBS analysis also showed that most of the differentially methylated tiles were located in DNA repeats, low CpG-density and intergenic regions. Ingenuity Pathway Analysis revealed that methylation of promoter regions was altered in several genes involved in cancer and neurobehavioral disorders including CBFA2T3, PTPN6, COL18A1, ALDH2, UBE4B, ERBB2, GABRB3, CNTNAP4 and NIPA1. Our data reveal alterations of the human sperm epigenome associated with high-dose folic acid supplementation, effects that were exacerbated by a common polymorphism in MTHFR. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Chemoraces and Habitat Specialization of Claviceps purpurea Populations

PubMed Central

Pažoutová, Sylvie; Olšovská, Jana; Linka, Marek; Kolínská, Renata; Flieger, Miroslav

2000-01-01

We studied genetic variability of 100 isolates of Claviceps purpurea by using randomly amplified polymorphic DNA (RAPD), an EcoRI restriction site polymorphism in the 5.8S ribosomal DNA (rDNA), the alkaloids produced, and conidial morphology. We identified three groups: (i) group G1 from fields and open meadows (57 isolates), (ii) group G2 from shady or wet habitats (41 isolates), and (iii) group G3 from Spartina anglica from salt marshes (2 isolates). The sclerotia of G1 isolates contained ergotamines and ergotoxines; G2 isolates produced ergosine and ergocristine along with small amounts of ergocryptine; and G3 isolates produced ergocristine and ergocryptine. The conidia of G1 isolates were 5 to 8 μm long, the conidia of G2 isolates were 7 to 10 μm long, and the conidia of G3 isolates were 10 to 12 μm long. Sclerotia of the G2 and G3 isolates floated on water. In the 5.8S rDNA analysis, an EcoRI site was found in G1 and G3 isolates but not in G2 isolates. The host preferences of the groups were not absolute, and there were host genera that were common to both G1 and G2; the presence of members of different groups in the same locality was rare. Without the use of RAPD or rDNA polymorphism, it was not possible to distinguish the three groups solely on the basis of phenotype, host, or habitat. In general, populations of C. purpurea are not host specialized, as previously assumed, but they are habitat specialized, and collecting strategies and toxin risk assessments should be changed to reflect this paradigm shift. PMID:11097923

Role of CYP1B1 in PAH-DNA Adduct Formation and Breast Cancer Risk

DTIC Science & Technology

2006-03-01

32 cases and 11 controls) undergoing surgery and analyzed these specimens for CYP1B1 gene expression, CYP1B1 genotype and PAH-DNA adducts. CYP1B1...quantitated and its purity determined by its 260/280 nm absorption. Samples were aliqoted for later measurements of CYP1B1 genotype and DNA adducts...19.78) 0.06 – 73.7 d. Perform CYP1B1 genotype analysis The CYP1B1 genotype at two polymorphic sites located in the catalytic side of the enzyme

Association Between Chloroplast DNA and Mitochondrial DNA Haplotypes in Prunus spinosa L. (Rosaceae) Populations across Europe

PubMed Central

MOHANTY, APARAJITA; MARTÍN, JUAN PEDRO; GONZÁLEZ, LUIS MIGUEL; AGUINAGALDE, ITZIAR

2003-01-01

Chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) were studied in 24 populations of Prunus spinosa sampled across Europe. The cpDNA and mtDNA fragments were amplified using universal primers and subsequently digested with restriction enzymes to obtain the polymorphisms. Combinations of all the polymorphisms resulted in 33 cpDNA haplotypes and two mtDNA haplotypes. Strict association between the cpDNA haplotypes and the mtDNA haplotypes was detected in most cases, indicating conjoint inheritance of the two genomes. The most frequent and abundant cpDNA haplotype (C20; frequency, 51 %) is always associated with the more frequent and abundant mtDNA haplotype (M1; frequency, 84 %). All but two of the cpDNA haplotypes associated with the less frequent mtDNA haplotype (M2) are private haplotypes. These private haplotypes are phylogenetically related but geographically unrelated. They form a separate cluster on the minimum‐length spanning tree. PMID:14534199

Meta-analysis demonstrates lack of association of the hOGG1 Ser326Cys polymorphism with bladder cancer risk.

PubMed

Zhong, D Y; Chu, H Y; Wang, M L; Ma, L; Shi, D N; Zhang, Z D

2012-09-26

The functional polymorphism Ser326Cys (rs1052133) in the human 8-oxoguanine DNA glycosylase (hOGG1) gene has been implicated in bladder cancer risk. However, reports of this association between the Ser326Cys polymorphism and bladder cancer risk are conflicting. In order to help clarify this relationship, we made a meta-analysis of seven case-control studies, summing 2521 cases and 2408 controls. We used odds ratios (ORs) with 95% confidence intervals (95%CIs) to assess the strength of the association. Overall, no significant association between the hOGG1 Ser326Cys polymorphism and bladder cancer risk was found for Cys/Cys vs Ser/Ser (OR = 1.10, 95%CI = 0.74-1.65), Ser/Cys vs Ser/Ser (OR = 1.07, 95%CI = 0.81-1.42), Cys/Cys + Ser/Cys vs Ser/Ser (OR = 1.08, 95%CI = 0.87-1.33), and Cys/Cys vs Ser/Cys + Ser/Ser (OR = 1.04, 95%CI = 0.65-1.69). Even when stratified by ethnicity, no significant association was observed. We concluded that the hOGG1 Ser326Cys polymorphism does not contribute to susceptibility to bladder cancer.

Lack of association of the two polymorphisms in alpha-2 macroglobulin with Alzheimer disease.

PubMed

Poduslo, S E; Shook, B; Drigalenko, E; Yin, X

2002-06-01

Alzheimer disease is a complex neurodegenerative disorder that is characterized by cognitive decline and distinct neuropathology. The gene for alpha-2 macroglobulin (A2M) on chromosome 12 has two polymorphisms that, in some cases, are associated with Alzheimer disease. We examined these two polymorphisms in our families in the DNA Bank (a collection of DNA samples from families with Alzheimer disease in Texas). Using both association studies and sib transmission/disequilibrium tests, we found no association of the two polymorphisms with the disease in our collection. In addition, we did not find an association of the disease with the two polymorphisms in A2M in a small subset of National Institute of Mental Health (NIMH) families. Thus, our studies do not support the A2M polymorphisms as a risk factor for Alzheimer disease. Copyright 2002 Wiley-Liss, Inc.

Molecular epidemiology of Pseudomonas aeruginosa.

PubMed

Speert, David P

2002-10-01

Pseudomonas aeruginosa is a serious opportunistic pathogen in certain compromised hosts, such as those with cystic fibrosis, thermal burns and cancer. It also causes less severe noninvasive disease, such as otitis externa and hot tub folliculitis, in normal hosts. P. aeruginosa is phenotypically very unstable, particularly in patients with chronic infection. Phenotypic typing techniques are useful for understanding the epidemiology of acute infections, but they are limited by their discriminatory power and by their inability to group isolates that are phenotypically unrelated but genetically homologous. Molecular typing techniques, developed over the past decade, are highly discriminatory and are useful for typing strains from patients with chronic infection where the bacterial phenotype is unstable; this is particularly true in cystic fibrosis, where patients often are infected with the same strain for several decades, but the bacteria undergo phenotypic alteration. Molecular typing techniques, which have proven useful in typing P. aeruginosa for epidemiological purposes, include pulsed field gel electrophoresis, restriction fragment length polymorphic DNA analysis, random amplified polymorphic DNA analysis, repetitive extrapalindromic PCR analysis, and multilocus sequence typing. These methods are generally only available in specialized laboratories, but they should be used when data from phenotypic typing analysis are ambiguous or when phenotypic methods are unreliable, such as in cystic fibrosis.

Isolation of stress responsive Psb A gene from rice (Oryza sativa l.) using differential display.

PubMed

Tyagi, Aruna; Chandra, Arti

2006-08-01

Differential display (DD) experiments were performed on drought-tolerant rice (Oryza sativa L.) genotype N22 to identify both upregulated and downregulated partial cDNAs with respect to moisture stress. DNA polymorphism was detected between drought-stressed and control leaf tissues on the DD gels. A partial cDNA showing differential expression, with respect to moisture stress was isolated from the gel. Northern blotting analysis was performed using this cDNA as a probe and it was observed that mRNA corresponding to this transcript was accumulated to high level in rice leaves under water deficit stress. At the DNA sequence level, the partial cDNA showed homology with psb A gene encoding for Dl protein.

Polymorphisms in RAI and in genes of nucleotide and base excision repair are not associated with risk of testicular cancer.

PubMed

Laska, Magdalena J; Nexø, Bjørn A; Vistisen, Kirsten; Poulsen, Henrik Enghusen; Loft, Steffen; Vogel, Ulla

2005-07-28

Testicular cancer has been suggested to be primed in utero and there is familiar occurrence, particularly brothers and sons of men with testicular cancer have increased risk. Although no specific causative genotoxic agents have been identified, variations in DNA repair capacity could be associated with the risk of testicular cancer. A case-control study of 184 testicular cancer cases and 194 population-based controls living in the Copenhagen Greater Area in Denmark was performed. We found that neither polymorphisms in several DNA repair genes nor alleles of several polymorphisms in the chromosomal of region 19q13.2-3, encompassing the genes ASE, ERCC1, RAI and XPD, were associated with risk of testicular cancer in Danish patients. This is in contrast to other cancers, where we reported strong associations between polymorphisms in ERCC1, ASE and RAI and occurrence of basal cell carcinoma, breast cancer and lung. To our knowledge this is the first study of DNA repair gene polymorphisms and risk of testicular cancer.

Application of AFLP fingerprint analysis for studying the biodiversity of Streptococcus thermophilus.

PubMed

Lazzi, Camilla; Bove, Claudio Giorgio; Sgarbi, Elisa; Gatti, Monica; Monica, Gatti; La Gioia, Federica; Torriani, Sandra; Sandra, Torriani; Neviani, Erasmo

2009-10-01

Streptococcus thermophilus is a lactic acid bacteria (LAB) widely used in milk fermentation processes as a starter culture. In this work the genetic diversity of S. thermophilus isolates from different sources was analyzed using Amplified Fragment Length Polymorphism fingerprinting (AFLP). Since this is the first report that indicates the application of AFLP in order to study genotypic polymorphism in S. thermophilus species, an optimization of experimental conditions was carried out to decide the optimal AFLP analysis protocol. Furthermore the fingerprinting resolutions of AFLP and RAPD (Random Amplified Polymorphic DNA) were evaluated and compared. The overall data suggest that genotypic characterization performed by AFLP provide a better view of microbial diversity of S. thermophilus, indicating that RAPD is less discriminating than AFLP. The successful use of AFLP analysis in the characterization of S. thermophilus strains reported in this study suggests the potential uses for this technique to define the whole-genome diversity of each specific strain, as an alternative to the fingerprinting methods used till now.

A technique for setting analytical thresholds in massively parallel sequencing-based forensic DNA analysis

PubMed Central

2017-01-01

Amplicon (targeted) sequencing by massively parallel sequencing (PCR-MPS) is a potential method for use in forensic DNA analyses. In this application, PCR-MPS may supplement or replace other instrumental analysis methods such as capillary electrophoresis and Sanger sequencing for STR and mitochondrial DNA typing, respectively. PCR-MPS also may enable the expansion of forensic DNA analysis methods to include new marker systems such as single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) that currently are assayable using various instrumental analysis methods including microarray and quantitative PCR. Acceptance of PCR-MPS as a forensic method will depend in part upon developing protocols and criteria that define the limitations of a method, including a defensible analytical threshold or method detection limit. This paper describes an approach to establish objective analytical thresholds suitable for multiplexed PCR-MPS methods. A definition is proposed for PCR-MPS method background noise, and an analytical threshold based on background noise is described. PMID:28542338

A technique for setting analytical thresholds in massively parallel sequencing-based forensic DNA analysis.

PubMed

Young, Brian; King, Jonathan L; Budowle, Bruce; Armogida, Luigi

2017-01-01

Amplicon (targeted) sequencing by massively parallel sequencing (PCR-MPS) is a potential method for use in forensic DNA analyses. In this application, PCR-MPS may supplement or replace other instrumental analysis methods such as capillary electrophoresis and Sanger sequencing for STR and mitochondrial DNA typing, respectively. PCR-MPS also may enable the expansion of forensic DNA analysis methods to include new marker systems such as single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) that currently are assayable using various instrumental analysis methods including microarray and quantitative PCR. Acceptance of PCR-MPS as a forensic method will depend in part upon developing protocols and criteria that define the limitations of a method, including a defensible analytical threshold or method detection limit. This paper describes an approach to establish objective analytical thresholds suitable for multiplexed PCR-MPS methods. A definition is proposed for PCR-MPS method background noise, and an analytical threshold based on background noise is described.

Ancient human mitochondrial DNA and radiocarbon analysis of archived quids from the Mule Spring Rockshelter, Nevada, USA.

PubMed

Hamilton-Brehm, Scott D; Hristova, Lidia T; Edwards, Susan R; Wedding, Jeffrey R; Snow, Meradeth; Kruger, Brittany R; Moser, Duane P

2018-01-01

Chewed and expectorated quids, indigestible stringy fibers from the roasted inner pulp of agave or yucca root, have proven resilient over long periods of time in dry cave environments and correspondingly, although little studied, are common in archaeological archives. In the late 1960s, thousands of quids were recovered from Mule Spring Rockshelter (Nevada, USA) deposits and stored without consideration to DNA preservation in a museum collection, remaining unstudied for over fifty years. To assess the utility of these materials as repositories for genetic information about past inhabitants of the region and their movements, twenty-one quids were selected from arbitrary excavation depths for detailed analysis. Human mitochondrial DNA sequences from the quids were amplified by PCR and screened for diagnostic single nucleotide polymorphisms. Most detected single nucleotide polymorphisms were consistent with recognized Native American haplogroup subclades B2a5, B2i1, C1, C1c, C1c2, and D1; with the majority of the sample set consistent with subclades C1, C1c, and C1c2. In parallel with the DNA analysis, each quid was radiocarbon dated, revealing a time-resolved pattern of occupancy from 347 to 977 calibrated years before present. In particular, this dataset reveals strong evidence for the presence of haplogroup C1/C1c at the Southwestern edge of the US Great Basin from ~670 to 980 cal YBP, which may temporally correspond with the beginnings of the so-called Numic Spread into the region. The research described here demonstrates an approach which combines targeted DNA analysis with radiocarbon age dating; thus enabling the genetic analysis of archaeological materials of uncertain stratigraphic context. Here we present a survey of the maternal genetic profiles from people who used the Mule Spring Rockshelter and the historic timing of their utilization of a key natural resource.

ERCC2/XPD Lys751Gln alter DNA repair efficiency of platinum-induced DNA damage through P53 pathway.

PubMed

Zhang, Guopei; Guan, Yangyang; Zhao, Yuejiao; van der Straaten, Tahar; Xiao, Sha; Xue, Ping; Zhu, Guolian; Liu, Qiufang; Cai, Yuan; Jin, Cuihong; Yang, Jinghua; Wu, Shengwen; Lu, Xiaobo

2017-02-01

Platinum-based treatment causes Pt-DNA adducts which lead to cell death. The platinum-induced DNA damage is recognized and repaired by the nucleotide excision repair (NER) system of which ERCC2/XPD is a critical enzyme. Single nucleotide polymorphisms in ERCC2/XPD have been found to be associated with platinum resistance. The aim of the present study was to investigate whether ERCC2/XPD Lys751Gln (rs13181) polymorphism is causally related to DNA repair capacity of platinum-induced DNA damage. First, cDNA clones expressing different genotypes of the polymorphism was transfected to an ERCC2/XPD defective CHO cell line (UV5). Second, all cells were treated with cisplatin. Cellular survival rate were investigated by MTT growth inhibition assay, DNA damage levels were investigated by comet assay and RAD51 staining. The distribution of cell cycle and the change of apoptosis rates were detected by a flow cytometric method (FCM). Finally, P53mRNA and phospho-P53 protein levels were further investigated in order to explore a possible explanation. As expected, there was a significantly increased in viability of UV5 ERCC2 (AA) as compared to UV5 ERCC2 (CC) after cisplatin treatment. The DNA damage level of UV5 ERCC2 (AA) was significant decreased compared to UV5 ERCC2 (CC) at 24 h of treatment. Mutation of ERCC2rs13181 AA to CC causes a prolonged S phase in cell cycle. UV5 ERCC2 (AA) alleviated the apoptosis compared to UV5 ERCC2 (CC) , meanwhile P53mRNA levels in UV ERCC2 (AA) was also lower when compared UV5 ERCC2 (CC) . It co-incides with a prolonged high expression of phospho-P53, which is relevant for cell cycle regulation, apoptosis, and the DNA damage response (DDR). We concluded that ERCC2/XPD rs13181 polymorphism is possibly related to the DNA repair capacity of platinum-induced DNA damage. This functional study provides some clues to clarify the relationship between cisplatin resistance and ERCC2/XPDrs13181 polymorphism. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Isolation and characterization of cDNA clones for human erythrocyte beta-spectrin.

PubMed Central

Prchal, J T; Morley, B J; Yoon, S H; Coetzer, T L; Palek, J; Conboy, J G; Kan, Y W

1987-01-01

Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical alpha (Mr 240,000) and beta (Mr 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. We report here the isolation and characterization of a human erythroid-specific beta-spectrin cDNA clone that encodes parts of the beta-9 through beta-12 repeat segments. This cDNA was used as a hybridization probe to assign the beta-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte beta-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the beta-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities. Images PMID:3478706

CTLA-4 gene polymorphisms and their influence on predisposition to autoimmune thyroid diseases (Graves’ disease and Hashimoto's thyroiditis)

PubMed Central

Pastuszak-Lewandoska, Dorota; Sewerynek, Ewa; Domańska, Daria; Gładyś, Aleksandra; Skrzypczak, Renata

2012-01-01

Introduction Autoimmune thyroid disease (AITD) is associated with both genetic and environmental factors which lead to the overactivity of immune system. Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) gene polymorphisms belong to the main genetic factors determining the susceptibility to AITD (Hashimoto's thyroiditis, HT and Graves' disease, GD) development. The aim of the study was to evaluate the relationship between CTLA-4 polymorphisms (A49G, 1822 C/T and CT60 A/G) and HT and/or GD in Polish patients. Material and methods Molecular analysis involved AITD group, consisting of HT (n=28) and GD (n=14) patients, and a control group of healthy persons (n=20). Genomic DNA was isolated from peripheral blood and CTLA-4 polymorphisms were assessed by polymerase chain reaction-restriction fragment length polymorphism method, using three restriction enzymes: Fnu4HI (A49G), BsmAI (1822 C/T) and BsaAI (CT60 A/G). Results Statistical analysis (χ2 test) confirmed significant differences between the studied groups concerning CTLA-4 A49G genotypes. CTLA-4 A/G genotype was significantly more frequent in AITD group and OR analysis suggested that it might increase the susceptibility to HT. In GD patients, OR analysis revealed statistically significant relationship with the presence of G allele. In controls, CTLA-4 A/A genotype frequency was significantly increased suggesting a protective effect. There were no statistically significant differences regarding frequencies of other genotypes and polymorphic alleles of the CTLA-4 gene (1822 C/T and CT60 A/G) between the studied groups. Conclusions CTLA-4 A49G polymorphism seems to be an important genetic determinant of the risk of HT and GD in Polish patients. PMID:22851994

CTLA-4 gene polymorphisms and their influence on predisposition to autoimmune thyroid diseases (Graves' disease and Hashimoto's thyroiditis).

PubMed

Pastuszak-Lewandoska, Dorota; Sewerynek, Ewa; Domańska, Daria; Gładyś, Aleksandra; Skrzypczak, Renata; Brzeziańska, Ewa

2012-07-04

Autoimmune thyroid disease (AITD) is associated with both genetic and environmental factors which lead to the overactivity of immune system. Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) gene polymorphisms belong to the main genetic factors determining the susceptibility to AITD (Hashimoto's thyroiditis, HT and Graves' disease, GD) development. The aim of the study was to evaluate the relationship between CTLA-4 polymorphisms (A49G, 1822 C/T and CT60 A/G) and HT and/or GD in Polish patients. Molecular analysis involved AITD group, consisting of HT (n=28) and GD (n=14) patients, and a control group of healthy persons (n=20). Genomic DNA was isolated from peripheral blood and CTLA-4 polymorphisms were assessed by polymerase chain reaction-restriction fragment length polymorphism method, using three restriction enzymes: Fnu4HI (A49G), BsmAI (1822 C/T) and BsaAI (CT60 A/G). Statistical analysis (χ(2) test) confirmed significant differences between the studied groups concerning CTLA-4 A49G genotypes. CTLA-4 A/G genotype was significantly more frequent in AITD group and OR analysis suggested that it might increase the susceptibility to HT. In GD patients, OR analysis revealed statistically significant relationship with the presence of G allele. In controls, CTLA-4 A/A genotype frequency was significantly increased suggesting a protective effect. There were no statistically significant differences regarding frequencies of other genotypes and polymorphic alleles of the CTLA-4 gene (1822 C/T and CT60 A/G) between the studied groups. CTLA-4 A49G polymorphism seems to be an important genetic determinant of the risk of HT and GD in Polish patients.

Terminal Restriction Fragment Length Polymorphism Analysis Program, a Web-Based Research Tool for Microbial Community Analysis

PubMed Central

Marsh, Terence L.; Saxman, Paul; Cole, James; Tiedje, James

2000-01-01

Rapid analysis of microbial communities has proven to be a difficult task. This is due, in part, to both the tremendous diversity of the microbial world and the high complexity of many microbial communities. Several techniques for community analysis have emerged over the past decade, and most take advantage of the molecular phylogeny derived from 16S rRNA comparative sequence analysis. We describe a web-based research tool located at the Ribosomal Database Project web site (http://www.cme.msu.edu/RDP/html/analyses.html) that facilitates microbial community analysis using terminal restriction fragment length polymorphism of 16S ribosomal DNA. The analysis function (designated TAP T-RFLP) permits the user to perform in silico restriction digestions of the entire 16S sequence database and derive terminal restriction fragment sizes, measured in base pairs, from the 5′ terminus of the user-specified primer to the 3′ terminus of the restriction endonuclease target site. The output can be sorted and viewed either phylogenetically or by size. It is anticipated that the site will guide experimental design as well as provide insight into interpreting results of community analysis with terminal restriction fragment length polymorphisms. PMID:10919828

C677T polymorphism in the methylenetetrahydrofolate reductase gene is associated with primary closed angle glaucoma

PubMed Central

Michael, Shazia; Qamar, Raheel; Akhtar, Farah; Khan, Wajid Ali

2008-01-01

Purpose To determine whether or not there is an association of the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism with disease in cohorts of primary open-angle glaucoma (POAG) and primary closed-angle glaucoma (PCAG) from Pakistan. Methods This was a prospective study consisting of 150 patients (90 POAG and 60 PCAG) and 70 control subjects. Genomic DNA was extracted from leukocytes of the peripheral blood. MTHFR C677T polymorphism analysis was performed by the polymerase chain reaction-restriction fragment length polymorphism (RFLP) technique. Results The prevalence of the MTHFR C/T genotype was 22.2% in POAG, 13.3% in PACG, and 18.6% in controls whereas the MTHFR T/T genotype was present solely in the PACG group (6.9%). The difference regarding the T/T genotype between PACG and controls was statistically significant (p<0.01). Conclusions The MTHFR C677T polymorphism was found to be associated with PCAG but not POAG in patients of Pakistani origin. PMID:18385801

Genomic biomarkers and clinical outcomes of physical activity.

PubMed

Izzotti, Alberto

2011-07-01

Clinical and experimental studies in humans provide evidence that moderate physical activity significantly decreases artery oxidative damage to nuclear DNA, DNA-adducts related to age and dyslipedemia, and mitochondrial DNA damage. Maintenance of adequate mitochondrial function is crucial for preventing lipid accumulation and peroxidation occurring in atherosclerosis. Studies performed on human muscle biopsies analyzing gene expression in living humans reveal that physically active subjects improve the expression of genes involved in mitochondrial function and of related microRNAs. The attenuation of oxidative damage to nuclear and mitochondrial DNA by physical activity resulted in beneficial effects due to polymorphisms of glutathione S-transferases genes. Subjects bearing null GSTM1/T1 polymorphisms have poor life expectancy in the case of being sedentary, which was increased 2.6-fold in case they performed physical activity. These findings indicate that the preventive effect of physical activity undergoes interindividual variation affected by genetic polymorphisms. © 2011 New York Academy of Sciences.

DETERMINATION OF GENETIC DIVERSITY AND PATERNITY IN THE GRAY-TAILED VOLE (MICROTUS CANICAUDUS) BY RAPD-PCR

EPA Science Inventory

Genetic relatedness of gray-tailed voles (Microtus canicaudus) was determined by random amplified polymorphic DNA (RAPD). This work is the first reported use of the RAPD method for pedigree analysis of M. canicaudus and demonstrates the feasibility of RAPD for assessing paternity...

Molecular Characterization of Herpes Simplex Virus 2 Strains by Analysis of Microsatellite Polymorphism

PubMed Central

Ait-Arkoub, Zaïna; Voujon, Delphine; Deback, Claire; Abrao, Emiliana P.; Agut, Henri; Boutolleau, David

2013-01-01

The complete 154-kbp linear double-stranded genomic DNA sequence of herpes simplex virus 2 (HSV-2), consisting of two extended regions of unique sequences bounded by a pair of inverted repeat elements, was published in 1998 and since then has been widely employed in a wide range of studies. Throughout the HSV-2 genome are scattered 150 microsatellites (also referred to as short tandem repeats) of 1- to 6-nucleotide motifs, mainly distributed in noncoding regions. Microsatellites are considered reliable markers for genetic mapping to differentiate herpesvirus strains, as shown for cytomegalovirus and HSV-1. The aim of this work was to characterize 12 polymorphic microsatellites within the HSV-2 genome by use of 3 multiplex PCR assays in combination with length polymorphism analysis for the rapid genetic differentiation of 56 HSV-2 clinical isolates and 2 HSV-2 laboratory strains (gHSV-2 and MS). This new system was applied to a specific new HSV-2 variant recently identified in HIV-1-infected patients originating from West Africa. Our results confirm that microsatellite polymorphism analysis is an accurate tool for studying the epidemiology of HSV-2 infections. PMID:23966512

DNA sequence divergence among derivatives of Escherichia coli K-12 detected by arbitrary primer PCR (random amplified polymorphic DNA) fingerprinting.

PubMed Central

Brikun, I; Suziedelis, K; Berg, D E

1994-01-01

Derivatives of Escherichia coli K-12 of known ancestry were characterized by random amplified polymorphic DNA (RAPD) fingerprinting to better understand genome evolution in this family of closely related strains. This sensitive method entails PCR amplification with arbitrary primers at low stringency and yields arrays of anonymous DNA fragments that are strain specific. Among 150 fragments scored, eight were polymorphic in that they were produced from some but not all strains. Seven polymorphic bands were chromosomal, and one was from the F-factor plasmid. Five of the six mapped polymorphic chromosomal bands came from just 7% of the genome, a 340-kb segment that includes the terminus of replication. Two of these were from the cryptic Rac prophage, and the inability to amplify them from strains was attributable to deletion (excision) or to rearrangement of Rac. Two other terminus-region segments that resulted in polymorphic bands appeared to have sustained point mutations that affected the ability to amplify them. Control experiments showed that RAPD bands from the 340-kb terminus-region segment and also from two plasmids (P1 and F) were represented in approximate proportion to their size. Optimization experiments showed that the concentration of thermostable polymerase strongly affected the arrays of RAPD products obtained. Comparison of RAPD polymorphisms and positions of strains exhibiting them in the pedigree suggests that many sequence changes occurred in these historic E. coli strains during their storage. We propose that the clustering of such mutations near the terminus reflects errors during completion of chromosome replication, possibly during slow growth in the stab cultures that were often used to store E. coli strains in the early years of bacterial genetics. Images PMID:8132463

Mismatch repair gene MSH3 polymorphism is associated with the risk of sporadic prostate cancer

PubMed Central

Hirata, Hiroshi; Hinoda, Yuji; Kawamoto, Ken; Kikuno, Nobuyuki; Suehiro, Yutaka; Okayama, Naoko; Tanaka, Yuichiro; Dahiya, Rajvir

2014-01-01

Purpose The mismatch repair (MMR) system is a DNA repair mechanism that corrects mispaired bases during DNA replication errors. Cancer cells deficient in the MMR proteins have a 102 –103-fold increase in the mutation rate. Single nucleotide polymorphisms (SNPs) of MMR genes have been shown to cause a reduction in DNA repair activity. We hypothesized that mismatch repair gene polymorphism could be a risk factor for prostate cancer (PC) and that p53 Pro/Pro genotype carriers could influence MSH3 and MSH6 polymorphisms. Material and Methods DNA samples from 110 cases of prostate cancer and healthy controls (n=110) were analyzed by SSCP and PCR-RFLP to determine the genotypic frequency of five different polymorphic loci on two MMR genes (MSH3 and MSH6) and p53 codon72. The chi-square test was applied to compare the genotype frequency between patients and controls. Results A significant increase in the G/A+A/A genotype of MSH3 Pro222Pro was observed in patients compared to controls (OR, 1.87; 95% CI, 1.0–3.5). The frequency of A/G + G/G genotypes of MSH3 exon23 Thr1036Ala also tended to increase in patients (OR, 1.57; 95% CI, 0.92–2.72). Among p53 codon72 Arg/Pro + Pro/Pro carriers, the frequency of the AG + GG genotype of MSH3 exon23 was significantly increased in patients compared to controls (OR = 2.1, 95% CI; 1.05–4.34). Conclusion This is the first report on the association of MSH3 gene polymorphisms in prostate cancer. These results suggest that the MSH3 polymorphism may be a risk factor for prostate cancer. PMID:18355840

Randomly amplified polymorphic DNA linkage relationships in different Norway spruce populations

Treesearch

M. Troggio; Thomas L. Kubisiak; G. Bucci; P. Menozzi

2001-01-01

We tested the constancy of linkage relationships of randomly amplified polymorphic DNA (RAPD) marker loci used to construct a population-based consensus map in material from an Italian stand of Picea abies (L.) Karst. in 29 individuals from three Norwegian populations. Thirteen marker loci linked in the Italian stand did show a consistent locus...

Analysis of Ethnic Admixture in Prostate Cancer

DTIC Science & Technology

2006-12-01

low penetrant genes have been identified as potential PCA suscept- ibility genes. These candidate genes include SRD5A2 (MIM 607306), CYP3A4 (MIM 124010...progression [13]. The CDH1gene is located at 16q22.1 and consists of 16 exons spanning approximately 100 kb of genomic DNA. Several polymorphisms, germline and...upstreamof theATGstart site and all 16 exons of CDH1 were screened for DNA sequence variation by denaturing high-performance liquid chro- matography

Assessment of Azorean ancestry by Alu insertion polymorphisms.

PubMed

Branco, Claudia C; Palla, Raquel; Lino, Sílvia; Pacheco, Paula R; Cabral, Rita; De Fez, Laura; Peixoto, Bernardo R; Mota-Vieira, Luisa

2006-01-01

Knowledge of population ancestry from genetic markers is essential, for example, to understand the history of human migration and to carry out admixture and association studies. Here we assess the genome ancestry of the Azorean population through analysis of six Alu polymorphic sites (TPA-25, ACE, APO, B65, PV92, and D1) in 65 Azoreans and 30 Portuguese unrelated blood donors and compare data for the Y-chromosome and mtDNA. Allele frequencies were calculated by direct counting. Statistical analysis was performed using Arlequin 2.0. Nei's genetic distance was calculated with DISPAN software, and trees were constructed by neighbor joining (NJ) using PHYLIP 3.63. The results show that all Alu insertions were polymorphic. APO is the closest to fixation. The less frequent insertions are PV92 and D1 in the Azores and Portugal, respectively. ACE and TPA-25 show the highest values of heterozygosity in both populations. Allele frequencies are very similar to those obtained in European populations. These results are validated by the Y-chromosome and mtDNA data, where the majority of the maternal and paternal lineages are European. Overall, these data are reflected in the phylogenetic tree, in which the Azoreans and the Portuguese branch with Catalans, Andalusians, Moroccans, and Algerians. We conclude that the population of the Azores shows no significant genetic differences from that of mainland Portugal and that it is an outbred population. Moreover, the data validate the use of Alu insertion polymorphisms to assess the origin and history of human populations. Am. J. Hum. Biol. 18:223-226, 2006. (c) 2006 Wiley-Liss, Inc.

Molecular epidemiology of Escherichia coli O157:H7 strains by bacteriophage lambda restriction fragment length polymorphism analysis: application to a multistate foodborne outbreak and a day-care center cluster.

PubMed

Samadpour, M; Grimm, L M; Desai, B; Alfi, D; Ongerth, J E; Tarr, P I

1993-12-01

Genomic DNAs prepared from 168 isolates of Escherichia coli O157:H7 were analyzed for restriction fragment length polymorphisms on Southern blots probed with bacteriophage lambda DNA. The isolates analyzed included strains from a recent large multistate outbreak of E. coli O157:H7 infection associated with consumption of poorly cooked beef in restaurants, a day-care center cluster, and temporally and geographically unrelated isolates. E. coli O157:H7 isolates recovered from the incriminated meat and from 61 (96.8%) of 63 patients from Washington and Nevada possessed identical lambda restriction fragment length patterns. The lambda restriction fragment length polymorphisms observed in 11 (91.7%) of 12 day-care center patients were identical, but they differed from that of the strain associated with the multistate outbreak. E. coli O157:H7 from 42 patients temporally or geographically unrelated to either cluster of infection possessed unique and different lambda restriction fragment length patterns, except for paired isolates from three separate clusters of infection. These data demonstrate that the hybridization of DNA digests of E. coli O157:H7 with radiolabelled bacteriophage lambda DNA can be a useful, stable, and discriminatory epidemiologic tool for analyzing the linkage between strains of E. coli O157:H7.

Identification of exotic genetic components and DNA methylation pattern analysis of three cotton introgression lines from Gossypium bickii.

PubMed

He, Shou-Pu; Sun, Jun-Ling; Zhang, Chao; Du, Xiong-Ming

2011-01-01

The impact of alien DNA fragments on plant genome has been studied in many species. However, little is known about the introgression lines of Gossypium. To study the consequences of introgression in Gossypium, we investigated 2000 genomic and 800 epigenetic sites in three typical cotton introgression lines, as well as their cultivar (Gossypium hirsutum) and wild parents (Gossypium bickii), by amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP). The results demonstrate that an average of 0.5% of exotic DNA segments from wild cotton is transmitted into the genome of each introgression line, with the addition of other forms of genetic variation. In total, an average of 0.7% of genetic variation sites is identified in introgression lines. Simultaneously, the overall cytosine methylation level in each introgression line is very close to that of the upland cotton parent (an average of 22.6%). Further dividing patterns reveal that both hypomethylation and hypermethylation occurred in introgression lines in comparison with the upland cotton parent. Sequencing of nine methylation polymorphism fragments showed that most (7 of 9) of the methylation alternations occurred in the noncoding sequences. The molecular evidence of introgression from wild cotton into introgression lines in our study is identified by AFLP. Moreover, the causes of petal variation in introgression lines are discussed.

Analysis of cytotoxic T lymphocyte associated antigen 4 gene polymorphisms in patients with ulcerative colitis.

PubMed

Lankarani, Kamran B; Karbasi, Ashraf; Kalantari, Tahereh; Yarmohammadi, Hooman; Saberi-Firoozi, Mehdi; Alizadeh-Naeeni, Mahvash; Taghavi, Ali R; Fattahi, Mahammad R; Ghaderi, Abbas

2006-02-01

Ulcerative colitis (UC) is a multifactorial disease associated with dysregulated immunity. Recently, cytotoxic T lymphocyte associated antigen 4 (CTLA-4) gene polymorphisms have been reported in association with several autoimmune diseases in several populations. In the present study, the possible implication of the CTLA-4 gene as a risk factor for UC in the Iranian population was investigated. One hundred UC patients and 100 healthy subjects were studied. CTLA-4 exon 1 position 49 (A/G: codon 17: Thr/Ala) polymorphisms were investigated by polymerase chain reaction single strand confirmation polymorphism method. Four of the patients and one of the healthy controls were excluded from the study because of incomplete DNA extraction. The allele frequencies of A and G in 96 patients (A: 66.1%; G: 33.9%) were not significantly different from the 99 control subjects (A: 63.1%; G: 36.9%, P > 0.05). No significant differences in the distribution of genotype frequencies were observed between A + 49G gene polymorphisms and UC in the Iranian population (P > 0.05). CTLA-4 polymorphism is not associated with UC in the Iranian population.

Genomic polymorphism, recombination, and linkage disequilibrium in human major histocompatibility complex-encoded antigen-processing genes.

PubMed Central

van Endert, P M; Lopez, M T; Patel, S D; Monaco, J J; McDevitt, H O

1992-01-01

Recently, two subunits of a large cytosolic protease and two putative peptide transporter proteins were found to be encoded by genes within the class II region of the major histocompatibility complex (MHC). These genes have been suggested to be involved in the processing of antigenic proteins for presentation by MHC class I molecules. Because of the high degree of polymorphism in MHC genes, and previous evidence for both functional and polypeptide sequence polymorphism in the proteins encoded by the antigen-processing genes, we tested DNA from 27 consanguineous human cell lines for genomic polymorphism by restriction fragment length polymorphism (RFLP) analysis. These studies demonstrate a strong linkage disequilibrium between TAP1 and LMP2 RFLPs. Moreover, RFLPs, as well as a polymorphic stop codon in the telomeric TAP2 gene, appear to be in linkage disequilibrium with HLA-DR alleles and RFLPs in the HLA-DO gene. A high rate of recombination, however, seems to occur in the center of the complex, between the TAP1 and TAP2 genes. Images PMID:1360671

mtDNAmanager: a Web-based tool for the management and quality analysis of mitochondrial DNA control-region sequences

PubMed Central

Lee, Hwan Young; Song, Injee; Ha, Eunho; Cho, Sung-Bae; Yang, Woo Ick; Shin, Kyoung-Jin

2008-01-01

Background For the past few years, scientific controversy has surrounded the large number of errors in forensic and literature mitochondrial DNA (mtDNA) data. However, recent research has shown that using mtDNA phylogeny and referring to known mtDNA haplotypes can be useful for checking the quality of sequence data. Results We developed a Web-based bioinformatics resource "mtDNAmanager" that offers a convenient interface supporting the management and quality analysis of mtDNA sequence data. The mtDNAmanager performs computations on mtDNA control-region sequences to estimate the most-probable mtDNA haplogroups and retrieves similar sequences from a selected database. By the phased designation of the most-probable haplogroups (both expected and estimated haplogroups), mtDNAmanager enables users to systematically detect errors whilst allowing for confirmation of the presence of clear key diagnostic mutations and accompanying mutations. The query tools of mtDNAmanager also facilitate database screening with two options of "match" and "include the queried nucleotide polymorphism". In addition, mtDNAmanager provides Web interfaces for users to manage and analyse their own data in batch mode. Conclusion The mtDNAmanager will provide systematic routines for mtDNA sequence data management and analysis via easily accessible Web interfaces, and thus should be very useful for population, medical and forensic studies that employ mtDNA analysis. mtDNAmanager can be accessed at . PMID:19014619

Polymorphic microsatellite DNA markers for the Florida manatee (Trichechus manatus latirostris)

USGS Publications Warehouse

Pause, K.C.; Nourisson, C.; Clark, A.; Kellogg, M.E.; Bonde, R.K.; McGuire, P.M.

2007-01-01

Florida manatees (Trichechus manatus latirostris) are marine mammals that inhabit the coastal waters and rivers of the southeastern USA, primarily Florida. Previous studies have shown that Florida manatees have low mitochondrial DNA variability, suggesting that nuclear DNA loci are necessary for discriminatory analyses. Here we report 10 polymorphic microsatellite loci with an average of 4.2 alleles per locus, and average heterozygosity of 50.1%. These loci have been developed for use in population studies, parentage assignment, and individual identification. ?? 2007 Blackwell Publishing Ltd.

Substitutions of short heterologous DNA segments of intragenomic or extragenomic origins produce clustered genomic polymorphisms

PubMed Central

Harms, Klaus; Lunnan, Asbjørn; Hülter, Nils; Mourier, Tobias; Vinner, Lasse; Andam, Cheryl P.; Marttinen, Pekka; Fridholm, Helena; Hansen, Anders Johannes; Hanage, William P.; Nielsen, Kaare Magne; Willerslev, Eske; Johnsen, Pål Jarle

2016-01-01

In a screen for unexplained mutation events we identified a previously unrecognized mechanism generating clustered DNA polymorphisms such as microindels and cumulative SNPs. The mechanism, short-patch double illegitimate recombination (SPDIR), facilitates short single-stranded DNA molecules to invade and replace genomic DNA through two joint illegitimate recombination events. SPDIR is controlled by key components of the cellular genome maintenance machinery in the gram-negative bacterium Acinetobacter baylyi. The source DNA is primarily intragenomic but can also be acquired through horizontal gene transfer. The DNA replacements are nonreciprocal and locus independent. Bioinformatic approaches reveal occurrence of SPDIR events in the gram-positive human pathogen Streptococcus pneumoniae and in the human genome. PMID:27956618

[Genetic polymorphism of flax Linum usitatissimum based on use of molecular cytogenetic markers].

PubMed

Rachinskaia, O A; Lemesh, V A; Muravenko, O V; Iurkevich, O Iu; Guzenko, E V; Bol'sheva, N L; Bogdanova, M V; Samatadze, T E; Popov, K V; Malyshev, S V; Shostak, N G; Heller, K; Khotyleva, L V; Zelenin, A V

2011-01-01

Using a set of approaches based on the use of molecular cytogenetic markers (DAPI/C-banding, estimation of the total area of DAPI-positive regions in prophase nuclei, FISH with 26S and 5S rDNA probes) and the microsatellite (SSR-PCR) assay, we studied genomic polymorphism in 15 flax (Linum usitatissimum L.) varieties from different geographic regions belonging to three directions of selection (oil, fiber, and intermediate flaxes) and in the k-37 x Viking hybrid. All individual chromosomes have been identified in the karyotypes of these varieties on the basis of the patterns of differential DAPI/C-banding and the distribution of 26S and 5S rDNA, and idiograms of the chromosomes have been generated. Unlike the oil flax varieties, the chromosomes in the karyotypes of the fiber flax varieties have, as a rule, pericentromeric and telomeric DAPI-positive bands of smaller size, but contain larger intercalary regions. Two chromosomal rearrangements (chromosome 3 inversions) were discovered in the variety Luna and in the k-37 x Viking hybrid. In both these forms, no colocalization of 26S rDNA and 5S rDNA on the satellite chromosome was detected. The SSR assay with the use of 20 polymorphic pairs of primers revealed 22 polymorphic loci. Based on the SSR data, we analyzed genetic similarity of the flax forms studied and constructed a genetic similarity dendrogram. The genotypes studied here form three clusters. The oil varieties comprise an independent cluster. The genetically related fiber flax varieties Vita and Luna, as well as the landrace Lipinska XIII belonging to the intermediate type, proved to be closer to the oil varieties than the remaining fiber flax varieties. The results of the molecular chromosomal analysis in the fiber and oil flaxes confirm their very close genetic similarity. In spite of this, the combined use of the chromosomal and molecular markers has opened up unique possibilities for describing the genotypes of flax varieties and creating their genetic passports.

[RDBH-method and big DyeTM terminator technology in accurate diagnosis of β-thalassemia and the allelic polymorphism of β-globin cluster].

PubMed

Akperova, G A

2014-11-01

IThe purpose of this study was to evaluate of the efficiency of RDBH-method and Big DyeTM Terminator technology in an accurate diagnosis of β-thalassemia and the allelic polymorphism of β-globin cluster. It was done a complete hematology analysis (HB, MCH, MCV, MCHC, RBC, Hct, HbA2, HbF, Serum iron, Serum ferritin at four children (males, 6-10 years old) and their parents. Molecular analysis included Reverse Dot-Blot Hybridization StripAssay (RDBH) and DNA sequencing on ABI PRISM Big DyeTM Terminator. Hematologic and molecular parameters were contradictory. The homozygosity for β0-thalassemia (β0IVS2.1[G>A] and β0codon 8[-AA]) at three boys with the mild clinical manifestation and heterozygosity of their parents for mutations, and the absence of β-globin mutations at parents and a boy who holds monthly transfusion was established by RDBH-analysis. DNA sequencing by technology Big DyeTM Terminator showed polymorphism at positions -551 and -521 of Cap5'-region (-650-250) - (AT)7(T)7 and (AT)8(T)5. Application of the integrated clinical-molecular approach is an ideal method for an accurate diagnosis, identification of asymptomatic carriers and a reduce of the risk of complications from β-thalassemia, moreover screening of γG-gene and the level of fetal hemoglobin in early childhood will help manage of β-thalassemia clinic and prevent heavy consequences of the disease.

Rapid single nucleotide polymorphism based method for hematopoietic chimerism analysis and monitoring using high-speed droplet allele-specific PCR and allele-specific quantitative PCR.

PubMed

Taira, Chiaki; Matsuda, Kazuyuki; Yamaguchi, Akemi; Uehara, Masayuki; Sugano, Mitsutoshi; Okumura, Nobuo; Honda, Takayuki

2015-05-20

Chimerism analysis is important for the evaluation of engraftment and predicting relapse following hematopoietic stem cell transplantation (HSCT). We developed a chimerism analysis for single nucleotide polymorphisms (SNPs), including rapid screening of the discriminable donor/recipient alleles using droplet allele-specific PCR (droplet-AS-PCR) pre-HSCT and quantitation of recipient DNA using AS-quantitative PCR (AS-qPCR) following HSCT. SNP genotyping of 20 donor/recipient pairs via droplet-AS-PCR and the evaluation of the informativity of 5 SNP markers for chimerism analysis were performed. Samples from six follow-up patients were analyzed to assess the chimerism via AS-qPCR. These results were compared with that determined by short tandem repeat PCR (STR-PCR). Droplet-AS-PCR could determine genotypes within 8min. The total informativity using all 5 loci was 95% (19/20). AS-qPCR provided the percentage of recipient DNA in all 6 follow-up patients without influence of the stutter peak or the amplification efficacy, which affected the STR-PCR results. The droplet-AS-PCR had an advantage over STR-PCR in terms of rapidity and simplicity for screening before HSCT. Furthermore, AS-qPCR had better accuracy than STR-PCR for quantification of recipient DNA following HSCT. The present chimerism assay compensates for the disadvantages of STR-PCR and is readily performable in clinical laboratories. Copyright © 2015 Elsevier B.V. All rights reserved.

Analysis of DNA methylation of perennial ryegrass under drought using the methylation-sensitive amplification polymorphism (MSAP) technique.

PubMed

Tang, Xiao-Mei; Tao, Xiang; Wang, Yan; Ma, Dong-Wei; Li, Dan; Yang, Hong; Ma, Xin-Rong

2014-12-01

Perennial ryegrass (Lolium perenne), an excellent grass for forage and turf, is widespread in temperate regions. Drought is an important factor that limits its growth, distribution, and yield. DNA methylation affects gene expression and plays an important role in adaptation to adverse environments. In this study, the DNA methylation changes in perennial ryegrass under drought stress were assessed using methylation-sensitive amplified polymorphism (MSAP). After 15 days of drought stress treatment, the plant height was less than half of the control, and the leaves were smaller and darker. Genome-wide, a total of 652 CCGG sites were detected by MSAP. The total methylation level was 57.67 and 47.39 % in the control and drought treatment, respectively, indicating a decrease of 10.28 % due to drought exposure. Fifteen differentially displayed DNA fragments in MSAP profiles were cloned for sequencing analysis. The results showed that most of the genes involved in stress responses. The relative expression levels revealed that three demethylated fragments were up-regulated. The expression of a predicted retrotransposon increased significantly, changing from hypermethylation to non-methylation. Although the extent of methylation in two other genes decreased, the sites of methylation remained, and the expression increased only slightly. All of these results suggested that drought stress decreased the total DNA methylation level in perennial ryegrass and demethylation up-regulated related gene expressions and that the extent of methylation was negatively correlated with expression. Overall, the induced epigenetic changes in genome probably are an important regulatory mechanism for acclimating perennial ryegrass to drought and possibly other environmental stresses.

Identification of SNP and SSR Markers in Finger Millet Using Next Generation Sequencing Technologies

PubMed Central

Gimode, Davis; Odeny, Damaris A.; de Villiers, Etienne P.; Wanyonyi, Solomon; Dida, Mathews M.; Mneney, Emmarold E.; Muchugi, Alice; Machuka, Jesse; de Villiers, Santie M.

2016-01-01

Finger millet is an important cereal crop in eastern Africa and southern India with excellent grain storage quality and unique ability to thrive in extreme environmental conditions. Since negligible attention has been paid to improving this crop to date, the current study used Next Generation Sequencing (NGS) technologies to develop both Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP) markers. Genomic DNA from cultivated finger millet genotypes KNE755 and KNE796 was sequenced using both Roche 454 and Illumina technologies. Non-organelle sequencing reads were assembled into 207 Mbp representing approximately 13% of the finger millet genome. We identified 10,327 SSRs and 23,285 non-homeologous SNPs and tested 101 of each for polymorphism across a diverse set of wild and cultivated finger millet germplasm. For the 49 polymorphic SSRs, the mean polymorphism information content (PIC) was 0.42, ranging from 0.16 to 0.77. We also validated 92 SNP markers, 80 of which were polymorphic with a mean PIC of 0.29 across 30 wild and 59 cultivated accessions. Seventy-six of the 80 SNPs were polymorphic across 30 wild germplasm with a mean PIC of 0.30 while only 22 of the SNP markers showed polymorphism among the 59 cultivated accessions with an average PIC value of 0.15. Genetic diversity analysis using the polymorphic SNP markers revealed two major clusters; one of wild and another of cultivated accessions. Detailed STRUCTURE analysis confirmed this grouping pattern and further revealed 2 sub-populations within wild E. coracana subsp. africana. Both STRUCTURE and genetic diversity analysis assisted with the correct identification of the new germplasm collections. These polymorphic SSR and SNP markers are a significant addition to the existing 82 published SSRs, especially with regard to the previously reported low polymorphism levels in finger millet. Our results also reveal an unexploited finger millet genetic resource that can be included in the regional breeding programs in order to efficiently optimize productivity. PMID:27454301

Identification of SNP and SSR Markers in Finger Millet Using Next Generation Sequencing Technologies.

PubMed

Gimode, Davis; Odeny, Damaris A; de Villiers, Etienne P; Wanyonyi, Solomon; Dida, Mathews M; Mneney, Emmarold E; Muchugi, Alice; Machuka, Jesse; de Villiers, Santie M

2016-01-01

Finger millet is an important cereal crop in eastern Africa and southern India with excellent grain storage quality and unique ability to thrive in extreme environmental conditions. Since negligible attention has been paid to improving this crop to date, the current study used Next Generation Sequencing (NGS) technologies to develop both Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP) markers. Genomic DNA from cultivated finger millet genotypes KNE755 and KNE796 was sequenced using both Roche 454 and Illumina technologies. Non-organelle sequencing reads were assembled into 207 Mbp representing approximately 13% of the finger millet genome. We identified 10,327 SSRs and 23,285 non-homeologous SNPs and tested 101 of each for polymorphism across a diverse set of wild and cultivated finger millet germplasm. For the 49 polymorphic SSRs, the mean polymorphism information content (PIC) was 0.42, ranging from 0.16 to 0.77. We also validated 92 SNP markers, 80 of which were polymorphic with a mean PIC of 0.29 across 30 wild and 59 cultivated accessions. Seventy-six of the 80 SNPs were polymorphic across 30 wild germplasm with a mean PIC of 0.30 while only 22 of the SNP markers showed polymorphism among the 59 cultivated accessions with an average PIC value of 0.15. Genetic diversity analysis using the polymorphic SNP markers revealed two major clusters; one of wild and another of cultivated accessions. Detailed STRUCTURE analysis confirmed this grouping pattern and further revealed 2 sub-populations within wild E. coracana subsp. africana. Both STRUCTURE and genetic diversity analysis assisted with the correct identification of the new germplasm collections. These polymorphic SSR and SNP markers are a significant addition to the existing 82 published SSRs, especially with regard to the previously reported low polymorphism levels in finger millet. Our results also reveal an unexploited finger millet genetic resource that can be included in the regional breeding programs in order to efficiently optimize productivity.

Molecular population genetics of the insulin/TOR signal transduction pathway: a network-level analysis in Drosophila melanogaster.

PubMed

Alvarez-Ponce, David; Guirao-Rico, Sara; Orengo, Dorcas J; Segarra, Carmen; Rozas, Julio; Aguadé, Montserrat

2012-01-01

The IT-insulin/target of rapamycin (TOR)-signal transduction pathway is a relatively well-characterized pathway that plays a central role in fundamental biological processes. Network-level analyses of DNA divergence in Drosophila and vertebrates have revealed a clear gradient in the levels of purifying selection along this pathway, with the downstream genes being the most constrained. Remarkably, this feature does not result from factors known to affect selective constraint such as gene expression, codon bias, protein length, and connectivity. The present work aims to establish whether the selective constraint gradient detected along the IT pathway at the between-species level can also be observed at a shorter time scale. With this purpose, we have surveyed DNA polymorphism in Drosophila melanogaster and divergence from D. simulans along the IT pathway. Our network-level analysis shows that DNA polymorphism exhibits the same polarity in the strength of purifying selection as previously detected at the divergence level. This equivalent feature detected both within species and between closely and distantly related species points to the action of a general mechanism, whose action is neither organism specific nor evolutionary time dependent. The detected polarity would be, therefore, intrinsic to the IT pathway architecture and function.

Complement component 3: characterization and association with mastitis resistance in Egyptian water buffalo and cattle.

PubMed

El-Halawany, Nermin; Abd-El-Monsif, Shawky A; Al-Tohamy Ahmed, F M; Hegazy, Lamees; Abdel-Shafy, Hamdy; Abdel-Latif, Magdy A; Ghazi, Yasser A; Neuhoff, Christiane; Salilew-Wondim, Dessie; Schellander, Karl

2017-03-01

Mastitis is an infectious disease of the mammary gland that leads to reduced milk production and change in milk composition. Complement component C3 plays a major role as a central molecule of the complement cascade involving in killing of microorganisms, either directly or in cooperation with phagocytic cells. C3 cDNA were isolated, from Egyptian buffalo and cattle, sequenced and characterized. The C3 cDNA sequences of buffalo and cattle consist of 5025 and 5019 bp, respectively. Buffalo and cattle C3 cDNAs share 99% of sequence identity with each other. The 4986 bp open reading frame in buffalo encodes a putative protein of 1661 amino acids-as in cattle-and includes all the functional domains. Further, analysis of the C3 cDNA sequences detected six novel single-nucleotide polymorphisms (SNPs) in buffalo and three novel SNPs in cattle. The association analysis of the detected SNPs with milk somatic cell score as an indicator of mastitis revealed that the most significant association in buffalo was found in the C>A substitution (ss: 1752816097) in exon 27, whereas in cattle it was in the C>T substitution (ss: 1752816085) in exon 12. Our findings provide preliminary information about the contribution of C3 polymorphisms to mastitis resistance in buffalo and cattle.

DNA repair gene XRCC1 polymorphisms, smoking, and bladder cancer risk.

PubMed

Stern, M C; Umbach, D M; van Gils, C H; Lunn, R M; Taylor, J A

2001-02-01

Bladder cancer is the sixth most common cancer in the United States. The main identified risk factor is cigarette smoking, which is estimated to contribute to up to 50% of new cases in men and 20% in women. Besides containing other carcinogens, cigarette smoke is a rich source of reactive oxygen species (ROS) that can induce a variety of DNA damage, some of which is repaired by the base excision repair (BER) pathway. The XRCC1 gene protein plays an important role in BER by serving as a scaffold for other repair enzymes and by recognizing single-strand DNA breaks. Three polymorphisms that induce amino acid changes have been found in codon 194 (exon 6), codon 280 (exon 9), and codon 399 (exon 10) of this gene. We tested whether polymorphisms in XRCC1 were associated with bladder cancer risk and whether this association was modified by cigarette smoking. Therefore, we genotyped for the three polymorphisms in 235 bladder cancer cases and 213 controls who had been frequency matched to cases on age, sex, and ethnicity. We found no evidence of an association between the codon 280 variant and bladder cancer risk [odds ratio (OR), 1.2; 95% confidence interval (CI), 0.6-2.6]. We found some evidence of a protective effect for subjects that carried at least one copy of the codon 194 variant allele relative to those homozygous for the common allele (OR, 0.59; 95% CI, 0.3-1.0). The combined analysis with smoking history suggested a possible gene-exposure interaction; however, the results were not statistically significant. Similarly, for the codon 399 polymorphism, our data suggested a protective effect of the homozygous variant genotype relative to carriers of either one or two copies of the common allele (OR, 0.70; 95% CI, 0.4-1.3), and provided limited evidence, albeit not statistically significant, for a gene-smoking interaction.

Ancestral Polymorphisms and Sex-Biased Migration Shaped the Demographic History of Brown Bears and Polar Bears

PubMed Central

Nakagome, Shigeki; Mano, Shuhei; Hasegawa, Masami

2013-01-01

Recent studies have reported discordant gene trees in the evolution of brown bears and polar bears. Genealogical histories are different among independent nuclear loci and between biparentally inherited autosomal DNA (aDNA) and matrilineal mitochondrial DNA (mtDNA). Based on multi-locus genomic sequences from aDNA and mtDNA, we inferred the population demography of brown and polar bears and found that brown bears have 6 times (aDNA) or more than 14 times (mtDNA) larger population sizes than polar bears and that polar bear lineage is derived from within brown bear diversity. In brown bears, the effective population size ratio of mtDNA to aDNA was at least 0.62, which deviated from the expected value of 0.25, suggesting matriarchal population due to female philopatry and male-biased migration. These results emphasize that ancestral polymorphisms and sex-biased migration may have contributed to conflicting branching patterns in brown and polar bears across aDNA genes and mtDNA. PMID:24236053

Ancestral polymorphisms and sex-biased migration shaped the demographic history of brown bears and polar bears.

PubMed

Nakagome, Shigeki; Mano, Shuhei; Hasegawa, Masami

2013-01-01

Recent studies have reported discordant gene trees in the evolution of brown bears and polar bears. Genealogical histories are different among independent nuclear loci and between biparentally inherited autosomal DNA (aDNA) and matrilineal mitochondrial DNA (mtDNA). Based on multi-locus genomic sequences from aDNA and mtDNA, we inferred the population demography of brown and polar bears and found that brown bears have 6 times (aDNA) or more than 14 times (mtDNA) larger population sizes than polar bears and that polar bear lineage is derived from within brown bear diversity. In brown bears, the effective population size ratio of mtDNA to aDNA was at least 0.62, which deviated from the expected value of 0.25, suggesting matriarchal population due to female philopatry and male-biased migration. These results emphasize that ancestral polymorphisms and sex-biased migration may have contributed to conflicting branching patterns in brown and polar bears across aDNA genes and mtDNA.

Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids.

PubMed

Kerman, Kagan; Saito, Masato; Tamiya, Eiichi

2008-08-01

Here we report an electrochemical biosensor that would allow for simple and rapid analysis of nucleic acids in combination with nuclease activity on nucleic acids and electroactive bionanoparticles. The detection of single-nucleotide polymorphisms (SNPs) using PNA probes takes advantage of the significant structural and physicochemical differences between the full hybrids and SNPs in PNA/DNA and DNA/DNA duplexes. Ferrocene-conjugated chitosan nanoparticles (Chi-Fc) were used as the electroactive indicator of hybridization. Chi-Fc had no affinity towards the neutral PNA probe immobilized on a gold electrode (AuE) surface. When the PNA probe on the electrode surface hybridized with a full-complementary target DNA, Chi-Fc electrostatically attached to the negatively-charged phosphate backbone of DNA on the surface and gave rise to a high electrochemical oxidation signal from ferrocene at approximately 0.30 V. Exposing the surface to a single-stranded DNA specific nuclease, Nuclease S1, was found to be very effective for removing the nonspecifically adsorbed SNP DNA. An SNP in the target DNA to PNA made it susceptible to the enzymatic digestion. After the enzymatic digestion and subsequent exposure to Chi-Fc, the presence of SNPs was determined by monitoring the changes in the electrical current response of Chi-Fc. The method provided a detection limit of 1 fM (S/N = 3) for the target DNA oligonucleotide. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism (GMO) in standard Roundup Ready soybean samples. PNA-mediated PCR amplification of real DNA samples was performed to detect SNPs related to alcohol dehydrogenase (ALDH). Chitosan nanoparticles are promising biomaterials for various analytical and pharmaceutical applications.

Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

PubMed Central

Torriani, Sandra; Zapparoli, Giacomo; Dellaglio, Franco

1999-01-01

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412T, which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains. PMID:10508059

Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

PubMed

Torriani, S; Zapparoli, G; Dellaglio, F

1999-10-01

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.

Genetic diversity and classification of Tibetan yak populations based on the mtDNA COIII gene.

PubMed

Song, Q Q; Chai, Z X; Xin, J W; Zhao, S J; Ji, Q M; Zhang, C F; Ma, Z J; Zhong, J C

2015-03-13

To determine the level of genetic diversity and phylogenetic relationships among Tibetan yak populations, the mitochondrial DNA cytochrome c oxidase subunit 3 (COIII) genes of 378 yak individuals from 16 populations were analyzed in this study. The results showed that the length of cytochrome c oxidase subunit 3 gene sequences was 781 bp, with nucleotide frequencies of 29.2, 29.4, 26.1, and 15.2% for T, C, A, and G, respectively. A total of 26 haplotypes were identified, with 69 polymorphic sites, including 11 parsimony-informative sites and 58 single-nucleotide polymorphism sites. No deletions/insertions were found in sequence comparison, indicating that nucleotide mutation types were transitions and transversions. Haplotype and nucleotide diversities were 0.562 and 0.00138, respectively, indicating a high level of genetic diversity in Tibetan yak populations. Phylogenetic relationship analysis indicated that Tibetan yak populations are divided into 2 groups.

Frequency of distribution of inflammatory cytokines IL-1, IL-6 and TNF-alpha gene polymorphism in patients with obstructive sleep apnea.

PubMed

Popko, K; Gorska, E; Potapinska, O; Wasik, M; Stoklosa, A; Plywaczewski, R; Winiarska, M; Gorecka, D; Sliwinski, P; Popko, M; Szwed, T; Demkow, U

2008-12-01

Obesity is one of the most commonly identified factors for the obstructive sleep apnea syndrome (OSAS). Adipose tissue is the source of many cytokines, among them there are IL-6, IL-1, and TNF-alpha. The level of inflammatory cytokines increases in people with OSAS and obesity. The aim of this study was to evaluate the distribution of genotypes in inflammatory cytokine genes in people with obesity-related OSAS. The examined group consisted of 102 person with obesity related-OSAS and 77 normal weight person without OSAS. Genotyping of DNA sequence variation was carried out by restriction enzyme (IL-1: Taq I, IL-6: Lwe I, TNF-alpha: Nco I) analysis of PCR amplified DNA. The study revealed a significant correlation between polymorphism located in the promoter region of inflammatory cytokine genes and obesity-related OSAS.

Analysis of a p53 Mutation Associated with Cancer Susceptibility for Biochemistry and Genetic Laboratory Courses

ERIC Educational Resources Information Center

Soto-Cruz, Isabel; Legorreta-Herrera, Martha

2009-01-01

We have devised and implemented a module for an upper division undergraduate laboratory based on the amplification and analysis of a p53 polymorphism associated with cancer susceptibility. First, students collected a drop of peripheral blood cells using a sterile sting and then used FTA cards to extract the genomic DNA. The p53 region is then PCR…

Demonstration of Protein-Based Human Identification Using the Hair Shaft Proteome

PubMed Central

Leppert, Tami; Anex, Deon S.; Hilmer, Jonathan K.; Matsunami, Nori; Baird, Lisa; Stevens, Jeffery; Parsawar, Krishna; Durbin-Johnson, Blythe P.; Rocke, David M.; Nelson, Chad; Fairbanks, Daniel J.; Wilson, Andrew S.; Rice, Robert H.; Woodward, Scott R.; Bothner, Brian; Hart, Bradley R.; Leppert, Mark

2016-01-01

Human identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 single nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects’ DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European–American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). This study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts. PMID:27603779

Chloroplast DNA polymorphism and evolutional relationships between Asian cultivated rice (Oryza sativa) and its wild relatives (O. rufipogon).

PubMed

Li, W J; Zhang, B; Huang, G W; Kang, G P; Liang, M Z; Chen, L B

2012-12-17

We analyzed chloroplast DNA (cpDNA) polymorphism and phylogenic relationships between 6 typical indica rice, 4 japonica rice, 8 javanica rice, and 12 Asian common wild rice (Oryza rufipogon) strains collected from different latitudes in China by comparing polymorphism at 9 highly variable regions. One hundred and forty-four polymorphic bases were detected. The O. rufipogon samples had 117 polymorphic bases, showing rich genetic diversity. One hundred and thirty-one bases at 13 sites were identified with indica/japonica characteristics; they showed differences between the indica and japonica subspecies at these sites. The javanica strains and japonica shared similar bases at these 131 polymorphic sites, suggesting that javanica is closely related to japonica. On the basis of length analyses of the open reading frame (ORF)100 and (ORF)29-tRNA-Cys(GCA) (TrnC(GCA)) fragments, the O. rufipogon strains were classified into indica/japonica subgroups, which was consistent with the results of the phylogenic tree assay based on concatenated datasets. These results indicated that differences in indica and japonica also exist in the cpDNA genome of the O. rufipogon strains. However, these differences demonstrated a certain degree of primitiveness and incompleteness, as an O. rufipogon line may show different indica/ japonica attributes at different sites. Consequently, O. rufipogon cannot be simply classified into the indica/japonica types as O. sativa. Our data support the hypothesis that Asian cultivated rice, O. indica and O. japonica, separately evolved from Asian common wild rice (O. rufipogon) strains, which have different indica-japonica differentiation trends.

Database on natural polymorphisms and resistance-related non-synonymous mutations in thymidine kinase and DNA polymerase genes of herpes simplex virus types 1 and 2.

PubMed

Sauerbrei, Andreas; Bohn-Wippert, Kathrin; Kaspar, Marisa; Krumbholz, Andi; Karrasch, Matthias; Zell, Roland

2016-01-01

The use of genotypic resistance testing of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) is increasing because the rapid availability of results significantly improves the treatment of severe infections, especially in immunocompromised patients. However, an essential precondition is a broad knowledge of natural polymorphisms and resistance-associated mutations in the thymidine kinase (TK) and DNA polymerase (pol) genes, of which the DNA polymerase (Pol) enzyme is targeted by the highly effective antiviral drugs in clinical use. Thus, this review presents a database of all non-synonymous mutations of TK and DNA pol genes of HSV-1 and HSV-2 whose association with resistance or natural gene polymorphism has been clarified by phenotypic and/or functional assays. In addition, the laboratory methods for verifying natural polymorphisms or resistance mutations are summarized. This database can help considerably to facilitate the interpretation of genotypic resistance findings in clinical HSV-1 and HSV-2 strains. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Mitochondrial DNA polymorphisms associated with longevity in a Finnish population.

PubMed

Niemi, Anna-Kaisa; Hervonen, Antti; Hurme, Mikko; Karhunen, Pekka J; Jylhä, Marja; Majamaa, Kari

2003-01-01

Sequence variation in mitochondrial DNA (mtDNA) may cause slight differences both in the functioning of the respiratory chain and in free radical production, and an association between certain mtDNA haplogroups and longevity has been suggested. In order to determine further the role of mtDNA in longevity, we studied the frequencies of mtDNA haplogroups and haplogroup clusters among elderly subjects and controls in a Finnish population. Samples were obtained from 225 persons aged 90-91 years (Vitality 90+) and from 400 middle-aged controls and 257 infants. MtDNA haplogroups were determined by restriction fragment length polymorphism. The haplogroup frequencies of the Vitality 90+ group differed from both those of the middle-aged controls ( P=0.01) and the infants ( P=0.00005), haplogroup H being less frequent than among the middle-aged subjects ( P=0.001) and infants ( P=0.00001), whereas haplogroups U and J were more frequent. Haplogroup clusters also differed between Vitality 90+ and both the middle-aged subjects ( P=0.002) and infants ( P=0.00001), the frequency of haplogroup cluster HV being lower in the former and that of UK and WIX being higher. These data suggest an association between certain mtDNA haplogroups or haplogroup clusters and longevity. Furthermore, our data appear to favour the presence of advantageous polymorphisms and support a role for mitochondria and mtDNA in the degenerative processes involved in ageing.

XPD polymorphisms: effects on DNA repair proficiency.

PubMed

Lunn, R M; Helzlsouer, K J; Parshad, R; Umbach, D M; Harris, E L; Sanford, K K; Bell, D A

2000-04-01

XPD codes for a DNA helicase involved in transcription and nucleotide excision repair. Rare XPD mutations diminish nucleotide excision repair resulting in hypersensitivity to UV light and increased risk of skin cancer. Several polymorphisms in this gene have been identified but their impact on DNA repair is not known. We compared XPD genotypes at codons 312 and 751 with DNA repair proficiency in 31 women. XPD genotypes were measured by PCR-RFLP. DNA repair proficiency was assessed using a cytogenetic assay that detects X-ray induced chromatid aberrations (breaks and gaps). Chromatid aberrations were scored per 100 metaphase cells following incubation at 37 degrees C (1.5 h after irradiation) to allow for repair of DNA damage. Individuals with the Lys/Lys codon 751 XPD genotype had a higher number of chromatid aberrations (132/100 metaphase cells) than those having a 751Gln allele (34/100 metaphase cells). Individuals having greater than 60 chromatid breaks plus gaps were categorized as having sub-optimal repair. Possessing a Lys/Lys751 genotype increased the risk of sub-optimal DNA repair (odds ratio = 7.2, 95% confidence interval = 1.01-87.7). The Asp312Asn XPD polymorphism did not appear to affect DNA repair proficiency. These results suggest that the Lys751 (common) allele may alter the XPD protein product resulting in sub-optimal repair of X-ray-induced DNA damage.

Discovery of human inversion polymorphisms by comparative analysis of human and chimpanzee DNA sequence assemblies.

PubMed

Feuk, Lars; MacDonald, Jeffrey R; Tang, Terence; Carson, Andrew R; Li, Martin; Rao, Girish; Khaja, Razi; Scherer, Stephen W

2005-10-01

With a draft genome-sequence assembly for the chimpanzee available, it is now possible to perform genome-wide analyses to identify, at a submicroscopic level, structural rearrangements that have occurred between chimpanzees and humans. The goal of this study was to investigate chromosomal regions that are inverted between the chimpanzee and human genomes. Using the net alignments for the builds of the human and chimpanzee genome assemblies, we identified a total of 1,576 putative regions of inverted orientation, covering more than 154 mega-bases of DNA. The DNA segments are distributed throughout the genome and range from 23 base pairs to 62 mega-bases in length. For the 66 inversions more than 25 kilobases (kb) in length, 75% were flanked on one or both sides by (often unrelated) segmental duplications. Using PCR and fluorescence in situ hybridization we experimentally validated 23 of 27 (85%) semi-randomly chosen regions; the largest novel inversion confirmed was 4.3 mega-bases at human Chromosome 7p14. Gorilla was used as an out-group to assign ancestral status to the variants. All experimentally validated inversion regions were then assayed against a panel of human samples and three of the 23 (13%) regions were found to be polymorphic in the human genome. These polymorphic inversions include 730 kb (at 7p22), 13 kb (at 7q11), and 1 kb (at 16q24) fragments with a 5%, 30%, and 48% minor allele frequency, respectively. Our results suggest that inversions are an important source of variation in primate genome evolution. The finding of at least three novel inversion polymorphisms in humans indicates this type of structural variation may be a more common feature of our genome than previously realized.

High frequency of DD polymorphism of the angiotensin-converting enzyme gene in Turkish asthmatic patients.

PubMed

Urhan, Meral; Degirmenci, Irfan; Harmanci, Emel; Gunes, Hasan V; Metintas, Muzaffer; Basaran, Ayse

2004-01-01

The aim of this study is to detect the incidence of the angiotensin-converting enzyme (ACE) gene polymorphism in Turkish asthmatic patients and to examine whether there is an association between the disease and ACE gene polymorphism. In our study, the genomic DNA of 100 asthmatic patients and 88 healthy subjects was analyzed Genomic DNA was isolated from peripheral blood by using standard methods. The intron 16 of the ACE gene was amplified by the polymerase chain reaction (PCR) method using primers ACE and ACEX to examine the presence and absence of a 287-base pair (bp) DNA fragment that showed I/D polymorphism genotypes. PCR products were separated by agarose gel electrophoresis and were visualized by a charge-coupled device camera. Serum ACE activities were measured using an ACE kit. The results were evaluated statistically using the chi-square test and one-way analysis of variance. Although the population of patients with asthma was characterized by a higher frequency (30%) of the DD genotype of ACE, they were characterized by lower frequency (48%) of the ID genotype of ACE (DD, 16%, and ID, 64%, in healthy control subjects). The frequency of the I and D alleles of the ACE gene was not significantly different between asthmatic patients (0.46/0.54) and healthy controls (0.52/ 0.48). In addition, in both asthmatic patients and controls, there was a significant decrease of the levels of ACE activity in individuals that have II genotypes when compared with individuals that have DD genotypes. ACE activities were increased significantly in all asthmatic patients (67.20 +/- 1.95 IU/L) compared with all healthy controls (60.90 +/- 2.12 IU/L).

Genes tagging and molecular diversity of red rot susceptible/tolerant sugarcane hybrids using c-DNA and unigene derived markers.

PubMed

Singh, R K; Singh, R B; Singh, S P; Sharma, M L

2012-04-01

Sugarcane is an important international commodity as a valuable agricultural crop especially in tropical and subtropical countries. Two bulked DNA used to screen polymorphic primers from commercial hybrids (varieties) with moderately resistant and highly susceptible to red rot disease. Among 145 simple sequence repeat and unigene primers screened, 37 (25%) were found to be highly robust and polymorphic with Polymorphism Information Content values ranging from 0.50 to 1.00 with the mean value of 0.82. Among these microsatellites, twenty one were used in the study of genetic relationships and marker identification in sugarcane varieties for red rot resistance. A total of 105 polymorphic DNA bands were identified, with their fragment size ranging from 54 to 1,280 bp. Jaccard's similarity coefficient value recorded between closely related hybrids was 0.986 while lowest coefficient value of 0.341 was detected with distantly related hybrids. The average similarity coefficient among these hybrids was 0.663. Cluster analysis resulted in a dendrogram with two major clusters separating the moderately resistant varieties from highly susceptible varieties. Three group specific fragments amplified by unigene Saccharum microsatellite primers viz; two markers UGSM316(850) and UGSM316(60) were closely associated with moderately resistant varieties by appearing bands in this region but the bands were absent in highly susceptible varieties. Similarly UGSM316(400) marker was tightly linked with highly susceptible varieties by amplifying uniformly in sugarcane varieties showing highly susceptible reaction to red rot but it was absent in moderately resistant varietal groups. Validation of red rot resistance/susceptibility associated markers on a group of different mapping populations for red rot resistant/susceptible traits is in progress.

Genome-wide association study identifies phospholipase C zeta 1 (PLCz1) as a stallion fertility locus in Hanoverian warmblood horses.

PubMed

Schrimpf, Rahel; Dierks, Claudia; Martinsson, Gunilla; Sieme, Harald; Distl, Ottmar

2014-01-01

A consistently high level of stallion fertility plays an economically important role in modern horse breeding. We performed a genome-wide association study for estimated breeding values of the paternal component of the pregnancy rate per estrus cycle (EBV-PAT) in Hanoverian stallions. A total of 228 Hanoverian stallions were genotyped using the Equine SNP50 Beadchip. The most significant association was found on horse chromosome 6 for a single nucleotide polymorphism (SNP) within phospholipase C zeta 1 (PLCz1). In the close neighbourhood to PLCz1 is located CAPZA3 (capping protein (actin filament) muscle Z-line, alpha 3). The gene PLCz1 encodes a protein essential for spermatogenesis and oocyte activation through sperm induced Ca2+-oscillation during fertilization. We derived equine gene models for PLCz1 and CAPZA3 based on cDNA and genomic DNA sequences. The equine PLCz1 had four different transcripts of which two contained a premature termination codon. Sequencing all exons and their flanking sequences using genomic DNA samples from 19 Hanoverian stallions revealed 47 polymorphisms within PLCz1 and one SNP within CAPZA3. Validation of these 48 polymorphisms in 237 Hanoverian stallions identified three intronic SNPs within PLCz1 as significantly associated with EBV-PAT. Bioinformatic analysis suggested regulatory effects for these SNPs via transcription factor binding sites or microRNAs. In conclusion, non-coding polymorphisms within PLCz1 were identified as conferring stallion fertility and PLCz1 as candidate locus for male fertility in Hanoverian warmblood. CAPZA3 could be eliminated as candidate gene for fertility in Hanoverian stallions.

Genome-Wide Association Study Identifies Phospholipase C zeta 1 (PLCz1) as a Stallion Fertility Locus in Hanoverian Warmblood Horses

PubMed Central

Schrimpf, Rahel; Dierks, Claudia; Martinsson, Gunilla; Sieme, Harald; Distl, Ottmar

2014-01-01

A consistently high level of stallion fertility plays an economically important role in modern horse breeding. We performed a genome-wide association study for estimated breeding values of the paternal component of the pregnancy rate per estrus cycle (EBV-PAT) in Hanoverian stallions. A total of 228 Hanoverian stallions were genotyped using the Equine SNP50 Beadchip. The most significant association was found on horse chromosome 6 for a single nucleotide polymorphism (SNP) within phospholipase C zeta 1 (PLCz1). In the close neighbourhood to PLCz1 is located CAPZA3 (capping protein (actin filament) muscle Z-line, alpha 3). The gene PLCz1 encodes a protein essential for spermatogenesis and oocyte activation through sperm induced Ca2+-oscillation during fertilization. We derived equine gene models for PLCz1 and CAPZA3 based on cDNA and genomic DNA sequences. The equine PLCz1 had four different transcripts of which two contained a premature termination codon. Sequencing all exons and their flanking sequences using genomic DNA samples from 19 Hanoverian stallions revealed 47 polymorphisms within PLCz1 and one SNP within CAPZA3. Validation of these 48 polymorphisms in 237 Hanoverian stallions identified three intronic SNPs within PLCz1 as significantly associated with EBV-PAT. Bioinformatic analysis suggested regulatory effects for these SNPs via transcription factor binding sites or microRNAs. In conclusion, non-coding polymorphisms within PLCz1 were identified as conferring stallion fertility and PLCz1 as candidate locus for male fertility in Hanoverian warmblood. CAPZA3 could be eliminated as candidate gene for fertility in Hanoverian stallions. PMID:25354211

Screening of SHOX gene sequence variants in Saudi Arabian children with idiopathic short stature.

PubMed

Alharthi, Abdulla A; El-Hallous, Ehab I; Talaat, Iman M; Alghamdi, Hamed A; Almalki, Matar I; Gaber, Ahmed

2017-10-01

Short stature affects approximately 2%-3% of children, representing one of the most frequent disorders for which clinical attention is sought during childhood. Despite assumed genetic heterogeneity, mutations or deletions in the short stature homeobox-containing gene ( SHOX ) are frequently detected in subjects with short stature. Idiopathic short stature (ISS) refers to patients with short stature for various unknown reasons. The goal of this study was to screen all the exons of SHOX to identify related mutations. We screened all the exons of SHOX for mutations analysis in 105 ISS children patients (57 girls and 48 boys) living in Taif governorate, KSA using a direct DNA sequencing method. Height, arm span, and sitting height were recorded, and subischial leg length was calculated. A total of 30 of 105 ISS patients (28%) contained six polymorphic variants in exons 1, 2, 4, and 6. One mutation was found in the DNA domain binding region of exon 4. Three of these polymorphic variants were novel, while the others were reported previously. There were no significant differences in anthropometric measures in ISS patients with and without identifiable polymorphic variants in SHOX . In Saudi Arabia ISS patients, rather than SHOX , it is possible that new genes are involved in longitudinal growth. Additional molecular analysis is required to diagnose and understand the etiology of this disease.

Acute oral mucositis in nasopharyngeal carcinoma patients treated with radiotherapy: association with genetic polymorphism in DNA DSB repair genes.

PubMed

Ren, Jing-Hua; Dai, Xiao-Fang; Yan, Guo-Li; Jin, Min; Liu, Cui-Wei; Yang, Kun-Yu; Wu, Gang; Ma, C-M Charlie

2014-03-01

The aim of this study was to investigate the association between polymorphic variants of DNA repair genes with the susceptibility of acute oral mucositis (OM) in nasopharyngeal carcinoma (NPC) patients treated with radiotherapy. The study population consisted of 120 NPC patients treated with intensity-modulated radiation therapy (IMRT). Among them 70 patients also received concurrent chemotherapy. Genotypes in DNA repair genes Ku70 c.-1310C>G (rs2267437), Ku70 c.1781G> T (rs132788), Ku80 c.2099-2408G> A (rs3835), Ku80 c.*841G> A (rs2440) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) c.2888 + 713C> T (rs2213178) were determined by polymerase chain reaction combined with the restriction fragment length polymorphism (PCR-RFLP) technique. Mucositis was scored using the Common Terminology Criteria (CTC) for Adverse Events v.3.0 scale. The population was divided into the CTC0-2 group (CTC toxicity grade 0, 1 and 2) and the CTC3 + group (CTC toxicity grade 3 and above). Odd ratios (OR) and 95% confidence intervals (CI) were calculated using the multivariate logistic regression analysis. A significant difference in Ku70 c.1781G> T genotype distribution was observed between the CTC0-2 and CTC3 + groups for the 120 patients analyzed. The GG carriers were at higher risks for severe OM (CTC3+) compared with the TT homozygotes (OR = 3.000, 95% CI = 1.287-6.994, p = 0.011). No association was found between Ku70 (c.-1310C> G), Ku80 (c.2099-2408G> A, c.*841G> A), DNA-PKcs (c.2888 + 713 C > T) and the development of severe oral mucositis. Stratification analyses for the 50 patients treated with radiation alone further confirmed the association between the variant genotype of GG and severe OM (OR = 5.128, 95% CI = 1.183-22.238, p = 0.029). Concurrent radiochemotherapy increased the risk of severe OM for both the TT homozygotes and GG genotypes. Our study suggests that the Ku70 c.1781G> T polymorphism may be a susceptibility factor for radiation-induced oral mucositis in Chinese nasopharyngeal carcinoma patients.

Genetic polymorphisms in prehistoric Pacific islanders determined by analysis of ancient bone DNA.

PubMed

Hagelberg, E; Clegg, J B

1993-05-22

A previously characterized Asian-specific mitochondrial DNA (mtDNA) length mutation has been detected in DNA isolated from prehistoric human bones from Polynesia, including Hawaii, Chatham Islands and Society Islands. In contrast, the Asian mutation was absent in skeletal samples from the Melanesian archipelagos of New Britain and Vanuatu and in the oldest samples from Fiji, Tonga and Samoa in the central Pacific (2700-1600 years BP) although it was present in a more recent prehistoric sample from Tonga. These results, augmented by informative DNA sequence data from the hypervariable region of mtDNA, fail to support current views that the central Pacific was settled directly by voyagers from island Southeast Asia, the putative ancestors of modern Polynesians. An earlier occupation by peoples from the neighbouring Melanesian archipelagos seems more likely.

Fingerprinting of HLA class I genes for improved selection of unrelated bone marrow donors.

PubMed

Martinelli, G; Farabegoli, P; Buzzi, M; Panzica, G; Zaccaria, A; Bandini, G; Calori, E; Testoni, N; Rosti, G; Conte, R; Remiddi, C; Salvucci, M; De Vivo, A; Tura, S

1996-02-01

The degree of matching of HLA genes between the selected donor and recipient is an important aspect of the selection of unrelated donors for allogeneic bone marrow transplantation (UBMT). The most sensitive methods currently used are serological typing of HLA class I genes, mixed lymphocyte culture (MLC), IEF and molecular genotyping of HLA class II genes by direct sequencing of PCR products. Serological typing of class I antigenes (A, B and C) fails to detect minor differences demonstrated by direct sequencing of DNA polymorphic regions. Molecular genotyping of HLA class I genes by DNA analysis is costly and work-intensive. To improve compatibility between donor and recipient, we have set up a new rapid and non-radioisotopic application of the 'fingerprinting PCR' technique for the analysis of the polymorphic second exon of the HLA class I A, B and C genes. This technique is based on the formation of specific patterns (PCR fingerprints) of homoduplexes and heteroduplexes between heterologous amplified DNA sequences. After an electrophoretic run on non-denaturing polyacrylamide gel, different HLA class I types give allele-specific banding patterns. HLA class I matching is performed, after the gel has been soaked in ethidium bromide or silver-stained, by visual comparison of patients' fingerprints with those of donors. Identity can be confirmed by mixing donor and recipient DNAs in an amplification cross-match. To assess the technique, 10 normal samples, 22 related allogeneic bone marrow transplanted pairs and 10 unrelated HLA-A and HLA-B serologically matched patient-donor pairs were analysed for HLA class I polymorphic regions. In all the related pairs and in 1/10 unrelated pairs, matched donor-recipient patterns were identified. This new application of PCR fingerprinting may confirm the HLA class I serological selection of unrelated marrow donors.

Analysis of plastome and chondriome genome types in potato somatic hybrids from Solanum tuberosum × Solanum etuberosum.

PubMed

Tiwari, Jagesh K; Chandel, Poonam; Singh, Bir Pal; Bhardwaj, Vinay

2014-01-01

Cytoplasm types of the potato somatic hybrids from Solanum tuberosum × Solanum etuberosum were analysed using chloroplast (cp) and mitochondrial (mt) organelle genomes-specific markers. Of the 29 markers (15 cpDNA and 14 mtDNA) amplified in the 26 genotypes, 5 cpDNA (H3, NTCP4, NTCP8, NTCP9, and ALC1/ALC3) and 13 mtDNA markers showed polymorphism. The cluster analysis based on the mtDNA markers detected higher diversity compared with the cpDNA markers. Presence of new mtDNA fragments of the markers, namely, T11-2, Nsm1, pumD, Nsm3, and Nsm4, were observed, while monomorphic loci revealed highly conserved genomic regions in the somatic hybrids. The study revealed that the somatic hybrids had diverse cytoplasm types consisting predominantly of T-, W-, and C-, with a few A- and S-type cp genomes; and α-, β-, and γ-type mt genomes. Somatic hybridization has unique potential to widen the cytoplasm types of the cultivated gene pools from wild species through introgression by breeding methods.

Study on DNA Damage Induced by Neon Beam Irradiation in Saccharomyces Cerevisiae

NASA Astrophysics Data System (ADS)

Lu, Dong; Li, Wenjian; Wu, Xin; Wang, Jufang; Ma, Shuang; Liu, Qingfang; He, Jinyu; Jing, Xigang; Ding, Nan; Dai, Zhongying; Zhou, Jianping

2010-12-01

Yeast strain Saccharomyces cerevisiae was irradiated with different doses of 85 MeV/u 20Ne10+ to investigate DNA damage induced by heavy ion beam in eukaryotic microorganism. The survival rate, DNA double strand breaks (DSBs) and DNA polymorphic were tested after irradiation. The results showed that there were substantial differences in DNA between the control and irradiated samples. At the dose of 40 Gy, the yeast cell survival rate approached 50%, DNA double-strand breaks were barely detectable, and significant DNA polymorphism was observed. The alcohol dehydrogenase II gene was amplified and sequenced. It was observed that base changes in the mutant were mainly transversions of T→G and T→C. It can be concluded that heavy ion beam irradiation can lead to change in single gene and may be an effective way to induce mutation.

Genetic structure in contemporary south Tyrolean isolated populations revealed by analysis of Y-chromosome, mtDNA, and Alu polymorphisms.

PubMed

Pichler, Irene; Mueller, Jakob C; Stefanov, Stefan A; De Grandi, Alessandro; Volpato, Claudia Beu; Pinggera, Gerd K; Mayr, Agnes; Ogriseg, Martin; Ploner, Franz; Meitinger, Thomas; Pramstaller, Peter P

2006-08-01

Most of the inhabitants of South Tyrol in the eastern Italian Alps can be considered isolated populations because of their physical separation by mountain barriers and their sociocultural heritage. We analyzed the genetic structure of South Tyrolean populations using three types of genetic markers: Y-chromosome, mitochondrial DNA (mtDNA), and autosomal Alu markers. Using random samples taken from the populations of Val Venosta, Val Pusteria, Val Isarco, Val Badia, and Val Gardena, we calculated genetic diversity within and among the populations. Microsatellite diversity and unique event polymorphism diversity (on the Y chromosome) were substantially lower in the Ladin-speaking population of Val Badia compared to the neighboring German-speaking populations. In contrast, the genetic diversity of mtDNA haplotypes was lowest for the upper Val Venosta and Val Pusteria. These data suggest a low effective population size, or little admixture, for the gene pool of the Ladin-speaking population from Val Badia. Interestingly, this is more pronounced for Ladin males than for Ladin females. For the pattern of genetic Alu variation, both Ladin samples (Val Gardena and Val Badia) are among the samples with the lowest diversity. An admixture analysis of one German-speaking valley (Val Venosta) indicates a relatively high genetic contribution of Ladin origin. The reduced genetic diversity and a high genetic differentiation in the Rhaetoroman- and German-speaking South Tyrolean populations may constitute an important basis for future medical genetic research and gene mapping studies in South Tyrol.

Rapid genetic and epigenetic alterations under intergeneric genomic shock in newly synthesized Chrysanthemum morifolium x Leucanthemum paludosum hybrids (Asteraceae).

PubMed

Wang, Haibin; Jiang, Jiafu; Chen, Sumei; Qi, Xiangyu; Fang, Weimin; Guan, Zhiyong; Teng, Nianjun; Liao, Yuan; Chen, Fadi

2014-01-01

The Asteraceae family is at the forefront of the evolution due to frequent hybridization. Hybridization is associated with the induction of widespread genetic and epigenetic changes and has played an important role in the evolution of many plant taxa. We attempted the intergeneric cross Chrysanthemum morifolium × Leucanthemum paludosum. To obtain the success in cross, we have to turn to ovule rescue. DNA profiling of the amphihaploid and amphidiploid was investigated using amplified fragment length polymorphism, sequence-related amplified polymorphism, start codon targeted polymorphism, and methylation-sensitive amplification polymorphism (MSAP). Hybridization induced rapid changes at the genetic and the epigenetic levels. The genetic changes mainly involved loss of parental fragments and gaining of novel fragments, and some eliminated sequences possibly from the noncoding region of L. paludosum. The MSAP analysis indicated that the level of DNA methylation was lower in the amphiploid (∼45%) than in the parental lines (51.5-50.6%), whereas it increased after amphidiploid formation. Events associated with intergeneric genomic shock were a feature of C. morifolium × L. paludosum hybrid, given that the genetic relationship between the parental species is relatively distant. Our results provide genetic and epigenetic evidence for understanding genomic shock in wide crosses between species in Asteraceae and suggest a need to expand our current evolutionary framework to encompass a genetic/epigenetic dimension when seeking to understand wide crosses.

Simple Sequence Repeats in Escherichia coli: Abundance, Distribution, Composition, and Polymorphism

PubMed Central

Gur-Arie, Riva; Cohen, Cyril J.; Eitan, Yuval; Shelef, Leora; Hallerman, Eric M.; Kashi, Yechezkel

2000-01-01

Computer-based genome-wide screening of the DNA sequence of Escherichia coli strain K12 revealed tens of thousands of tandem simple sequence repeat (SSR) tracts, with motifs ranging from 1 to 6 nucleotides. SSRs were well distributed throughout the genome. Mononucleotide SSRs were over-represented in noncoding regions and under-represented in open reading frames (ORFs). Nucleotide composition of mono- and dinucleotide SSRs, both in ORFs and in noncoding regions, differed from that of the genomic region in which they occurred, with 93% of all mononucleotide SSRs proving to be of A or T. Computer-based analysis of the fine position of every SSR locus in the noncoding portion of the genome relative to downstream ORFs showed SSRs located in areas that could affect gene regulation. DNA sequences at 14 arbitrarily chosen SSR tracts were compared among E. coli strains. Polymorphisms of SSR copy number were observed at four of seven mononucleotide SSR tracts screened, with all polymorphisms occurring in noncoding regions. SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identification, and detection of loci affecting key phenotypes) and for evolutionary adaptation of microbes.[The sequence data described in this paper have been submitted to the GenBank data library under accession numbers AF209020–209030 and AF209508–209518.] PMID:10645951

Multiple correspondence analysis and random amplified polymorphic DNA molecular typing to assess the sources of Staphylococcus aureus contamination in alheira production lines.

PubMed

Esteves, A; Patarata, L; Aymerich, T; Garriga, M; Martins, C

2007-03-01

Sources and tracing of Staphylococcus aureus in alheira (garlic sausage) production were evaluated by multifactorial correspondence analysis (MCA) of occurrence data and a random amplified polymorphic DNA (RAPD) on S. aureus isolates. Samples from four production lines, four different production batches, and 14 different sampling sites (including raw material, different contact surfaces, and several stages of alheira manufacturing) were analyzed at four sampling times. From the 896 microbial analyses completed, a collection of 170 S. aureus isolates was obtained. Although analysis of the occurrence data alone was not elucidative enough, MCA and RAPD-PCR were able to assess the sources of contamination and to trace the spread of this microorganism along the production lines. MCA results indicated that the presence of S. aureus in alheira was related to its presence in the intermediate manufacturing stages after heat treatment but before stuffing in the casings. It was also possible to associate a cross-contamination path related to handler procedures. RAPD-PCR typing in accordance to MCA results confirmed the cross-contamination path between the raw material and casings and the role of handlers as an important cross-contamination vehicle.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, inter-serovar convergence, and evidence of attenuation in Leptospira reference collections.

PubMed

Tulsiani, S M; Craig, S B; Graham, G C; Cobbold, R C; Dohnt, M F; Burns, M-A; Jansen, C C; Leung, L K-P; Field, H E; Smythe, L D

2010-07-01

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD-HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD-HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD-HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD-HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.

[The estimation of possibilities for the application of the laser capture microdissection technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA].

PubMed

Ivanov, P L; Leonov, S N; Zemskova, E Iu

2012-01-01

The present study was designed to estimate the possibilities of application of the laser capture microdissection (LCM) technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA. The experimental method employed for the purpose was the multiplex multilocus analysis of autosomal DNA polymorphism in the preparations of buccal epitheliocytes obtained by LCM. The key principles of the study were the application of physical methods for contrast enhancement of the micropreparations (such as phase-contrast microscopy and dark-field microscopy) and PCR-compatible cell lysis. Genotyping was carried out with the use of AmpFISTR Minifiler TM PCR Amplification Kits ("Applied Biosynthesis", USA). It was shown that the technique employed in the present study ensures reliable genotyping of human chromosomal DNA in the pooled preparations containing 10-20 dissected diploid cells each. This result fairly well agrees with the calculated sensitivity of the method. A few practical recommendations are offered.

DNA origami-based shape IDs for single-molecule nanomechanical genotyping

NASA Astrophysics Data System (ADS)

Zhang, Honglu; Chao, Jie; Pan, Dun; Liu, Huajie; Qiang, Yu; Liu, Ke; Cui, Chengjun; Chen, Jianhua; Huang, Qing; Hu, Jun; Wang, Lianhui; Huang, Wei; Shi, Yongyong; Fan, Chunhai

2017-04-01

Variations on DNA sequences profoundly affect how we develop diseases and respond to pathogens and drugs. Atomic force microscopy (AFM) provides a nanomechanical imaging approach for genetic analysis with nanometre resolution. However, unlike fluorescence imaging that has wavelength-specific fluorophores, the lack of shape-specific labels largely hampers widespread applications of AFM imaging. Here we report the development of a set of differentially shaped, highly hybridizable self-assembled DNA origami nanostructures serving as shape IDs for magnified nanomechanical imaging of single-nucleotide polymorphisms. Using these origami shape IDs, we directly genotype single molecules of human genomic DNA with an ultrahigh resolution of ~10 nm and the multiplexing ability. Further, we determine three types of disease-associated, long-range haplotypes in samples from the Han Chinese population. Single-molecule analysis allows robust haplotyping even for samples with low labelling efficiency. We expect this generic shape ID-based nanomechanical approach to hold great potential in genetic analysis at the single-molecule level.

DNA origami-based shape IDs for single-molecule nanomechanical genotyping

PubMed Central

Zhang, Honglu; Chao, Jie; Pan, Dun; Liu, Huajie; Qiang, Yu; Liu, Ke; Cui, Chengjun; Chen, Jianhua; Huang, Qing; Hu, Jun; Wang, Lianhui; Huang, Wei; Shi, Yongyong; Fan, Chunhai

2017-01-01

Variations on DNA sequences profoundly affect how we develop diseases and respond to pathogens and drugs. Atomic force microscopy (AFM) provides a nanomechanical imaging approach for genetic analysis with nanometre resolution. However, unlike fluorescence imaging that has wavelength-specific fluorophores, the lack of shape-specific labels largely hampers widespread applications of AFM imaging. Here we report the development of a set of differentially shaped, highly hybridizable self-assembled DNA origami nanostructures serving as shape IDs for magnified nanomechanical imaging of single-nucleotide polymorphisms. Using these origami shape IDs, we directly genotype single molecules of human genomic DNA with an ultrahigh resolution of ∼10 nm and the multiplexing ability. Further, we determine three types of disease-associated, long-range haplotypes in samples from the Han Chinese population. Single-molecule analysis allows robust haplotyping even for samples with low labelling efficiency. We expect this generic shape ID-based nanomechanical approach to hold great potential in genetic analysis at the single-molecule level. PMID:28382928

[The joint applications of DNA chips and single nucleotide polymorphisms in forensic science].

PubMed

Bai, Peng; Tian, Li; Zhou, Xue-ping

2005-05-01

DNA chip technology, being a new high-technology, shows its vigorous life and rapid growth. Single Nucleotide Polymorphisms (SNPs) is the most common diversity in the human genome. It provides suitable genetic markers which play a key role in disease linkage study, pharmacogenomics, forensic medicine, population evolution and immigration study. Their advantage such as being analyzed with DNA chips technology, is predicted to play an important role in the field of forensic medicine, especially in paternity test and individual identification. This report mainly reviews the characteristics of DNA chip and SNPs, and their joint applications in the practice of forensic medicine.

Detection of DNA "fingerprints" of cultivated rice by hybridization with a human minisatellite DNA probe.

PubMed

Dallas, J F

1988-09-01

A human minisatellite DNA probe detects several restriction fragment length polymorphisms in cultivars of Asian and African rice. Certain fragments appear to be inherited in a Mendelian fashion and may represent unlinked loci. The hybridization patterns appear to be cultivar-specific and largely unchanged after the regeneration of plants from tissue culture. The results suggest that these regions of the rice genome may be used to generate cultivar-specific DNA fingerprints. The demonstration of similarity between a human minisatellite sequence and polymorphic regions in the rice genome suggests that such regions also occur in the genomes of many other plant species.

Using Harry Potter to Introduce Students to DNA Fingerprinting & Forensic Science

ERIC Educational Resources Information Center

Palmer, Laura K.

2010-01-01

This lesson uses characters from the Harry Potter series of novels as a "hook" to stimulate students' interest in introductory forensic science. Students are guided through RFLP (restriction fragment length polymorphism) analysis using inexpensive materials and asked to interpret data from a mock crime scene. Importantly, the lesson provides an…

PRELIMINARY STUDIES ON THE POPULATION GENETICS OF THE CENTRAL STONEROLLER (CAMPOSTOMA ANOMALUM) FROM THE GREAT MIAMI RIVER BASIN, OHIO

EPA Science Inventory

Molecular approaches are particularly useful for measuring genetic diversity and were applied to samples of central stonerollers obtained from sites along tributaries to the Great Miami River in Ohio. We used Random Amplified Polymorphic DNA (RAPD) analysis to assess the level of...

PRELIMINARY STUDIES ON THE POPULATION GENETICS OF THE CENTRAL STONEROLLER (COMPOSTOMA ANOMALUM) FROM THE GREAT MIAMI RIVER BASIN, OHIO

EPA Science Inventory

Molecular approaches are particularly useful for measuring genetic diversity and were applied to samples of central stonerollers obtained from sites along tributaries to the Great Miami River in Ohio. We used Random Amplified Polymorphic DNA (RAPD) analysis to assess the level o...

A Laboratory Exercise to Determine Human ABO Blood Type by Noninvasive Methods

ERIC Educational Resources Information Center

Martin, Michael P.; Detzel, Stephen M.

2008-01-01

Analysis of single-nucleotide polymorphisms and their association with diseases and nondisease phenotypes is of growing importance in human biology studies. In this laboratory exercise, students determine the genetic basis for their ABO blood type; however, no blood is drawn. Students isolate genomic DNA from buccal mucosa cells that are present…

Identification of cultivars and validation of genetic relationships in Mangifera indica L. using RAPD markers.

PubMed

Schnell, R J; Ronning, C M; Knight, R J

1995-02-01

Twenty-five accessions of mango were examined for random amplified polymorphic DNA (RAPD) genetic markers with 80 10-mer random primers. Of the 80 primers screened, 33 did not amplify, 19 were monomorphic, and 28 gave reproducible, polymorphic DNA amplification patterns. Eleven primers were selected from the 28 for the study. The number of bands generated was primer- and genotype-dependent, and ranged from 1 to 10. No primer gave unique banding patterns for each of the 25 accessions; however, ten different combinations of 2 primer banding patterns produced unique fingerprints for each accession. A maternal half-sib (MHS) family was included among the 25 accessions to see if genetic relationships could be detected. RAPD data were used to generate simple matching coefficients, which were analyzed phenetically and by means of principal coordinate analysis (PCA). The MHS clustered together in both the phenetic and the PCA while the randomly selected accessions were scattered with no apparent pattern. The uses of RAPD analysis for Mangifera germ plasm classification and clonal identification are discussed.

Extensive 5.8S nrDNA polymorphism in Mammillaria (Cactaceae) with special reference to the identification of pseudogenic internal transcribed spacer regions.

PubMed

Harpke, Doerte; Peterson, Angela

2008-05-01

The internal transcribed spacer (ITS) region (ITS1, 5.8S rDNA, ITS2) represents the most widely applied nuclear marker in eukaryotic phylogenetics. Although this region has been assumed to evolve in concert, the number of investigations revealing high degrees of intra-individual polymorphism connected with the presence of pseudogenes has risen. The 5.8S rDNA is the most important diagnostic marker for functionality of the ITS region. In Mammillaria, intra-individual 5.8S rDNA polymorphisms of up to 36% and up to nine different types have been found. Twenty-eight of 30 cloned genomic Mammillaria sequences were identified as putative pseudogenes. For the identification of pseudogenic ITS regions, in addition to formal tests based on substitution rates, we attempted to focus on functional features of the 5.8S rDNA (5.8S motif, secondary structure). The importance of functional data for the identification of pseudogenes is outlined and discussed. The identification of pseudogenes is essential, because they may cause erroneous phylogenies and taxonomic problems.

Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma.

PubMed

Ho, Sherry Sze Yee; Barrett, Angela; Thadani, Henna; Asibal, Cecille Laureano; Koay, Evelyn Siew-Chuan; Choolani, Mahesh

2015-07-01

Prenatal diagnosis of sex-linked disorders requires invasive procedures, carrying a risk of miscarriage of up to 1%. Cell-free fetal DNA (cffDNA) present in cell-free DNA (cfDNA) from maternal plasma offers a non-invasive source of fetal genetic material for analysis. Detection of Y-chromosome sequences in cfDNA indicates presence of a male fetus; in the absence of a Y-chromosome signal a female fetus is inferred. We aimed to validate the clinical utility of insertion-deletion polymorphisms (INDELs) to confirm presence of a female fetus using cffDNA. Quantitative real-time PCR (qPCR) for the Y-chromosome-specific sequence, SRY, was performed on cfDNA from 82 samples at 6-39 gestational weeks. In samples without detectable SRY, qPCRs for eight INDELs were performed on maternal genomic DNA and cfDNA. Detection of paternally inherited fetal alleles in cfDNA negative for SRY confirmed a female fetus. Fetal sex was correctly determined in 77/82 (93.9%) cfDNA samples. SRY was detected in all 39 samples from male-bearing pregnancies, and none of the 43 female-bearing pregnancies (sensitivity and specificity of SRY qPCR is therefore 100%; 95% CI 91%-100%). Paternally inherited fetal alleles were detected in 38/43 samples with no SRY signal, confirming the presence of a female fetus (INDEL assay sensitivity is therefore 88.4%; 95% CI 74.1%-95.6%). Since paternally inherited fetal INDELs were not used in women bearing male fetuses, the specificity of INDELs cannot be calculated. Five cfDNA samples were negative for both SRY and INDELS. We have validated a non-invasive prenatal test to confirm fetal sex as early as 6 gestational weeks using cffDNA from maternal plasma.

Restriction fragment length polymorphism within the class I gene loci of the equine major histocompatibility complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alexander, A.J.; Bailey, E.; Woodward, J.G.

1986-03-05

Fourteen standard bred horses were serotyped as homozygous for 1 of 6 Equine Leukocyte Antigen (ELA) specificities. DNA was purified from peripheral leukocytes and digested with Hind III or Pvu II. Southern blot hybridization analysis was carried out using a /sup 32/P-labeled mouse cDNA probe (PH2IIa) specific for class I MHC genes. Both enzymes generated blots that contained a large number of bands (23 to 30) per horse. Significant polymorphism existed among most fragment sizes, while a dozen highly conserved band sizes suggested the presence of Qa/tla - like genes. Only 2 animals (both W6's) showed identical band patterns. Polymorphismmore » was greatest between horses of different serotypes and was significantly decreased within serotypes. Unique bands were present on both blots for both W1's and W6's and may account for the serologic specificity seen in ELA W1 and W6 horses. This study is consistent with the findings in other higher vertebrates and implies that the MHC of the horse includes a highly polymorphic class I multigene family.« less

Mismatch cleavage by single-strand specific nucleases

PubMed Central

Till, Bradley J.; Burtner, Chris; Comai, Luca; Henikoff, Steven

2004-01-01

We have investigated the ability of single-strand specific (sss) nucleases from different sources to cleave single base pair mismatches in heteroduplex DNA templates used for mutation and single-nucleotide polymorphism analysis. The TILLING (Targeting Induced Local Lesions IN Genomes) mismatch cleavage protocol was used with the LI-COR gel detection system to assay cleavage of amplified heteroduplexes derived from a variety of induced mutations and naturally occurring polymorphisms. We found that purified nucleases derived from celery (CEL I), mung bean sprouts and Aspergillus (S1) were able to specifically cleave nearly all single base pair mismatches tested. Optimal nicking of heteroduplexes for mismatch detection was achieved using higher pH, temperature and divalent cation conditions than are routinely used for digestion of single-stranded DNA. Surprisingly, crude plant extracts performed as well as the highly purified preparations for this application. These observations suggest that diverse members of the S1 family of sss nucleases act similarly in cleaving non-specifically at bulges in heteroduplexes, and single-base mismatches are the least accessible because they present the smallest single-stranded region for enzyme binding. We conclude that a variety of sss nucleases and extracts can be effectively used for high-throughput mutation and polymorphism discovery. PMID:15141034

Identification of three randomly amplified polymorphic DNA-polymerase chain reaction markers for distinguishing Asian and North American Gypsy Moths (Lepidoptera: Lymantriidae)

Treesearch

David E. Schreiber; Karen J. Garner; James M. Slavicek

1997-01-01

Gypsy moths originating in Asia have recently been introduced into North America, making it necessary to develop markers for distinguishing the Asian strain from the established North American population. We have identified 3 randomly amplified polymorphic DNA-polymerase chain reaction generated (RAPD-PCR) markers which are specific for either Asian or North American...

Prevalence of K13-propeller gene polymorphisms among Plasmodium falciparum parasites isolated from adult symptomatic patients in northern Uganda.

PubMed

Ocan, Moses; Bwanga, Freddie; Okeng, Alfred; Katabazi, Fred; Kigozi, Edgar; Kyobe, Samuel; Ogwal-Okeng, Jasper; Obua, Celestino

2016-08-19

In the absence of an effective vaccine, malaria treatment and eradication is still a challenge in most endemic areas globally. This is especially the case with the current reported emergence of resistance to artemisinin agents in Southeast Asia. This study therefore explored the prevalence of K13-propeller gene polymorphisms among Plasmodium falciparum parasites in northern Uganda. Adult patients (≥18 years) presenting to out-patients department of Lira and Gulu regional referral hospitals in northern Uganda were randomly recruited. Laboratory investigation for presence of plasmodium infection among patients was done using Plasmodium falciparum exclusive rapid diagnostic test, histidine rich protein-2 (HRP2) (Pf). Finger prick capillary blood from patients with a positive malaria test was spotted on a filter paper Whatman no. 903. The parasite DNA was extracted using chelex resin method and sequenced for mutations in K13-propeller gene using Sanger sequencing. PCR DNA sequence products were analyzed using in DNAsp 5.10.01software, data was further processed in Excel spreadsheet 2007. A total of 60 parasite DNA samples were sequenced. Polymorphisms in the K13-propeller gene were detected in four (4) of the 60 parasite DNA samples sequenced. A non-synonymous polymorphism at codon 533 previously detected in Cambodia was found in the parasite DNA samples analyzed. Polymorphisms at codon 522 (non-synonymous) and codon 509 (synonymous) were also found in the samples analyzed. The study found evidence of positive selection in the Plasmodium falciparum population in northern Uganda (Tajima's D = -1.83205; Fu and Li's D = -1.82458). Polymorphism in the K13-propeller gene previously reported in Cambodia has been found in the Ugandan Plasmodium falciparum parasites. There is need for continuous surveillance for artemisinin resistance gene markers in the country.

Genotyping of Plant and Animal Samples without Prior DNA Purification

PubMed Central

Chum, Pak Y.; Haimes, Josh D.; André, Chas P.; Kuusisto, Pia K.; Kelley, Melissa L.

2012-01-01

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors. PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size. PMID:23051689

Gender-related survival differences associated with EGFR polymorphisms in metastatic colon cancer.

PubMed

Press, Oliver A; Zhang, Wu; Gordon, Michael A; Yang, Dongyun; Lurje, Georg; Iqbal, Syma; El-Khoueiry, Anthony; Lenz, Heinz-Josef

2008-04-15

Evidence is accumulating supporting gender-related differences in the development of colonic carcinomas. Sex steroid hormone receptors are expressed in the colon and interact with epidermal growth factor receptor (EGFR), a gene widely expressed in colonic tissue. Increased EGFR expression is linked with poor prognosis in colon cancer. Within the EGFR gene there are two functional polymorphisms of interest: a polymorphism located at codon 497 (HER-1 R497K) and a dinucleotide (CA)(n) repeat polymorphism located within intron 1. These germ-line polymorphisms of EGFR were analyzed in genomic DNA from 318 metastatic colon cancer patients, 177 males and 141 females, collected from 1992 to 2003. Gender-related survival differences were associated with the HER-1 R497K polymorphism (P(interaction) = 0.003). Females with the HER-1 497 Arg/Arg variant had better overall survival (OS) when compared with the Lys/Lys and/or Lys/Arg variants. In males the opposite was true. The EGFR dinucleotide (CA)(n) repeat also trended with a gender-related OS difference (P(interaction) = 0.11). Females with both short <20 (CA)(n) repeat alleles had better OS than those with any long >or=20 (CA)(n) repeats. In males the opposite was true. Combination analysis of the two polymorphisms taken together also revealed the same gender-related survival difference (P(interaction) = 0.002). These associations were observed using multivariable analysis. The two polymorphisms were not in linkage disequilibrium and are independent of one another. This study supports the role of functional EGFR polymorphisms as independent prognostic markers in metastatic colon cancer. As a prognostic factor, these variants had opposite prognostic implications based on gender.

Association of UGT2B7 and UGT1A4 Polymorphisms with Serum Concentration of Antiepileptic Drugs in Children.

PubMed

Du, Zhongliang; Jiao, Yukun; Shi, Lianting

2016-10-31

BACKGROUND This study aimed to analyze the relationship of UGT2B7 and UGT1A4 polymorphisms with metabolism of valproic acid (VPA) and lamotrigine (LTG) in epileptic children. MATERIAL AND METHODS We administered VPA (102) and LTG (102) to 204 children with epilepsy. Blood samples were collected before the morning dose. Serum concentration of LTG was measured by high-performance liquid chromatography (HPLC). Serum VPA concentration was tested by fluorescence polarization immunoassay. UGT2B7 A268G, C802T, and G211T polymorphisms, as well as UGT1A4 L48V polymorphism, were assayed by direct automated DNA sequencing after PCR. Evaluation of efficacy was conducted using the Engel method. RESULTS The adjusted serum concentration of VPA was 4.26 μg/mL per mg/kg and LTG was 1.56 μg/mL per mg/kg. Multiple linear regression analysis revealed that VPA or LTG adjusted concentration showed a good linear relation with sex and age. UGT2B7 A268G and C802T polymorphisms were demonstrated to affect the serum concentration of VPA (F=3.147, P=0.047; F=22.754, P=0.000). UGT1A4 L48V polymorphism was not related with the serum concentration of LTG (F=5.328, P=0.006). In the efficacy analysis, we found that C802T polymorphism exerted strong effects on efficacy of VPA (χ²=9.265, P=0.010). L48V polymorphism also showed effects on efficacy of LTG (χ²=17.397, P=0.001). CONCLUSIONS UGT2B7, UGT1A4 polymorphisms play crucial roles in metabolism of VPA and LTG.

VDR polymorphisms are associated with bone mineral density in post-menopausal Mayan-Mestizo women.

PubMed

Canto-Cetina, Thelma; Cetina Manzanilla, José Antonio; González Herrera, Lizbeth; Rojano-Mejía, David; Coral-Vázquez, Ramón Mauricio; Coronel, Agustín; Canto, Patricia

2015-01-01

Osteoporosis is characterized by low bone mineral density (BMD), which is determined by an interaction of genetic, metabolic and environmental factors. To analyse the association between two polymorphisms of VDR as well as their haplotypes with BMD in post-menopausal Maya-Mestizo women. This study comprised 600 post-menopausal Maya-Mestizo women. A structured questionnaire for risk factors was applied and BMD was assessed at the lumbar spine (LS) and total hip (TH) by dual-energy X-ray absorptiometry. DNA was extracted from blood leukocytes. Two single-nucleotide polymorphisms of VDR (rs731236 and rs2228570) were studied using real-time PCR allelic discrimination for genotyping. Differences between the means of the BMDs according to the genotype were analysed with covariance. Haplotype analysis was conducted. TT genotype of rs731236 of VDR had higher BMD at total hip and femoral neck (FN), and one haplotype formed by the two polymorphisms was associated with only TH-BMD variations. This difference was statistically significant after adjustment for confounders. The genotype of rs2228570 of VDR analysis showed no significant differences with BMD variations. The results showed that the TT genotype of rs731236 of VDR and one haplotype formed by rs731236 and rs2228570 polymorphisms were associated with higher BMD at TH and FN.

Analysis of short tandem repeat polymorphisms using infrared fluorescence with M18 tailed primers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oetting, W.S.; Wiesner, G.; Laken, S.

The use of short tandem repeat polymorphisms (STRPs) are becoming increasingly important as markers for linkage analysis due to their large numbers of the human genome and their high degree of polymorphism. Fluorescence based detection of the STRP pattern using the LI-COR model 4000S automated DNA sequencer eliminates the need for radioactivity and produces a digitized image that can be used for the analysis of the polymorphisms. In an effort to reduce the cost of STRP analysis, we have synthesized primers with a 19 bp extension complementary to the sequence of the M13 primer on the 5{prime} end of onemore » of the two primers used in the amplification of the STRP instead of using primers with direct conjugation of the infrared fluorescent dye. Up to 5 primer pairs can be multiplexed together with the M13 primer-dye conjugate as the sole primer conjugated to the fluorescent dye. Comparisons between primers that have been directly conjugated to the fluor with those having the M13 sequence extension show no difference in the ability to determine the STRP pattern. At present, the entire Weber 4A set of STRP markers is available with the M13 5{prime} extension. We are currently using this technique for linkage analysis of familial breast cancer and asthma. The combination of STRP analysis using fluorescence detection will allow this technique to be fully automated for allele scoring and linkage analysis.« less

Mitochondrial genome diversity in the Tubalar, Even, and Ulchi: contribution to prehistory of native Siberians and their affinities to Native Americans.

PubMed

Sukernik, Rem I; Volodko, Natalia V; Mazunin, Ilya O; Eltsov, Nikolai P; Dryomov, Stanislav V; Starikovskaya, Elena B

2012-05-01

To fill remaining gaps in mitochondrial DNA diversity in the least surveyed eastern and western flanks of Siberia, 391 mtDNA samples (144 Tubalar from Altai, 87 Even from northeastern Siberia, and 160 Ulchi from the Russian Far East) were characterized via high-resolution restriction fragment length polymorphism/single nucleotide polymorphisms analysis. The subhaplogroup structure was extended through complete sequencing of 67 mtDNA samples selected from these and other related native Siberians. Specifically, we have focused on the evolutionary histories of the derivatives of M and N haplogroups, putatively reflecting different phases of settling Siberia by early modern humans. Population history and phylogeography of the resulting mtDNA genomes, combined with those from previously published data sets, revealed a wide range of tribal- and region-specific mtDNA haplotypes that emerged or diversified in Siberia before or after the last glacial maximum, ∼18 kya. Spatial distribution and ages of the "east" and "west" Eurasian mtDNA haploclusters suggest that anatomically modern humans that originally colonized Altai derived from macrohaplogroup N and came from Southwest Asia around 38,000 years ago. The derivatives of macrohaplogroup M, which largely emerged or diversified within the Russian Far East, came along with subsequent migrations to West Siberia millennia later. The last glacial maximum played a critical role in the timing and character of the settlement of the Siberian subcontinent. Copyright © 2012 Wiley Periodicals, Inc.

Endothelial nitric oxide synthase polymorphism and prognosis in systolic heart failure patients.

PubMed

Azzam, Naiel; Zafrir, Barak; Fares, Fuad; Smith, Yoav; Salman, Nabeeh; Nevzorov, Roman; Amir, Offer

2015-05-01

The endothelial nitric oxide synthase (eNOS) gene single nucleotide polymorphism G894T is associated with thrombotic vascular diseases. However, its functional significance is controversial and data are scarce concerning its influence in heart failure (HF). We studied 215 patients with chronic systolic HF. DNA was analyzed for eNOS gene G894T polymorphism using PCR and DNA sequencing. Evaluation of clinical characteristics and analysis of factors associated with 2-year mortality were performed for the homozygous G-allele G894T variant (GG), relative to the TT and GT variants. The genotype distributions of eNOS G894T alleles were: GG 135 patients (63%) and TT/GT 80 (37%). Two-year mortality was significantly higher in the GG variant (48%) than the combined TT/GT group (32%). The usage of nitrates was associated with increased 2-year mortality (HR 2.0, 95% CI 1.28-3.17; p = 0.003), which was most significant in the GG group treated with nitrates (73.5%) in comparison to the TT/GT group not treated with nitrates (34%); HR 2.75, 95% CI 1.57-4.79, P < 0.001. Homozygosity for the G allele of the eNOS G894T polymorphism was associated with worse survival in systolic HF patients, especially in those treated with nitrates. ENOS polymorphism may result in different mechanistic interactions in HF than in thrombotic vascular diseases, suggesting that overexpression of NO may be associated with deleterious effects in systolic HF. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of five genetic variants as novel determinants of type 2 diabetes mellitus in Japanese by exome-wide association studies.

PubMed

Yamada, Yoshiji; Sakuma, Jun; Takeuchi, Ichiro; Yasukochi, Yoshiki; Kato, Kimihiko; Oguri, Mitsutoshi; Fujimaki, Tetsuo; Horibe, Hideki; Muramatsu, Masaaki; Sawabe, Motoji; Fujiwara, Yoshinori; Taniguchi, Yu; Obuchi, Shuichi; Kawai, Hisashi; Shinkai, Shoji; Mori, Seijiro; Arai, Tomio; Tanaka, Masashi

2017-10-06

We performed exome-wide association studies to identify single nucleotide polymorphisms that either influence fasting plasma glucose level or blood hemoglobin A 1c content or confer susceptibility to type 2 diabetes mellitus in Japanese. Exome-wide association studies were performed with the use of Illumina Human Exome-12 DNA Analysis or Infinium Exome-24 BeadChip arrays and with 11,729 or 8635 subjects for fasting plasma glucose level or blood hemoglobin A 1c content, respectively, or with 14,023 subjects for type 2 diabetes mellitus (3573 cases, 10,450 controls). The relation of genotypes of 41,265 polymorphisms to fasting plasma glucose level or blood hemoglobin A 1c content was examined by linear regression analysis. After Bonferroni's correction, 41 and 17 polymorphisms were significantly ( P < 1.21 × 10 -6 ) associated with fasting plasma glucose level or blood hemoglobin A 1c content, respectively, with two polymorphisms (rs139421991, rs189305583) being associated with both. Examination of the relation of allele frequencies to type 2 diabetes mellitus with Fisher's exact test revealed that 87 polymorphisms were significantly ( P < 1.21 × 10 -6 ) associated with type 2 diabetes mellitus. Subsequent multivariable logistic regression analysis with adjustment for age and sex showed that four polymorphisms (rs138313632, rs76974938, rs139012426, rs147317864) were significantly ( P < 1.44 × 10 -4 ) associated with type 2 diabetes mellitus, with rs138313632 and rs139012426 also being associated with fasting plasma glucose and rs76974938 with blood hemoglobin A 1c . Five polymorphisms-rs139421991 of CAT , rs189305583 of PDCL2 , rs138313632 of RUFY1 , rs139012426 of LOC100505549 , and rs76974938 of C21orf59 -may be novel determinants of type 2 diabetes mellitus.

Identification of rs7350481 at chromosome 11q23.3 as a novel susceptibility locus for metabolic syndrome in Japanese individuals by an exome-wide association study.

PubMed

Yamada, Yoshiji; Sakuma, Jun; Takeuchi, Ichiro; Yasukochi, Yoshiki; Kato, Kimihiko; Oguri, Mitsutoshi; Fujimaki, Tetsuo; Horibe, Hideki; Muramatsu, Masaaki; Sawabe, Motoji; Fujiwara, Yoshinori; Taniguchi, Yu; Obuchi, Shuichi; Kawai, Hisashi; Shinkai, Shoji; Mori, Seijiro; Arai, Tomio; Tanaka, Masashi

2017-06-13

We have performed exome-wide association studies to identify genetic variants that influence body mass index or confer susceptibility to obesity or metabolic syndrome in Japanese. The exome-wide association study for body mass index included 12,890 subjects, and those for obesity and metabolic syndrome included 12,968 subjects (3954 individuals with obesity, 9014 controls) and 6817 subjects (3998 individuals with MetS, 2819 controls), respectively. Exome-wide association studies were performed with Illumina HumanExome-12 DNA Analysis BeadChip or Infinium Exome-24 BeadChip arrays. The relation of genotypes of single nucleotide polymorphisms to body mass index was examined by linear regression analysis, and that of allele frequencies of single nucleotide polymorphisms to obesity or metabolic syndrome was evaluated with Fisher's exact test. The exome-wide association studies identified six, 11, and 40 single nucleotide polymorphisms as being significantly associated with body mass index, obesity (P <1.21 × 10-6), or metabolic syndrome (P <1.20 × 10-6), respectively. Subsequent multivariable logistic regression analysis with adjustment for age and sex revealed that three and five single nucleotide polymorphisms were related (P < 0.05) to obesity or metabolic syndrome, respectively, with one of these latter polymorphisms-rs7350481 (C/T) at chromosome 11q23.3-also being significantly (P < 3.13 × 10-4) associated with metabolic syndrome. The polymorphism rs7350481 may thus be a novel susceptibility locus for metabolic syndrome in Japanese. In addition, single nucleotide polymorphisms in three genes (CROT, TSC1, RIN3) and at four loci (ANKK1, ZNF804B, CSRNP3, 17p11.2) were implicated as candidate determinants of obesity and metabolic syndrome, respectively.

Association between DNA methyltransferase gene polymorphism and Parkinson's disease.

PubMed

Pezzi, Julio Carlos; de Bem, Cintia Monique Boschmann Ens; da Rocha, Tatiane Jacobsen; Schumacher-Schuh, Artur F; Chaves, Marcia Lorena Fagundes; Rieder, Carlos Roberto; Hutz, Mara H; Fiegenbaum, Marilu; Camozzato, Ana Luiza

2017-02-03

Parkinson's disease (PD) is a common and complex neurodegenerative disorder, the second most prevalent, only behind Alzheimer's disease. Recent studies suggest that environmental factors may contribute for neurodegeneration through induction of epigenetic modifications, such as DNA methylation, that is carried out by enzymes, such as DNMT1 and DNMT3B. This present study targeted to investigate the association among DNMT1 and DNMT3B polymorphisms with PD. Five hundred and twenty-two participants (214 PD patients following UK Brain Bank criteria and 308 healthy individuals) were evaluated. DNA was obtained from whole blood and genotypes were detected by an allelic discrimination assay using TaqMan ® MGB probes on a real-time PCR system. The polymorphisms studied were rs2162560 and rs759920 (DNMT1) and rs2424913, rs998382 and rs2424932 (DNMT3B). Was found association between DNMT3B rs2424913 in T allele carriers with PD. The presence of the T allele was associated with PD (OR=1.80, 95% CI 1.16-2.81, p=0.009). No significant difference was observed for others DNMT3B SNPs. Also, no association between PD and the control group were observed for DNMT1 polymorphisms. This is the first study addressing an association between DNMT3B polymorphism and PD. The polymorphism may play a role in the pathogenesis of PD. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Association of the hOGG1 Ser326Cys polymorphism with sporadic amyotrophic lateral sclerosis.

PubMed

Coppedè, Fabio; Mancuso, Michelangelo; Lo Gerfo, Annalisa; Carlesi, Cecilia; Piazza, Selina; Rocchi, Anna; Petrozzi, Lucia; Nesti, Claudia; Micheli, Dario; Bacci, Andrea; Migliore, Lucia; Murri, Luigi; Siciliano, Gabriele

2007-06-13

Amyotropic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disease causing the loss of motoneurons of the brain and the spinal cord. The etiology of ALS is still uncertain, but males are at increased risk for the disease than females. Several studies have suggested that motoneurons in ALS might be subjected to the double insult of increased DNA oxidative damage and deficiencies in DNA repair systems. Particularly, increased levels of 8-oxoguanine and impairments of the DNA base excision repair system have been observed in neurons of ALS patients. There is evidence that the Ser326Cys polymorphism of the human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene is associated with a reduced DNA repair activity. To evaluate the role of the hOGG1 Ser326Cys polymorphism in sporadic ALS (sALS), we screened 136 patients and 129 matched controls. In the total population, we observed association between both the Cys326 allele (p=0.02) and the combined Ser326Cys+Cys326Cys genotype (OR=1.65, 95% CI=1.06-2.88) and increased risk of disease. After stratification by gender, the Cys326 allele (p=0.01), both the Ser326Cys genotype (OR=2.14, 95% CI=1.09-4.19) and the combined Ser326Cys+Cys326Cys genotype (OR=2.15, 95% CI=1.16-4.01) were associated with sALS risk only in males. No significant association between the Ser326Cys polymorphism and disease phenotype, including age and site of onset and disease progression, was observed. Present results suggest a possible involvement of the hOGG1 Ser326Cys polymorphism in sALS pathogenesis.

hOGG1 Ser326Cys polymorphism and G:C-to-T:A mutations: no evidence for a role in tobacco-related non small cell lung cancer.

PubMed

Hu, Ying Chuan; Ahrendt, Steven A

2005-04-10

Human 8-oxoguanine DNA glycosylase 1 (hOGG1) plays a major role in the repair of 8-hydroxyguanine, one of the major forms of DNA damage generated by reactive oxygen species in tobacco smoke. If left unrepaired by hOGG1, 8-hydroxyguanine can produce G:C-to-T:A transversions. Recent studies have suggested that the hOGG1 Ser326Cys polymorphism is associated with both a decrease in enzyme activity and an increased risk of lung cancer. To define the interaction between tobacco carcinogens, hOGG1-mediated DNA repair and DNA damage, we examined the role of the hOGG1 Ser326Cys polymorphism in mutation of the p53 gene in non small cell lung cancer (NSCLC). Tumor and nonneoplastic DNA were collected from 141 cigarette smokers with NSCLC. p53 mutations were detected by direct dideoxy sequencing and/or the GeneChip p53 assay in 74 of the 141 (52%) tumors. hOGG1 codon 326 polymorphisms were identified by polymerase chain reaction-restriction fragment length polymorphism analysis. The distribution of hOGG1 codon 326 genotypes was Ser/Ser, 90 of 141 (64%); Ser/Cys, 45 of 141 (32%); and Cys/Cys, 6 of 141 (4%). p53 mutations were significantly (p = 0.04) less common in NSCLC from patients with codon 326 Ser/Cys or Cys/Cys genotypes (21 of 51; 41%) than in NSCLC from Ser/Ser homozygotes (53 of 90; 59%). The decrease in p53 mutation frequency among carriers of the Cys allele was more evident in lung squamous cell cancer [7 of 17 (41%) for Cys/Cys and Ser/Cys vs. 27 of 38 (71%) for Ser/Ser; p = 0.04] than in nonbronchoalveolar adenocarcinoma [11 of 26 (42%) for Cys/Cys and Ser/Cys vs. 20 of 35 (57%) for Ser/Ser; p = 0.25]. The prevalence of G:C-to-T:A transversions was similar among hOGG1 codon 326 genotypes. In summary, the hOGG1 codon 326 Cys allele was associated with a decrease in p53 mutations and no effect on G:C-to-T:A transversions in NSCLC. This decrease in p53 mutations in vivo is not consistent with a decrease in the repair of 8-hydroxyguanine among carriers of the hOGG1 codon 326 Cys allele in vitro. (c) 2004 Wiley-Liss, Inc.

A semester-long project for teaching basic techniques in molecular biology such as restriction fragment length polymorphism analysis to undergraduate and graduate students.

PubMed

DiBartolomeis, Susan M

2011-01-01

Several reports on science education suggest that students at all levels learn better if they are immersed in a project that is long term, yielding results that require analysis and interpretation. I describe a 12-wk laboratory project suitable for upper-level undergraduates and first-year graduate students, in which the students molecularly locate and map a gene from Drosophila melanogaster called dusky and one of dusky's mutant alleles. The mapping strategy uses restriction fragment length polymorphism analysis; hence, students perform most of the basic techniques of molecular biology (DNA isolation, restriction enzyme digestion and mapping, plasmid vector subcloning, agarose and polyacrylamide gel electrophoresis, DNA labeling, and Southern hybridization) toward the single goal of characterizing dusky and the mutant allele dusky(73). Students work as individuals, pairs, or in groups of up to four students. Some exercises require multitasking and collaboration between groups. Finally, results from everyone in the class are required for the final analysis. Results of pre- and postquizzes and surveys indicate that student knowledge of appropriate topics and skills increased significantly, students felt more confident in the laboratory, and students found the laboratory project interesting and challenging. Former students report that the lab was useful in their careers.

A Semester-Long Project for Teaching Basic Techniques in Molecular Biology Such as Restriction Fragment Length Polymorphism Analysis to Undergraduate and Graduate Students

PubMed Central

DiBartolomeis, Susan M.

2011-01-01

Several reports on science education suggest that students at all levels learn better if they are immersed in a project that is long term, yielding results that require analysis and interpretation. I describe a 12-wk laboratory project suitable for upper-level undergraduates and first-year graduate students, in which the students molecularly locate and map a gene from Drosophila melanogaster called dusky and one of dusky's mutant alleles. The mapping strategy uses restriction fragment length polymorphism analysis; hence, students perform most of the basic techniques of molecular biology (DNA isolation, restriction enzyme digestion and mapping, plasmid vector subcloning, agarose and polyacrylamide gel electrophoresis, DNA labeling, and Southern hybridization) toward the single goal of characterizing dusky and the mutant allele dusky73. Students work as individuals, pairs, or in groups of up to four students. Some exercises require multitasking and collaboration between groups. Finally, results from everyone in the class are required for the final analysis. Results of pre- and postquizzes and surveys indicate that student knowledge of appropriate topics and skills increased significantly, students felt more confident in the laboratory, and students found the laboratory project interesting and challenging. Former students report that the lab was useful in their careers. PMID:21364104

Geographical markers for Saccharomyces cerevisiae strains with similar technological origins domesticated for rice-based ethnic fermented beverages production in North East India.

PubMed

Jeyaram, Kumaraswamy; Tamang, Jyoti Prakash; Capece, Angela; Romano, Patrizia

2011-11-01

Autochthonous strains of Saccharomyces cerevisiae from traditional starters used for the production of rice-based ethnic fermented beverage in North East India were examined for their genetic polymorphism using mitochondrial DNA-RFLP and electrophoretic karyotyping. Mitochondrial DNA-RFLP analysis of S. cerevisiae strains with similar technological origins from hamei starter of Manipur and marcha starter of Sikkim revealed widely separated clusters based on their geographical origin. Electrophoretic karyotyping showed high polymorphism amongst the hamei strains within similar mitochondrial DNA-RFLP cluster and one unique karyotype of marcha strain was widely distributed in the Sikkim-Himalayan region. We conceptualized the possibility of separate domestication events for hamei strains in Manipur (located in the Indo-Burma biodiversity hotspot) and marcha strains in Sikkim (located in Himalayan biodiversity hotspot), as a consequence of less homogeneity in the genomic structure between these two groups, their clear separation being based on geographical origin, but not on technological origin and low strain level diversity within each group. The molecular markers developed based on HinfI-mtDNA-RFLP profile and the chromosomal doublets in chromosome VIII position of Sikkim-Himalayan strains could be effectively used as geographical markers for authenticating the above starter strains and differentiating them from other commercial strains.

A tangled tale of two teal: Population history of the grey Anas gracilis and chestnut teal a. castanea of Australia

USGS Publications Warehouse

Joseph, L.; Adcock, G.J.; Linde, C.; Omland, K.E.; Heinsohn, R.; Terry, Chesser R.; Roshier, D.

2009-01-01

Two Australian species of teal (Anseriformes: Anatidae: Anas), the grey teal Anas gracilis and the chestnut teal A. castanea, are remarkable for the zero or near-zero divergence recorded between them in earlier surveys of mitochondrial DNA (mtDNA) diversity. We confirmed this result through wider geographical and population sampling as well as nucleotide sampling in the more rapidly evolving mtDNA control region. Any data set where two species share polymorphism as is the case here can be explained by a model of gene flow through hybridization on one hand or by incomplete lineage sorting on the other hand. Ideally, analysis of such shared polymorphism would simultaneously estimate the likelihood of both phenomena. To do this, we used the underlying principle of the IMa package to explore ramifications to understanding population histories of A. gracilis and A. castanea. We cannot reject that hybridization occurs between the two species but an equally or more plausible finding for their nearly zero divergence is incomplete sorting following very recent divergence between the two, probably in the mid-late Pleistocene. Our data add to studies that explore intermediate stages in the evolution of reciprocal monophyly and paraphyletic or polyphyletic relationships in mtDNA diversity among widespread Australian birds. ?? 2009 J. Avian Biol.

Molecular and biochemical studies on the effect of gamma rays on lead toxicity in cowpea (Vigna sinensis) plants.

PubMed

Mohamed, Heba Ibrahim

2011-12-01

The effect of lead acetate in the presence or absence of cowpea seeds irradiated with gamma rays on morphological criteria, protein electrophoresis, isozymes, and random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) of leaves was investigated. A highly significant decrease in shoot and root length was observed upon lead acetate exposure (300 and 600 μM). On the other hand, in seeds irradiated with gamma rays (2, 5, and 8 krad), these morphological parameters were increased after lead acetate treatments. Meanwhile, all treatments (lead acetate and gamma rays) caused variations in number, intensity, and/or density of SDS electrophoretic bands of proteins. In addition, electrophoretic studies of esterase, acid phosphatase, peroxidase, polyphenol oxidase, catalase, and superoxide dismutase isozyme activities were increased with increasing the concentrations of lead acetate and gamma ray doses. The variation in DNA profile in response to lead acetate and gamma irradiation treatments was detected by RAPD-PCR technique. The result of RAPD analysis using the five primers indicated the appearance and disappearance of DNA polymorphic bands at all treatments (gamma rays and lead stress). The relatively high concentrations of lead acetate (600 μM) induced more changes in genomic DNA pattern.

Population-scale whole genome sequencing identifies 271 highly polymorphic short tandem repeats from Japanese population.

PubMed

Hirata, Satoshi; Kojima, Kaname; Misawa, Kazuharu; Gervais, Olivier; Kawai, Yosuke; Nagasaki, Masao

2018-05-01

Forensic DNA typing is widely used to identify missing persons and plays a central role in forensic profiling. DNA typing usually uses capillary electrophoresis fragment analysis of PCR amplification products to detect the length of short tandem repeat (STR) markers. Here, we analyzed whole genome data from 1,070 Japanese individuals generated using massively parallel short-read sequencing of 162 paired-end bases. We have analyzed 843,473 STR loci with two to six basepair repeat units and cataloged highly polymorphic STR loci in the Japanese population. To evaluate the performance of the cataloged STR loci, we compared 23 STR loci, widely used in forensic DNA typing, with capillary electrophoresis based STR genotyping results in the Japanese population. Seventeen loci had high correlations and high call rates. The other six loci had low call rates or low correlations due to either the limitations of short-read sequencing technology, the bioinformatics tool used, or the complexity of repeat patterns. With these analyses, we have also purified the suitable 218 STR loci with four basepair repeat units and 53 loci with five basepair repeat units both for short read sequencing and PCR based technologies, which would be candidates to the actual forensic DNA typing in Japanese population.

Prenatal diagnosis of de novo t(2;18;14)(q33.1;q12.2;q31.2), dup(5)(q34q34), del(7)(p21.1p21.1), and del(10)(q25.3q25.3) and a review of the prenatally ascertained de novo apparently balanced complex and multiple chromosomal rearrangements.

PubMed

Chen, Chih-Ping; Chern, Schu-Rern; Lee, Chen-Chi; Lin, Chyi-Chyang; Li, Yueh-Chun; Hsieh, Lie-Jiau; Chen, Wen-Lin; Wang, Wayseen

2006-02-01

To present the prenatal diagnosis of a de novo complex chromosomal rearrangement (CCR) associated with de novo interstitial deletions and duplication and to review the literature. Amniocentesis was performed at 18 weeks' gestation because of an increased risk for Down syndrome based on maternal serum alpha-fetoprotein and human chorionic gonadotrophin screening. Amniocentesis revealed a karyotype of 46,XY,t(2;18;14)(q33.1;q12.2;q31.2),dup(5)(q34q34),del(7)(p21.1p21.1), del(10)(q25.3q25.3). The parental karyotypes were normal. The pregnancy was terminated. The fetus manifested facial dysmorphism, clinodactyly of both hands, and hypoplasia of the left great toe. Spectral karyotyping (SKY), cytogenetic polymorphism, and polymorphic DNA markers were used to investigate the imbalances and the origin of the de novo aberrant chromosomes. SKY showed a three-way CCR. Cytogenetic polymorphism investigation of the derivative chromosome 14 of the fetus and the parental chromosomes 14 determined the maternal origin of the translocation. Polymorphic DNA marker analysis confirmed the maternal origin of the de novo interstitial deletions and duplication. No cryptic imbalance at or near the breakpoints of the CCR was detected by the molecular analysis. De novo apparently balanced CCRs may be associated with imbalances in other chromosomes. We suggest further investigation and re-evaluation of cryptic or subtle imbalances in all cases classified as de novo apparently balanced CCRs. Copyright 2006 John Wiley & Sons, Ltd.

Trends in plant research using molecular markers.

PubMed

Garrido-Cardenas, Jose Antonio; Mesa-Valle, Concepción; Manzano-Agugliaro, Francisco

2018-03-01

A deep bibliometric analysis has been carried out, obtaining valuable parameters that facilitate the understanding around the research in plant using molecular markers. The evolution of the improvement in the field of agronomy is fundamental for its adaptation to the new exigencies that the current world context raises. In addition, within these improvements, this article focuses on those related to the biotechnology sector. More specifically, the use of DNA markers that allow the researcher to know the set of genes associated with a particular quantitative trait or QTL. The use of molecular markers is widely extended, including: restriction fragment length polymorphism, random-amplified polymorphic DNA, amplified fragment length polymorphism, microsatellites, and single-nucleotide polymorphisms. In addition to classical methodology, new approaches based on the next generation sequencing are proving to be fundamental. In this article, a historical review of the molecular markers traditionally used in plants, since its birth and how the new molecular tools facilitate the work of plant breeders is carried out. The evolution of the most studied cultures from the point of view of molecular markers is also reviewed and other parameters whose prior knowledge can facilitate the approach of researchers to this field of research are analyzed. The bibliometric analysis of molecular markers in plants shows that top five countries in this research are: US, China, India, France, and Germany, and from 2013, this research is led by China. On the other hand, the basic research using Arabidopsis is deeper in France and Germany, while other countries focused its efforts in their main crops as the US for wheat or maize, while China and India for wheat and rice.

Demonstration of protein-based human identification using the hair shaft proteome [Protein-based human identification: A proof of concept using the hair shaft proteome

DOE PAGES

Parker, Glendon J.; Leppert, Tami; Anex, Deon S.; ...

2016-09-07

Human identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 singlemore » nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects’ DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European–American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). Furthermore, this study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts.« less

Demonstration of protein-based human identification using the hair shaft proteome [Protein-based human identification: A proof of concept using the hair shaft proteome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parker, Glendon J.; Leppert, Tami; Anex, Deon S.

Human identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 singlemore » nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects’ DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European–American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). Furthermore, this study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts.« less

Complex Relationships between Occupation, Environment, DNA Adducts, Genetic Polymorphisms and Bladder Cancer in a Case-Control Study Using a Structural Equation Modeling

PubMed Central

Porru, Stefano; Pavanello, Sofia; Carta, Angela; Arici, Cecilia; Simeone, Claudio; Izzotti, Alberto; Mastrangelo, Giuseppe

2014-01-01

DNA adducts are considered an integrate measure of carcinogen exposure and the initial step of carcinogenesis. Their levels in more accessible peripheral blood lymphocytes (PBLs) mirror that in the bladder tissue. In this study we explore whether the formation of PBL DNA adducts may be associated with bladder cancer (BC) risk, and how this relationship is modulated by genetic polymorphisms, environmental and occupational risk factors for BC. These complex interrelationships, including direct and indirect effects of each variable, were appraised using the structural equation modeling (SEM) analysis. Within the framework of a hospital-based case/control study, study population included 199 BC cases and 213 non-cancer controls, all Caucasian males. Data were collected on lifetime smoking, coffee drinking, dietary habits and lifetime occupation, with particular reference to exposure to aromatic amines (AAs) and polycyclic aromatic hydrocarbons (PAHs). No indirect paths were found, disproving hypothesis on association between PBL DNA adducts and BC risk. DNA adducts were instead positively associated with occupational cumulative exposure to AAs (p = 0.028), whereas XRCC1 Arg 399 (p<0.006) was related with a decreased adduct levels, but with no impact on BC risk. Previous findings on increased BC risk by packyears (p<0.001), coffee (p<0.001), cumulative AAs exposure (p = 0.041) and MnSOD (p = 0.009) and a decreased risk by MPO (p<0.008) were also confirmed by SEM analysis. Our results for the first time make evident an association between occupational cumulative exposure to AAs with DNA adducts and BC risk, strengthening the central role of AAs in bladder carcinogenesis. However the lack of an association between PBL DNA adducts and BC risk advises that these snapshot measurements are not representative of relevant exposures. This would envisage new scenarios for biomarker discovery and new challenges such as repeated measurements at different critical life stages. PMID:24722645

Genetic susceptibility to bone and soft tissue sarcomas: a field synopsis and meta-analysis.

PubMed

Benna, Clara; Simioni, Andrea; Pasquali, Sandro; De Boni, Davide; Rajendran, Senthilkumar; Spiro, Giovanna; Colombo, Chiara; Virgone, Calogero; DuBois, Steven G; Gronchi, Alessandro; Rossi, Carlo Riccardo; Mocellin, Simone

2018-04-06

The genetic architecture of bone and soft tissue sarcomas susceptibility is yet to be elucidated. We aimed to comprehensively collect and meta-analyze the current knowledge on genetic susceptibility in these rare tumors. We conducted a systematic review and meta-analysis of the evidence on the association between DNA variation and risk of developing sarcomas through searching PubMed, The Cochrane Library, Scopus and Web of Science databases. To evaluate result credibility, summary evidence was graded according to the Venice criteria and false positive report probability (FPRP) was calculated to further validate result noteworthiness. Integrative analysis of genetic and eQTL (expression quantitative trait locus) data was coupled with network and pathway analysis to explore the hypothesis that specific cell functions are involved in sarcoma predisposition. We retrieved 90 eligible studies comprising 47,796 subjects (cases: 14,358, 30%) and investigating 1,126 polymorphisms involving 320 distinct genes. Meta-analysis identified 55 single nucleotide polymorphisms (SNPs) significantly associated with disease risk with a high (N=9), moderate (N=38) and low (N=8) level of evidence, findings being classified as noteworthy basically only when the level of evidence was high. The estimated joint population attributable risk for three independent SNPs (rs11599754 of ZNF365/EGR2 , rs231775 of CTLA4 , and rs454006 of PRKCG ) was 37.2%. We also identified 53 SNPs significantly associated with sarcoma risk based on single studies.Pathway analysis enabled us to propose that sarcoma predisposition might be linked especially to germline variation of genes whose products are involved in the function of the DNA repair machinery. We built the first knowledgebase on the evidence linking DNA variation to sarcomas susceptibility, which can be used to generate mechanistic hypotheses and inform future studies in this field of oncology.

Variation in Ribosomal DNA among Isolates of the Mycorrhizal Fungus Cenococcum Geophilum FR.

NASA Astrophysics Data System (ADS)

Lobuglio, Katherine Frances

1990-01-01

Cenococcum geophilum Fr., a cosmopolitan mycorrhizal fungus, is well-known for its extremely wide host and habitat range. The ecological diversity of C. geophilum sharply contrasts its present taxonomic status as a monotypic form -genus. Restriction fragment length polymorphisms (RFLPs) in nuclear ribosomal DNA (rDNA) was used to assess the degree of genetic variation among 72 isolates of C. geophilum. The probe used in this study was the rDNA repeat cloned from C. geophilum isolate A145 (pCG15). Length of the rDNA repeat was approximately 9 kb. The rDNA clone was mapped for 5 restriction endonucleases. Hybridization with cloned Saccharomyces cerevisiae rDNA (pSR118, and pSR125 containing the 18S, and 5.8-25S rRNA genes respectively), and alignment of restriction endonuclease sites conserved in the rDNA genes of other fungi, were used to position the corresponding rDNAs of C. geophilum. Southern hybridizations with EcoRI, HindIII, XhoI, and PstI digested DNAs indicated extensive variation among the C. geophilum isolates, greater than has been previously reported to occur within a fungal species. Most of the rDNA polymorphisms occurred in the IGS region. Restriction endonuclease site and length polymorphisms were also observed in the 5.8S-26S genic regions. Sixteen size categories of length mutations, 6 restriction endonuclease site additions, and 4 restriction endonuclease site deletions were determined using isolate A145 as a reference. The rDNA repeat length among the isolates varied from approximately 8.5 to 10.2 kb. RFLPs were also observed in the mitochondrial (mt) 24S rRNA gene and flanking regions of HindIII digested DNAs of C. geophilum isolates representing both geographically distinct and similar origins. Among the C. geophilum isolates analyzed there were fewer RFLPs in mt-DNA than in nuclear rDNA. EcoRI rDNA phenotypes between C. geophilum and Elaphomyces anthracinus, its proposed teleomorph or sexual state, did not correspond. In addition, the four Elaphomyces species examined had smaller rDNA repeat sizes (approximately 7.3 to 8.0 kb) than that observed among C. geophilum isolates. UPGMA cluster analysis grouped C. geophilum isolates, on the basis of shared nuclear rDNA phenotypes, into a broad range of clusters ranging from 100% to 44% similarity. Limited correlation was observed among nuclear rDNA phenotypes and culture morphology, mycorrhizal characteristics, or geographic origins of the isolates. The amount of genetic variation demonstrated in this study indicates that C. geophilum is either an extremely heterogenous species or it represents more than one species and possibly more than one genus.

Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives.

PubMed

Fregene, M A; Vargas, J; Ikea, J; Angel, F; Tohme, J; Asiedu, R A; Akoroda, M O; Roca, W M

1994-11-01

Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crop's present day variability.

Comparison of Mediterranean Pistacia lentiscus genotypes by random amplified polymorphic DNA, chemical, and morphological analyses.

PubMed

Barazani, Oz; Dudai, Nativ; Golan-Goldhirsh, Avi

2003-08-01

Characterization of the genetic variability of Mediterranean Pistacia lentiscus genotypes by RAPD, composition of essential oils, and morphology is presented. High polymorphism in morphological parameters was found among accessions, with no significant differences in relation to geographical origin, or to gender. GC-MS analysis of leaves extracted by t-butyl methyl ether, showed 12 monoterpenes, seven sesquiterpenes, and one linear nonterpenic compound. Cluster analysis divided the accessions into two main groups according to the relative content of the major compounds, with no relation to their geographical origin. In contrast, a dendrogram based on RAPD analysis gave two main clusters according to their geographical origins. Low correlation was found between genetic and essential oil content matrices. High morphological and chemical variability on one hand, and genotypic polymorphism on the other, provide ecological advantages that might explain the distribution of Pistacia lentiscus over a wide range of habitats. The plants under study were grown together in the same climatic and environmental conditions, thus pointing to the plausible genetic basis of the observed phenotypic differences.

Gene amplification of the Hps locus in Glycine max

PubMed Central

Gijzen, Mark; Kuflu, Kuflom; Moy, Pat

2006-01-01

Background Hydrophobic protein from soybean (HPS) is an 8 kD cysteine-rich polypeptide that causes asthma in persons allergic to soybean dust. HPS is synthesized in the pod endocarp and deposited on the seed surface during development. Past evidence suggests that the protein may mediate the adherence or dehiscence of endocarp tissues during maturation and affect the lustre, or glossiness of the seed surface. Results A comparison of soybean germplasm by genomic DNA blot hybridization shows that the copy number and structure of the Hps locus is polymorphic among soybean cultivars and related species. Changes in Hps gene copy number were also detected by comparative genomic DNA hybridization using cDNA microarrays. The Hps copy number polymorphisms co-segregated with seed lustre phenotype and HPS surface protein in a cross between dull- and shiny-seeded soybeans. In soybean cultivar Harosoy 63, a minimum of 27 ± 5 copies of the Hps gene were estimated to be present in each haploid genome. The isolation and analysis of genomic clones indicates that the core Hps locus is comprised of a tandem array of reiterated units, with each 8.6 kb unit containing a single HPS open reading frame. Conclusion This study shows that polymorphisms at the Hps locus arise from changes in the gene copy number via gene amplification. We present a model whereby Hps copy number modulates protein expression levels and seed lustre, and we suggest that gene amplification may result from selection pressures imposed on crop plants. PMID:16536872

Mycelial Propagation and Molecular Phylogenetic Relationships of Commercially Cultivated Agrocybe cylindracea based on ITS Sequences and RAPD

PubMed Central

Alam, Nuhu; Kim, Jeong Hwa; Shim, Mi Ja; Lee, U Youn

2010-01-01

This study evaluated the optimal vegetative growth conditions and molecular phylogenetic relationships of eleven strains of Agrocybe cylindracea collected from different ecological regions of Korea, China and Taiwan. The optimal temperature and pH for mycelial growth were observed at 25℃ and 6. Potato dextrose agar and Hennerberg were the favorable media for vegetative growth, whereas glucose tryptone was unfavorable. Dextrin, maltose, and fructose were the most effective carbon sources. The most suitable nitrogen sources were arginine and glycine, whereas methionine, alanine, histidine, and urea were least effective for the mycelial propagation of A. cylindracea. The internal transcribed spacer (ITS) regions of rDNA were amplified using PCR. The sequence of ITS2 was more variable than that of ITS1, while the 5.8S sequences were identical. The reciprocal homologies of the ITS sequences ranged from 98 to 100%. The strains were also analyzed by random amplification of polymorphic DNA (RAPD) using 20 arbitrary primers. Fifteen primers efficiently amplified the genomic DNA. The average number of polymorphic bands observed per primer was 3.8. The numbers of amplified bands varied based on the primers and strains, with polymorphic fragments ranging from 0.1 to 2.9 kb. The results of RAPD analysis were similar to the ITS region sequences. The results revealed that RAPD and ITS techniques were well suited for detecting the genetic diversity of all A. cylindracea strains tested. PMID:23956633

Polymorphisms within the FANCA gene associate with premature ovarian failure in Korean women.

PubMed

Pyun, Jung-A; Kim, Sunshin; Cha, Dong Hyun; Kwack, KyuBum

2014-05-01

This study investigated whether polymorphisms within the Fanconi anemia complementation group A (FANCA) gene contribute to the increased risk of premature ovarian failure (POF) in Korean women. Ninety-eight women with POF and 218 controls participated in this study. Genomic DNA from peripheral blood was isolated, and GoldenGate genotyping assay was used to identify single nucleotide polymorphisms (SNPs) within the FANCA gene. Two significant SNPs (rs1006547 and rs2239359; P < 0.05) were identified by logistic regression analysis, but results were insignificant after Bonferroni correction. Six SNPs formed a linkage disequilibrium block, and three main haplotypes were found. Two of three haplotypes (AAAGAA and GGGAGG) distributed highly in the POF group, whereas the remaining haplotype (GGAAGG) distributed highly in the control group by logistic regression analysis (highest odds ratio, 2.515; 95% CI, 1.515-4.175; P = 0.00036). Our observations suggest that genetic variations in the FANCA gene may increase the risk for POF in Korean women.

Development and Characterization of Somatic Hybrids of Ulva reticulata Forsskål (×) Monostroma oxyspermum (Kutz.)Doty

PubMed Central

Gupta, Vishal; Kumari, Puja; Reddy, CRK

2015-01-01

Ulvophycean species with diverse trait characteristics provide an opportunity to create novel allelic recombinant variants. The present study reports the development of seaweed variants with improved agronomic traits through protoplast fusion between Monostroma oxyspermum (Kutz.) Doty and Ulva reticulata Forsskål. A total of 12 putative hybrids were screened based on the variations in morphology and total DNA content over the fusion partners. DNA-fingerprinting by inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) analysis confirmed genomic introgression in the hybrids. The DNA fingerprint revealed sharing of parental alleles in regenerated hybrids and a few alleles that were unique to hybrids. The epigenetic variations in hybrids estimated in terms of DNA methylation polymorphism also revealed sharing of methylation loci with both the fusion partners. The functional trait analysis for growth showed a hybrid with heterotic trait (DGR% = 36.7 ± 1.55%) over the fusion partners U. reticulata (33.2 ± 2.6%) and M. oxyspermum (17.8 ± 1.77%), while others were superior to the mid-parental value (25.2 ± 2.2%) (p < 0.05). The fatty acid (FA) analysis of hybrids showed notable variations over fusion partners. Most hybrids showed increased polyunsaturated FAs (PUFAs) compared to saturated FAs (SFAs) and mainly includes the nutritionally important linoleic acid, α-linolenic acid, oleic acid, stearidonic acid, and docosahexaenoic acid. The other differences observed include superior cellulose content and antioxidative potential in hybrids over fusion partners. The hybrid varieties with superior traits developed in this study unequivocally demonstrate the significance of protoplast fusion technique in developing improved varients of macroalgae. PMID:25688248

Single-Nucleotide Polymorphisms of Genes Involved in Repair of Oxidative DNA Damage and the Risk of Recurrent Depressive Disorder.

PubMed

Czarny, Piotr; Kwiatkowski, Dominik; Toma, Monika; Gałecki, Piotr; Orzechowska, Agata; Bobińska, Kinga; Bielecka-Kowalska, Anna; Szemraj, Janusz; Berk, Michael; Anderson, George; Śliwiński, Tomasz

2016-11-20

BACKGROUND Depressive disorder, including recurrent type (rDD), is accompanied by increased oxidative stress and activation of inflammatory pathways, which may induce DNA damage. This thesis is supported by the presence of increased levels of DNA damage in depressed patients. Such DNA damage is repaired by the base excision repair (BER) pathway. BER efficiency may be influenced by polymorphisms in BER-related genes. Therefore, we genotyped nine single-nucleotide polymorphisms (SNPs) in six genes encoding BER proteins. MATERIAL AND METHODS Using TaqMan, we selected and genotyped the following SNPs: c.-441G>A (rs174538) of FEN1, c.2285T>C (rs1136410) of PARP1, c.580C>T (rs1799782) and c.1196A>G (rs25487) of XRCC1, c.*83A>C (rs4796030) and c.*50C>T (rs1052536) of LIG3, c.-7C>T (rs20579) of LIG1, and c.-468T>G (rs1760944) and c.444T>G (rs1130409) of APEX1 in 599 samples (288 rDD patients and 311 controls). RESULTS We found a strong correlation between rDD and both SNPs of LIG3, their haplotypes, as well as a weaker association with the c.-468T>G of APEXI which diminished after Nyholt correction. Polymorphisms of LIG3 were also associated with early onset versus late onset depression, whereas the c.-468T>G polymorphism showed the opposite association. CONCLUSIONS The SNPs of genes involved in the repair of oxidative DNA damage may modulate rDD risk. Since this is an exploratory study, the results should to be treated with caution and further work needs to be done to elucidate the exact involvement of DNA damage and repair mechanisms in the development of this disease.

Single-Nucleotide Polymorphisms of Genes Involved in Repair of Oxidative DNA Damage and the Risk of Recurrent Depressive Disorder

PubMed Central

Czarny, Piotr; Kwiatkowski, Dominik; Toma, Monika; Gałecki, Piotr; Orzechowska, Agata; Bobińska, Kinga; Bielecka-Kowalska, Anna; Szemraj, Janusz; Berk, Michael; Anderson, George; Śliwiński, Tomasz

2016-01-01

Background Depressive disorder, including recurrent type (rDD), is accompanied by increased oxidative stress and activation of inflammatory pathways, which may induce DNA damage. This thesis is supported by the presence of increased levels of DNA damage in depressed patients. Such DNA damage is repaired by the base excision repair (BER) pathway. BER efficiency may be influenced by polymorphisms in BER-related genes. Therefore, we genotyped nine single-nucleotide polymorphisms (SNPs) in six genes encoding BER proteins. Material/Methods Using TaqMan, we selected and genotyped the following SNPs: c.-441G>A (rs174538) of FEN1, c.2285T>C (rs1136410) of PARP1, c.580C>T (rs1799782) and c.1196A>G (rs25487) of XRCC1, c.*83A>C (rs4796030) and c.*50C>T (rs1052536) of LIG3, c.-7C>T (rs20579) of LIG1, and c.-468T>G (rs1760944) and c.444T>G (rs1130409) of APEX1 in 599 samples (288 rDD patients and 311 controls). Results We found a strong correlation between rDD and both SNPs of LIG3, their haplotypes, as well as a weaker association with the c.-468T>G of APEXI which diminished after Nyholt correction. Polymorphisms of LIG3 were also associated with early onset versus late onset depression, whereas the c.-468T>G polymorphism showed the opposite association. Conclusions The SNPs of genes involved in the repair of oxidative DNA damage may modulate rDD risk. Since this is an exploratory study, the results should to be treated with caution and further work needs to be done to elucidate the exact involvement of DNA damage and repair mechanisms in the development of this disease. PMID:27866211

Traceability of plant contribution in olive oil by amplified fragment length polymorphisms.

PubMed

Pafundo, Simona; Agrimonti, Caterina; Marmiroli, Nelson

2005-09-07

Application of DNA molecular markers to traceability of foods is thought to bring new benefit to consumer's protection. Even in a complex matrix such as olive oil, DNA could be traced with PCR markers such as the amplified fragment length polymorphisms (AFLPs). In this work, fluorescent AFLPs were optimized for the characterization of olive oil DNA, to obtain highly reproducible, high-quality fingerprints, testing different parameters: the concentrations of dNTPs and labeled primer, the kind of Taq DNA polymerase and thermal cycler, and the quantity of DNA employed. It was found that correspondence of fingerprinting by comparing results in oils and in plants was close to 70% and that the DNA extraction from olive oil was the limiting step for the reliability of AFLP profiles, due to the complex matrix analyzed.

T-cell receptor V sub. alpha. and C sub. alpha. alleles associated with multiple sclerosis and myasthenia gravis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oksenberg, J.R.; Cavalli-Sforza, L.L.; Steinman, L.

1989-02-01

Polymorphic markers in genes encoding the {alpha} chain of the human T-cell receptor (TcR) have been detected by Southern blot analysis in Pss I digests. Polymorphic bands were observed at 6.3 and 2.0 kilobases (kb) with frequencies of 0.30 and 0.44, respectively, in the general population. Using the polymerase chain reaction (PCR) method, the authors amplified selected sequences derived from the full-length TcR {alpha} cDNA probe. These PcR products were used as specific probes to demonstrate that the 6.3-kb polymorphic fragment hybridizes to the variable (V)-region probe and the 2.0-kb fragment hybridizes to the constant (C)-region probe. Segregation of themore » polymorphic bands was analyzed in family studies. To look for associations between these markers and autoimmune diseases, the authors have studied the restriction fragment length polymorphism distribution of the Pss I markers in patients with multiple sclerosis, myasthenia gravis, and Graves disease. Significant differences in the frequency of the polymorphic V{sub {alpha}} and C{sub {alpha}} markers were identified between patients and healthy individuals.« less

Methylenetetrahydrofolate reductase C677T and A1298C gene polymorphisms and therapy-related toxicity in children treated for acute lymphoblastic leukemia and non-Hodgkin lymphoma.

PubMed

Kantar, Mehmet; Kosova, Buket; Cetingul, Nazan; Gumus, Sevinc; Toroslu, Ertug; Zafer, Nur; Topcuoglu, Nejat; Aksoylar, Serap; Cinar, Mehtap; Tetik, Asli; Eroglu, Zuhal

2009-06-01

This study aimed to investigate the association of the methylenetetrahydrofolate reductase (MTHFR) gene C677T and A1298C polymorphisms with serum drug levels and toxicities after high-dose methotrexate (MTX) infusion. The study included 37 children with acute lymphoblastic leukemia or non-Hodgkin lymphoma. Serum MTX levels and toxicities of bone marrow, liver and kidney were analysed. Genotype analysis of the C677T and A1298C gene polymorphisms from genomic DNA of the subjects was performed by real-time PCR. Subjects with MTHFR polymorphism for C677T (CT, TT) had significantly higher MTX levels at 24 h (p = 0.009), and these genotypes did not seem to cause toxicity. Subjects with MTHFR polymorphism for A1298C (AC, CC) had significantly higher MTX levels at 48 h (p = 0.02), and had more grade III/IV anemia (p = 0.02), thrombocytopenia (p = 0.0001), elevated AST levels (p = 0.04) and frequent febrile neutropenic episodes (p = 0.004). The present study suggests that A1298C gene, but not C677T polymorphism is associated with MTX-related toxicity.

The CYP3A4 *1B polymorphism and prostate cancer susceptibility in a Portuguese population.

PubMed

Nogal, Ana; Coelho, Ana; Catarino, Raquel; Morais, António; Lobo, Francisco; Medeiros, Rui

2007-09-01

Testosterone exposure has been implicated in prostate carcinogenesis, and genes that alter its metabolism, such as CYP3A4, have been associated with prostate cancer susceptibility. The aim of our study was to assess the relationship between the CYP3A4 *1B polymorphism and its possible role in the development of prostate cancer. DNA samples obtained from the peripheral blood cells of 414 individuals diagnosed with prostate cancer and 337 healthy male donors were used in this case-control study. The CYP3A4*1B polymorphism was analyzed by polymerase chain reaction-restriction fragment length polymorphism methodology. We found no statistically significant differences in the distribution of the CYP3A4*1B genotypes between cases and controls (P = 0.470; odds ratio = 1.191; 95% confidence interval=0.740-1.918), as well as after the stratification of our analysis, according to important clinicopathologic parameters of prostate cancer. Our results suggest that the CYP3A4*1B polymorphism is not associated with prostate cancer risk within the Portuguese population.

DNA pooling: a comprehensive, multi-stage association analysis of ACSL6 and SIRT5 polymorphisms in schizophrenia.

PubMed

Chowdari, K V; Northup, A; Pless, L; Wood, J; Joo, Y H; Mirnics, K; Lewis, D A; Levitt, P R; Bacanu, S-A; Nimgaonkar, V L

2007-04-01

Many candidate gene association studies have evaluated incomplete, unrepresentative sets of single nucleotide polymorphisms (SNPs), producing non-significant results that are difficult to interpret. Using a rapid, efficient strategy designed to investigate all common SNPs, we tested associations between schizophrenia and two positional candidate genes: ACSL6 (Acyl-Coenzyme A synthetase long-chain family member 6) and SIRT5 (silent mating type information regulation 2 homologue 5). We initially evaluated the utility of DNA sequencing traces to estimate SNP allele frequencies in pooled DNA samples. The mean variances for the DNA sequencing estimates were acceptable and were comparable to other published methods (mean variance: 0.0008, range 0-0.0119). Using pooled DNA samples from cases with schizophrenia/schizoaffective disorder (Diagnostic and Statistical Manual of Mental Disorders edition IV criteria) and controls (n=200, each group), we next sequenced all exons, introns and flanking upstream/downstream sequences for ACSL6 and SIRT5. Among 69 identified SNPs, case-control allele frequency comparisons revealed nine suggestive associations (P<0.2). Each of these SNPs was next genotyped in the individual samples composing the pools. A suggestive association with rs 11743803 at ACSL6 remained (allele-wise P=0.02), with diminished evidence in an extended sample (448 cases, 554 controls, P=0.062). In conclusion, we propose a multi-stage method for comprehensive, rapid, efficient and economical genetic association analysis that enables simultaneous SNP detection and allele frequency estimation in large samples. This strategy may be particularly useful for research groups lacking access to high throughput genotyping facilities. Our analyses did not yield convincing evidence for associations of schizophrenia with ACSL6 or SIRT5.

A 3' untranslated region polymorphism rs2304277 in the DNA repair pathway gene OGG1 is a novel risk modulator for urothelial bladder carcinoma.

PubMed

Ahmed, Tayyaba; Nawaz, Saira; Noreen, Rabia; Bangash, Kashif Sardar; Rauf, Abdur; Younis, Muhammad; Anwar, Khursheed; Khawaja, Muhammad Athar; Azam, Maleeha; Qureshi, Abid Ali; Akhter, Saeed; Kiemeney, Lambertus A; Qamar, Raheel; Ali, Syeda Hafiza Benish

2018-03-01

Altered DNA repair capacity may affect an individual's susceptibility to cancers due to compromised genomic integrity. This study was designed to elucidate the association of selected polymorphisms in DNA repair genes with urothelial bladder carcinoma (UBC). OGG1 rs1052133 and rs2304277, XRCC1 rs1799782 and rs25487, XRCC3 rs861539, XPC rs2228001, and XPD rs13181 were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 200 UBC cases and 200 controls. We found association of OGG1 rs2304277 [odds ratio (OR) GG = 3.55, 95% confidence interval (CI) = 1.79-7.06] and XPC rs2228001 (OR AC = 2.38, 95% CI = 1.43-3.94) with UBC. In stratified analysis with respect to smoking status, OGG1 rs2304277 and XPC rs2228001 exhibited increased risk in smokers [(rs2304277 OR GG = 4.96, 95% CI = 1.51-16.30) (rs2228001 OR AC = 2.19, 95% CI = 1.02-4.72)] as well as nonsmokers [(rs2304277 OR GG = 2.95, 95% CI = 1.26-6.90) (rs2228001 OR AC = 2.57, 95% CI = 1.31-5.04)]. These polymorphisms were also associated with both low-grade [(rs2304277 OR GG = 3.73, 95% CI = 1.72-8.09) (rs2228001 OR AC = 2.18, 95% CI = 1.21-3.92)] and high-grade tumors [(rs2304277 OR GG = 3.45, 95% CI = 1.52-7.80) (rs2228001 OR AC = 2.81, 95% CI = 1.48-5.33)] as well as with non-muscle-invasive bladder cancer [(rs2304277 OR GG = 4.03, 95% CI = 1.87-8.67) (rs2228001 OR AC = 2.14, 95% CI = 1.20-3.81)] and muscle-invasive bladder cancer [(rs2304277 OR GG = 3.06, 95%CI = 1.31-7.13) (rs2228001 OR AC = 2.95, 95%CI = 1.51-5.75)]. This is the first study on DNA repair gene polymorphisms and UBC in the Pakistani population. It identifies OGG1 rs2304277 and replicates XPC rs2228001 as significant modulators of UBC susceptibility. © 2017 John Wiley & Sons Ltd/University College London.

Analysis of p53 gene mutations in human gliomas by polymerase chain reaction-based single-strand conformation polymorphism and DNA sequencing.

PubMed

Sarkar, F H; Kupsky, W J; Li, Y W; Sreepathi, P

1994-03-01

Mutations in the p53 gene have been recognized in brain tumors, and clonal expansion of p53 mutant cells has been shown to be associated with glioma progression. However, studies on the p53 gene have been limited by the need for frozen tissues. We have developed a method utilizing polymerase chain reaction (PCR) for the direct analysis of p53 mutation by single-strand conformation polymorphism (SSCP) and by direct DNA sequencing of the p53 gene using a single 10-microns paraffin-embedded tissue section. We applied this method to screen for p53 gene mutations in exons 5-8 in human gliomas utilizing paraffin-embedded tissues. Twenty paraffin blocks containing tumor were selected from surgical specimens from 17 different adult patients. Tumors included six anaplastic astrocytomas (AAs), nine glioblastomas (GBs), and two mixed malignant gliomas (MMGs). The tissue section on the stained glass slide was used to guide microdissection of an unstained adjacent tissue section to ensure > 90% of the tumor cell population for p53 mutational analysis. Simultaneously, microdissection of the tissue was also carried out to obtain normal tissue from adjacent areas as a control. Mutations in the p53 gene were identified in 3 of 17 (18%) patients by PCR-SSCP analysis and subsequently confirmed by PCR-based DNA sequencing. Mutations in exon 5 resulting in amino acid substitution were found in one thalamic AA (codon 158, CGC > CTT: Arg > Leu) and one cerebral hemispheric GB (codon 151, CCG > CTG: Pro > Leu).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the mechanism underlying a human chimera by SNP array analysis.

PubMed

Shin, So Youn; Yoo, Han-Wook; Lee, Beom Hee; Kim, Kun Suk; Seo, Eul-Ju

2012-09-01

Human chimerism resulting from the fusion of two different zygotes is a rare phenomenon. Two mechanisms of chimerism have been hypothesized: dispermic fertilization of an oocyte and its second polar body and dispermic fertilization of two identical gametes from parthenogenetic activation, and these can be identified and discriminated using DNA polymorphism. In the present study we describe a patient with chimerism presenting as a true hermaphrodite and applied single nucleotide polymorphism array analysis to demonstrate dispermic fertilization of two identical gametes from parthenogenetic activation as the underlying mechanism at the whole chromosome level. We suggest that application of genotyping array analysis to the diagnostic process in patients with disorders of sex development will help identify more human chimera patients and increase our understanding of the underlying mechanisms. Copyright © 2012 Wiley Periodicals, Inc.

Lactobacillus fabifermentans sp. nov. and Lactobacillus cacaonum sp. nov., isolated from Ghanaian cocoa fermentations.

PubMed

De Bruyne, Katrien; Camu, Nicholas; De Vuyst, Luc; Vandamme, Peter

2009-01-01

Two Gram-positive bacterial strains, LMG 24284T and LMG 24285T, were isolated from different spontaneous cocoa bean heap fermentations in Ghana. Analysis of their 16S rRNA gene sequences indicated that they were members of the Lactobacillus plantarum and Lactobacillus salivarius species groups, respectively. DNA-DNA hybridization experiments with their nearest phylogenetic neighbours demonstrated that both strains represented novel species that could be differentiated from their nearest neighbours by pheS sequence analysis, whole-cell protein electrophoresis, fluorescent amplified fragment length polymorphism analysis and biochemical characterization. Therefore, two novel Lactobacillus species are proposed, Lactobacillus fabifermentans sp. nov. (type strain LMG 24284T =DSM 21115T) and Lactobacillus cacaonum sp. nov. (type strain LMG 24285T =DSM 21116T).

A new polymorphic and multicopy MHC gene family related to nonmammalian class I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leelayuwat, C.; Degli-Esposti, M.A.; Abraham, L.J.

1994-12-31

The authors have used genomic analysis to characterize a region of the central major histocompatibility complex (MHC) spanning {approximately} 300 kilobases (kb) between TNF and HLA-B. This region has been suggested to carry genetic factors relevant to the development of autoimmune diseases such as myasthenia gravis (MG) and insulin dependent diabetes mellitus (IDDM). Genomic sequence was analyzed for coding potential, using two neural network programs, GRAIL and GeneParser. A genomic probe, JAB, containing putative coding sequences (PERB11) located 60 kb centromeric of HLA-B, was used for northern analysis of human tissues. Multiple transcripts were detected. Southern analysis of genomic DNAmore » and overlapping YAC clones, covering the region from BAT1 to HLA-F, indicated that there are at least five copies of PERB11, four of which are located within this region of the MHC. The partial cDNA sequence of PERB11 was obtained from poly-A RNA derived from skeletal muscle. The putative amino acid sequence of PERB11 shares {approximately} 30% identity to MHC class I molecules from various species, including reptiles, chickens, and frogs, as well as to other MHC class I-like molecules, such as the IgG FcR of the mouse and rat and the human Zn-{alpha}2-glycoprotein. From direct comparison of amino acid sequences, it is concluded that PERB11 is a distinct molecule more closely related to nonmammalian than known mammalian MHC class I molecules. Genomic sequence analysis of PERB11 from five MHC ancestral haplotypes (AH) indicated that the gene is polymorphic at both DNA and protein level. The results suggest that the authors have identified a novel polymorphic gene family with multiple copies within the MHC. 48 refs., 10 figs., 2 tabs.« less

Buccal DNA collection: comparison of buccal swabs with FTA cards.

PubMed

Milne, Elizabeth; van Bockxmeer, Frank M; Robertson, Laila; Brisbane, Joanna M; Ashton, Lesley J; Scott, Rodney J; Armstrong, Bruce K

2006-04-01

Collection and analysis of DNA, most commonly from blood or buccal cells, is becoming more common in epidemiologic studies. Buccal samples, which are painless to take and relatively easily collected, are often the preferred source. There are several buccal cell collection methods: swabs, brushes, mouthwash, and treated cards, such as FTA or IsoCode cards. Few studies have systematically compared methods of buccal cell collection with respect to DNA yield and amplification success under similar conditions. We compared buccal DNA collection and amplification using buccal swabs and FTA cards in 122 control subjects from our Australian case-control study of childhood acute lymphoblastic leukaemia. Buccal DNA was quantified using a real-time PCR for beta-actin and genotyped at the loci of three polymorphisms (MTHFR 677C>T, ACE I/D, and XPD 1012G>A). PCR was successful with DNA from buccal swabs for 62% to 89% of subjects and from FTA cards for 83% to 100% of subjects, depending on the locus. The matched pair odds ratios (95% confidence interval) comparing success of FTA cards with buccal swabs are as follows: MTHFR 677C>T using PCR-RFLP, 12.5 (11.6-13.5) and using real-time PCR, 130.0 (113.1-152.8); ACE I/D using PCR-amplified fragment length polymorphism, 3.36 (3.2-3.5); XPD 1012G>A using real-time PCR, 150.0 (132.7-172.3). FTA cards are a robust DNA collection method and generally produce DNA suitable for PCR more reliably than buccal swabs. There are, however, technical challenges in handling discs punched from FTA cards that intending users should be aware of.

Novel quantitative real-time LCR for the sensitive detection of SNP frequencies in pooled DNA: method development, evaluation and application.

PubMed

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-19

Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food.

Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

PubMed Central

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-01

Background Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. Methods The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. Conclusions The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. Significance The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food. PMID:21283808

MTHFR polymorphisms in Puerto Rican children with isolated congenital heart disease and their mothers

PubMed Central

García-Fragoso, Lourdes; García-García, Inés; Leavitt, Gloria; Renta, Jessicca; Ayala, Miguel A.; Cadilla, Carmen L.

2010-01-01

Congenital heart defects (CHD) are among the most common birth defects. There is evidence suggesting that polymorphisms in folate metabolism could alter susceptibility to CHD. The MTHFR 677TT genotype has been associated with the development of structural congenital heart malformations. The objective of this study was to identify common polymorphisms in the MTHFR gene in children with isolated CHD and their mothers. The DNA analysis for the C677T and A1298C mutations was performed. The study group included 27 mothers, 27 children with CHD, and 220 controls. The prevalence of the TT polymorphism was higher in mothers (22%) than in controls (10%). Compound heterozygosity for both polymorphisms was 3.7 times more common in children with CHD than in the newborn controls. Mothers of children with CHD were more likely to be compound heterozygotes. The higher prevalence of C677T polymorphisms in mothers of children with CHD and of compound heterozygosity for both polymorphisms suggests the possible role of folic acid in the prevention of CHD. Due to the relation of this enzyme to folate metabolism, current folate recommendations for women in childbearing age in Puerto Rico to reduce neural tube defects may need to be extended to the prevention of CHD. PMID:20657745

MTHFR polymorphisms in Puerto Rican children with isolated congenital heart disease and their mothers.

PubMed

García-Fragoso, Lourdes; García-García, Inés; Leavitt, Gloria; Renta, Jessicca; Ayala, Miguel A; Cadilla, Carmen L

2010-03-01

Congenital heart defects (CHD) are among the most common birth defects. There is evidence suggesting that polymorphisms in folate metabolism could alter susceptibility to CHD. The MTHFR 677TT genotype has been associated with the development of structural congenital heart malformations. The objective of this study was to identify common polymorphisms in the MTHFR gene in children with isolated CHD and their mothers. The DNA analysis for the C677T and A1298C mutations was performed. The study group included 27 mothers, 27 children with CHD, and 220 controls. The prevalence of the TT polymorphism was higher in mothers (22%) than in controls (10%). Compound heterozygosity for both polymorphisms was 3.7 times more common in children with CHD than in the newborn controls. Mothers of children with CHD were more likely to be compound heterozygotes. The higher prevalence of C677T polymorphisms in mothers of children with CHD and of compound heterozygosity for both polymorphisms suggests the possible role of folic acid in the prevention of CHD. Due to the relation of this enzyme to folate metabolism, current folate recommendations for women in childbearing age in Puerto Rico to reduce neural tube defects may need to be extended to the prevention of CHD.

Levels of intra-specific AFLP diversity in tuber-bearing potato species with different breeding systems and ploidy levels

USDA-ARS?s Scientific Manuscript database

DNA-based marker analysis of plant genebank material has become a useful tool in the evaluation of levels of genetic diversity and for the informed use and maintenance of germplasm. In this study we quantify levels of Amplified Fragment Length Polymorphism (AFLP) in representative accessions of wild...

Random Amplified Polymorphic DNA PCR in the Teaching of Molecular Epidemiology

ERIC Educational Resources Information Center

Reinoso, Elina B.; Bettera, Susana G.

2016-01-01

In this article, we describe a basic practical laboratory designed for fifth-year undergraduate students of Microbiology as part of the Epidemiology course. This practice provides the students with the tools for molecular epidemiological analysis of pathogenic microorganisms using a rapid and simple PCR technique. The aim of this work was to assay…

Detection of Variation in Long-Term Micropropagated Mature Pistachio via DNA-Based Molecular Markers.

PubMed

Akdemir, Hülya; Suzerer, Veysel; Tilkat, Engin; Onay, Ahmet; Çiftçi, Yelda Ozden

2016-12-01

Determination of genetic stability of in vitro-grown plantlets is needed for safe and large-scale production of mature trees. In this study, genetic variation of long-term micropropagated mature pistachio developed through direct shoot bud regeneration using apical buds (protocol A) and in vitro-derived leaves (protocol B) was assessed via DNA-based molecular markers. Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) were employed, and the obtained PIC values from RAPD (0.226), ISSR (0.220), and AFLP (0.241) showed that micropropagation of pistachio for different periods of time resulted in "reasonable polymorphism" among donor plant and its 18 clones. Mantel's test showed a consistence polymorphism level between marker systems based on similarity matrices. In conclusion, this is the first study on occurrence of genetic variability in long-term micropropagated mature pistachio plantlets. The obtained results clearly indicated that different marker approaches used in this study are reliable for assessing tissue culture-induced variations in long-term cultured pistachio plantlets.

Association study of estrogen receptor alpha gene polymorphisms with spontaneous abortion: is this a possible reason for unexplained spontaneous abortion?

PubMed

Anousha, Negin; Hossein-Nezhad, Arash; Biramijamal, Firouzeh; Rahmani, Ali; Maghbooli, Zhila; Aghababaei, Elahe; Nemati, Shahram

2013-01-01

Estrogen plays a crucial role in fetal and placental development through estrogen receptors. Association of estrogen receptor alpha gene (ESR1) polymorphisms with spontaneous abortion has been shown in some studies. Our main goal was to study the potential association of spontaneous abortion with the ESR1 gene variations (PvuII and XbaI) in fetal tissue. Totally, 161 samples were recruited including 80 samples of formalin-fixed paraffin-embedded fetal tissue from spontaneous abortion and 81 samples of normal term placental tissue. The restriction fragment length polymorphism (RFLP) method was performed for genotyping the rs2234693 (A/G XbaI) and rs9340799 (T/C PvuII) single nucleotide polymorphisms located in intron 1 of ESR1. The results have been confirmed by DNA sequencing analysis. The different genotypes distribution was detected in two study groups. Haplotype analysis indicated that ppxx is protective genotype against spontaneous abortion (P = 0.01). In conclusion, the potential role of ESR1 genetic variation in spontaneous abortion might be valuable in high-risk subjects, and that needs to be confirmed with future studies.

Association Study of Estrogen Receptor Alpha Gene Polymorphisms with Spontaneous Abortion: Is This a Possible Reason for Unexplained Spontaneous Abortion?

PubMed Central

Anousha, Negin; Hossein-Nezhad, Arash; Biramijamal, Firouzeh; Rahmani, Ali; Maghbooli, Zhila; Aghababaei, Elahe; Nemati, Shahram

2013-01-01

Estrogen plays a crucial role in fetal and placental development through estrogen receptors. Association of estrogen receptor alpha gene (ESR1) polymorphisms with spontaneous abortion has been shown in some studies. Our main goal was to study the potential association of spontaneous abortion with the ESR1 gene variations (PvuII and XbaI) in fetal tissue. Totally, 161 samples were recruited including 80 samples of formalin-fixed paraffin-embedded fetal tissue from spontaneous abortion and 81 samples of normal term placental tissue. The restriction fragment length polymorphism (RFLP) method was performed for genotyping the rs2234693 (A/G XbaI) and rs9340799 (T/C PvuII) single nucleotide polymorphisms located in intron 1 of ESR1. The results have been confirmed by DNA sequencing analysis. The different genotypes distribution was detected in two study groups. Haplotype analysis indicated that ppxx is protective genotype against spontaneous abortion (P = 0.01). In conclusion, the potential role of ESR1 genetic variation in spontaneous abortion might be valuable in high-risk subjects, and that needs to be confirmed with future studies. PMID:24228243

Methylenetetrahydrofolate reductase gene polymorphisms and acute lymphoblastic leukemia risk: a meta-analysis based on 28 case-control studies.

PubMed

Tong, Na; Sheng, Xiaojing; Wang, Meilin; Fang, Yongjun; Shi, Danni; Zhang, Zhizhong; Zhang, Zhengdong

2011-10-01

Methylenetetrahydrofolate reductase (MTHFR) is involved in DNA methylation and nucleotide synthesis. Accumulated evidence has demonstrated that C677T and A1298C polymorphisms of the MTHFR gene are associated with acute lymphoblastic leukemia (ALL) risk, but the results have been inconclusive. To determine a more precise estimation, we performed a meta-analysis of 28 studies with 4240 cases and 9289 controls. We found that the 677TT genotype showed a reduced risk of ALL compared with the 677CC genotype in the overall population (odds ratio [OR] 0.76, 95% confidence interval [CI] 0.61-0.92). The reduced risk was pronounced only among the Caucasian population (OR 0.68, 95% CI 0.51-0.90), not the Asian (OR 0.89, 95% CI 0.75-1.05). For the MTHFR A1298C polymorphism, no significant association with ALL susceptibility was observed in the pooled analyses. However, significantly increased ALL risk was found in childhood in the comparison of 1298CA versus AA genotype. This study provides evidence that MTHFR polymorphisms may play an important role in the development of ALL.

XRCC3 polymorphism is associated with hypertension-induced left ventricular hypertrophy.

PubMed

Ariyandy, Andi; Sakai, Chiemi; Ishida, Mari; Mizuta, Ryusei; Miyagawa, Kiyoshi; Tashiro, Satoshi; Kinomura, Aiko; Hiraaki, Koji; Ueda, Keitaro; Yoshizumi, Masao; Ishida, Takafumi

2018-06-01

Deficiency of X-ray repair cross-complementing protein 3 (XRCC3), a DNA-damage repair molecule, and the 241Met variant of XRCC3 have been reported to increase endoreduplication, which induces polyploidy. The aims of this study were to determine the impact of the XRCC3 polymorphism on the incidence of hypertension-induced left ventricular hypertrophy (LVH) and to investigate the mechanisms underlying any potential relationship. Patients undergoing chronic hemodialysis (n = 77) were genotyped to assess for the XRCC3 Thr241Met polymorphism. The XRCC3 241Thr/Met genotype was more frequent in the LVH (+) group than in the LVH (-) group (42.3 vs. 13.7%, χ2 = 7.85, p = 0.0051). To investigate possible mechanisms underlying these observations, human XRCC3 cDNA of 241Thr or that of 241Met was introduced into cultured CHO cells. The surface area of CHO cells expressing XRCC3 241Met was larger than that expressing 241Thr. Spontaneous DNA double-strand breaks accumulated to a greater degree in NIH3T3 cells expressing 241Met (3T3-241Met) than in those expressing 241Thr (3T3-241Thr). DNA damage caused by radiation induced cell senescence more frequently in 3T3-241Met. The levels of basal and TNF-α-stimulated MCP-1 mRNA and protein secretion were higher in 3T3-241Met. Finally, FACS analysis revealed that the cell percentage in G2/M phase including polyploidy was significantly higher in 3T3-241Met than in 3T3-241Thr. Furthermore, the basal level of MCP-1 mRNA positively correlated with the cell percentage in G2/M phase and polyploidy. These data suggest that the XRCC3 241Met increases the risk of LVH via accumulation of DNA damage, thereby altering cell cycle progression and inducing cell senescence and a proinflammatory phenotype.

Genetic polymorphisms of genes involved in DNA repair and metabolism influence micronucleus frequencies in human peripheral blood lymphocytes.

PubMed

Dhillon, Varinderpal S; Thomas, Philip; Iarmarcovai, G; Kirsch-Volders, Micheline; Bonassi, Stefano; Fenech, Michael

2011-01-01

The cytokinesis-block micronucleus cytome (CBMNCyt) assay is a widely used technique for measuring DNA damage in human populations. The formation of micronuclei (MN) in dividing cells can result from chromosome breakage due to unrepaired or mis-repaired DNA lesions or chromosome malsegregation due to mitotic malfunction. The sensitivity of the MN assay to polymorphisms in various genes involved in DNA repair, activation/deactivation of carcinogens/chemicals/drugs/alcohol, folate metabolism pathway and micronutrient transport has been extensively reported in the literature. MN frequency is also an important index for determining DNA repair efficiency phenotype (including mis-repair), response to environmental exposure and identifying various dietary factors required for optimal genome stability. The aim of the present study is to review the reported in vivo associations between genotype and MN frequency in humans taking into considerations the presence of interactions with nutrients levels and/or exposure to genotoxins. One hundred and eleven publications linking MN frequency in peripheral blood lymphocytes to gene polymorphism were retrieved from PubMed. After applying exclusion criteria, only 37 studies were evaluated in the present review. Polymorphisms in XRCC1 (Arg280His), ERCC2 (Lys751Gln), CYP2E1 (c1/c2) and MTR (A2756G) were consistently associated with the MN formation. These results contribute substantial evidence to the hypothesis that genotype may influence MN frequency in human cells.

Extremely low nucleotide polymorphism in Pinus krempfii Lecomte, a unique flat needle pine endemic to Vietnam

PubMed Central

Wang, Baosheng; Khalili Mahani, Marjan; Ng, Wei Lun; Kusumi, Junko; Phi, Hai Hong; Inomata, Nobuyuki; Wang, Xiao-Ru; Szmidt, Alfred E

2014-01-01

Pinus krempfii Lecomte is a morphologically and ecologically unique pine, endemic to Vietnam. It is regarded as vulnerable species with distribution limited to just two provinces: Khanh Hoa and Lam Dong. Although a few phylogenetic studies have included this species, almost nothing is known about its genetic features. In particular, there are no studies addressing the levels and patterns of genetic variation in natural populations of P. krempfii. In this study, we sampled 57 individuals from six natural populations of P. krempfii and analyzed their sequence variation in ten nuclear gene regions (approximately 9 kb) and 14 mitochondrial (mt) DNA regions (approximately 10 kb). We also analyzed variation at seven chloroplast (cp) microsatellite (SSR) loci. We found very low haplotype and nucleotide diversity at nuclear loci compared with other pine species. Furthermore, all investigated populations were monomorphic across all mitochondrial DNA (mtDNA) regions included in our study, which are polymorphic in other pine species. Population differentiation at nuclear loci was low (5.2%) but significant. However, structure analysis of nuclear loci did not detect genetically differentiated groups of populations. Approximate Bayesian computation (ABC) using nuclear sequence data and mismatch distribution analysis for cpSSR loci suggested recent expansion of the species. The implications of these findings for the management and conservation of P. krempfii genetic resources were discussed. PMID:25360263

The Flavin-Containing Monooxygenase 3 Gene and Essential Hypertension: The Joint Effect of Polymorphism E158K and Cigarette Smoking on Disease Susceptibility

PubMed Central

Bushueva, Olga; Solodilova, Maria; Churnosov, Mikhail; Ivanov, Vladimir; Polonikov, Alexey

2014-01-01

Gene encoding flavin-containing monooxygenase 3 (FMO3), a microsomal antioxidant defense enzyme, has been suggested to contribute to essential hypertension (EH). The present study was designed to investigate whether common functional polymorphism E158K (rs2266782) of the FMO3 gene is associated with EH susceptibility in a Russian population. A total of 2 995 unrelated subjects from Kursk (1 362 EH patients and 843 healthy controls) and Belgorod (357 EH patients and 422 population controls) regions of Central Russia were recruited for this study. DNA samples from all study participants were genotyped for the FMO3 gene polymorphism through PCR followed by RFLP analysis. We found that the polymorphism E158K is associated with increased risk of essential hypertension in both discovery population from Kursk region (OR 1.36 95% CI 1.09–1.69, P = 0.01) and replication population from Belgorod region (OR 1.54 95% CI 1.07–1.89, P = 0.02) after adjustment for gender and age using logistic regression analysis. Further analysis showed that the increased hypertension risk in carriers of genotype 158KK gene occurred in cigarette smokers, whereas nonsmoker carriers of this genotype did not show the disease risk. This is the first study reporting the association of the FMO3 gene polymorphism and the risk of essential hypertension. PMID:25243081

Genetic Diversity of Sheep Breeds from Albania, Greece, and Italy Assessed by Mitochondrial DNA and Nuclear Polymorphisms (SNPs)

PubMed Central

Pariset, Lorraine; Mariotti, Marco; Gargani, Maria; Joost, Stephane; Negrini, Riccardo; Perez, Trinidad; Bruford, Michael; Ajmone Marsan, Paolo; Valentini, Alessio

2011-01-01

We employed mtDNA and nuclear SNPs to investigate the genetic diversity of sheep breeds of three countries of the Mediterranean basin: Albania, Greece, and Italy. In total, 154 unique mtDNA haplotypes were detected by means of D-loop sequence analysis. The major nucleotide diversity was observed in Albania. We identified haplogroups, A, B, and C in Albanian and Greek samples, while Italian individuals clustered in groups A and B. In general, the data show a pattern reflecting old migrations that occurred in postneolithic and historical times. PCA analysis on SNP data differentiated breeds with good correspondence to geographical locations. This could reflect geographical isolation, selection operated by local sheep farmers, and different flock management and breed admixture that occurred in the last centuries. PMID:22125424

[Application of multiple polymorphism genetic markers in determination of half sibling sharing a same mother].

PubMed

Que, Ting-zhi; Zhao, Shu-min; Li, Cheng-tao

2010-08-01

Determination strategies for half sibling sharing a same mother were investigated through the detection of autosomal and X-chromosomal STR (X-STR) loci and polymorphisms on hypervariable (HV) region of mitochondrial DNA (mtDNA). Genomic DNA were extracted from blood stain samples of the 3 full siblings and one dubious half sibling sharing the same mother with them. Fifteen autosomal STR loci were genotyped by Sinofiler kit, and 19 X-STR loci were genotyped by Mentype Argus X-8 kit and 16 plex in-house system. Polymorphisms of mtDNA HV-I and HV-II were also detected with sequencing technology. Full sibling relationship between the dubious half sibling and each of the 3 full siblings were excluded based on the results of autosomal STR genotyping and calculation of full sibling index (FSI) and half sibling index (HIS). Results of sequencing for mtDNA HV-I and HV-II showed that all of the 4 samples came from a same maternal line. X-STR genotyping results determined that the dubious half sibling shared a same mother with the 3 full siblings. It is reliable to combine three different genotyping technologies including autosomal STR, X-STR and sequencing of mtDNA HV-I and HV-II for determination of half sibling sharing a same mother.

Identification of apple cultivars on the basis of simple sequence repeat markers.

PubMed

Liu, G S; Zhang, Y G; Tao, R; Fang, J G; Dai, H Y

2014-09-12

DNA markers are useful tools that play an important role in plant cultivar identification. They are usually based on polymerase chain reaction (PCR) and include simple sequence repeats (SSRs), inter-simple sequence repeats, and random amplified polymorphic DNA. However, DNA markers were not used effectively in the complete identification of plant cultivars because of the lack of known DNA fingerprints. Recently, a novel approach called the cultivar identification diagram (CID) strategy was developed to facilitate the use of DNA markers for separate plant individuals. The CID was designed whereby a polymorphic maker was generated from each PCR that directly allowed for cultivar sample separation at each step. Therefore, it could be used to identify cultivars and varieties easily with fewer primers. In this study, 60 apple cultivars, including a few main cultivars in fields and varieties from descendants (Fuji x Telamon) were examined. Of the 20 pairs of SSR primers screened, 8 pairs gave reproducible, polymorphic DNA amplification patterns. The banding patterns obtained from these 8 primers were used to construct a CID map. Each cultivar or variety in this study was distinguished from the others completely, indicating that this method can be used for efficient cultivar identification. The result contributed to studies on germplasm resources and the seedling industry in fruit trees.

Combination of DNA-based and conventional methods to detect human leukocyte antigen polymorphism and its use for paternity testing.

PubMed

Kereszturya, László; Rajczya, Katalin; Lászikb, András; Gyódia, Eva; Pénzes, Mária; Falus, András; Petrányia, Gyõzõ G

2002-03-01

In cases of disputed paternity, the scientific goal is to promote either the exclusion of a falsely accused man or the affiliation of the alleged father. Until now, in addition to anthropologic characteristics, the determination of genetic markers included human leukocyte antigen gene variants; erythrocyte antigens and serum proteins were used for that reason. Recombinant DNA techniques provided a new set of highly variable genetic markers based on DNA nucleotide sequence polymorphism. From the practical standpoint, the application of these techniques to paternity testing provides greater versatility than do conventional genetic marker systems. The use of methods to detect the polymorphism of human leukocyte antigen loci significantly increases the chance of validation of ambiguous results in paternity testing. The outcome of 2384 paternity cases investigated by serologic and/or DNA-based human leukocyte antigen typing was statistically analyzed. Different cases solved by DNA typing are presented involving cases with one or two accused men, exclusions and nonexclusions, and tests of the paternity of a deceased man. The results provide evidence for the advantage of the combined application of various techniques in forensic diagnostics and emphasizes the outstanding possibilities of DNA-based assays. Representative examples demonstrate the strength of combined techniques in paternity testing.

Screening of cytoplasmic DNA diversity between and within Lupinus mutabilis Sweet and Lupinus albus sensu lato by restriction fragment length polymorphism (RFLP).

PubMed

Olczak, T; Rurek, M; Jańska, H; Augustyniak, H; Sawicka-Sienkiewicz, E J

2001-01-01

Seven populations and five mutant lines of the Andean lupin and four species from the section Albus were screened for their mitochondrial and chloroplast polymorphisms. For this purpose the RFLP method with EcoRI as a restriction enzyme was used. Lupinus luteus, Lupinus albus and Phaseolus vulgaris organellar clones as well as amplified fragments were used as probes. We found that mitochondrial probes were more suitable than chloroplast probes for identification of inter- and intra-specific variations within the examined material. Most mitochondrial probes differentiate the two species investigated. A high level of mitochondrial polymorphism was observed among the populations of L. mutabilis in contrast to monomorphism among the species in the section Albus. A limited polymorphism was detected between the mutant lines of L. mutabilis. We conclude from this study that the mitochondrial RFLP analysis is a valuable tool for identification of variability among Andean lupin populations.

Analysis of vitamin D receptor gene polymorphisms in patients with chronic periodontitis.

PubMed

Gunes, Sezgin; Sumer, A Pinar; Keles, Gonca Cayir; Kara, Nurten; Koprulu, Hulya; Bagci, Hasan; Bek, Yuksel

2008-01-01

Genetic polymorphisms in the vitamin D receptor (VDR) gene are related to bone mineral density, bone turnover, and diseases with bone loss. Alveolar bone loss is a key feature in periodontitis. The aim of this study was to determine whether severe generalized chronic periodontitis (CP) in a Turkish population was associated with polymorphisms in the VDR gene. Samples of venous blood and DNA were obtained from 72 patients with severe generalized chronic periodontitis and 102 healthy controls. The polymorphic regions were amplified using PCR followed by digestion with restriction enzymes BsmI A/G(rs1544410), ApaI G/T(rs11168271), TaqI T/C(rs731236), and analyzed electrophoretically. Genotype and allele frequencies were calculated. There were no statistically significant differences in the frequencies of VDR BsmI, ApaI, TaqI genotypes between the CP patients and healthy controls. The GTT haplotype, constructed from the three adjacent restriction fragment length polymorphisms was found to be over-represented among CP cases. This corresponded an OR of 2.4 (95% confidence interval, 1.12-5.18) for heterozygous carriers and 2.27 (95% confidence interval, 0.95-5.4) for homozygous carrier of the risk haplotype. The present findings indicated that BsmI, ApaI, TaqI polymorphisms of the VDR gene were not associated with the severe generalized CP in the studied Turkish patients. Moreover, the VDR genotypes based on haplotype analysis may be associated with chronic periodontitis. In the future, diagnostic periodontal risk assessments like polymorphisms may be useful in detection of individuals susceptible for periodontitis.

Analysis of genetic variation in Ganoderma Lucidum after space flight

NASA Astrophysics Data System (ADS)

Qi, Jian-Jun; Ma, Rong-Cai; Chen, Xiang-Dong; Lan, Jin

A modified CTAB method was used in the extraction of total cellular DNA of Ganoderma lucidum. Four strains Cx, Ch, C3 and C4, and their counterparts, four space flown strains Sx, Xh, S3 and S4, were analysed by amplified fragment length polymorphism (AFLP) with several primer combinations. Polymorphic bands were detected between Sx and Cx, S3 and C3, respectively. Somatic incompatibility tests further confirmed their heterogeneity. However, no disparity between Sh and Ch, S4 and C4 was detectable. The results suggest that spaceflight may be used to accelerate breeding of Ganoderma lucidum strains for commercial cultivation.

MCT1, MCT4 and CD147 gene polymorphisms in healthy horses and horses with myopathy.

PubMed

Mykkänen, A K; Koho, N M; Reeben, M; McGowan, C M; Pösö, A R

2011-12-01

Polymorphisms in human lactate transporter proteins (monocarboxylate transporters; MCTs), especially the MCT1 isoform, can affect lactate transport activity and cause signs of exercise-induced myopathy. Muscles express MCT1, MCT4 and CD147, an ancillary protein, indispensable for the activity of MCT1 and MCT4. We sequenced the coding sequence (cDNA) of horse MCT4 for the first time and examined polymorphisms in the cDNA of MCT1, MCT4 and CD147 of 16 healthy horses. To study whether signs of myopathy are linked to the polymorphisms, biopsy samples were taken from 26 horses with exercise-induced recurrent myopathy. Two polymorphisms that cause a change in amino acid sequence were found in MCT1 (Val(432)Ile and Lys(457)Gln) and one in CD147 (Met(125)Val). All polymorphisms in MCT4 were silent. Mutations in MCT1 or CD147 in equine muscle were not associated with myopathy. In the future, a functional study design is needed to evaluate the physiological role of the polymorphisms found. Copyright © 2010 Elsevier Ltd. All rights reserved.

A Cross-Cancer Genetic Association Analysis of the DNA Repair and DNA Damage Signaling Pathways for Lung, Ovary, Prostate, Breast, and Colorectal Cancer.

PubMed

Scarbrough, Peter M; Weber, Rachel Palmieri; Iversen, Edwin S; Brhane, Yonathan; Amos, Christopher I; Kraft, Peter; Hung, Rayjean J; Sellers, Thomas A; Witte, John S; Pharoah, Paul; Henderson, Brian E; Gruber, Stephen B; Hunter, David J; Garber, Judy E; Joshi, Amit D; McDonnell, Kevin; Easton, Doug F; Eeles, Ros; Kote-Jarai, Zsofia; Muir, Kenneth; Doherty, Jennifer A; Schildkraut, Joellen M

2016-01-01

DNA damage is an established mediator of carcinogenesis, although genome-wide association studies (GWAS) have identified few significant loci. This cross-cancer site, pooled analysis was performed to increase the power to detect common variants of DNA repair genes associated with cancer susceptibility. We conducted a cross-cancer analysis of 60,297 single nucleotide polymorphisms, at 229 DNA repair gene regions, using data from the NCI Genetic Associations and Mechanisms in Oncology (GAME-ON) Network. Our analysis included data from 32 GWAS and 48,734 controls and 51,537 cases across five cancer sites (breast, colon, lung, ovary, and prostate). Because of the unavailability of individual data, data were analyzed at the aggregate level. Meta-analysis was performed using the Association analysis for SubSETs (ASSET) software. To test for genetic associations that might escape individual variant testing due to small effect sizes, pathway analysis of eight DNA repair pathways was performed using hierarchical modeling. We identified three susceptibility DNA repair genes, RAD51B (P < 5.09 × 10(-6)), MSH5 (P < 5.09 × 10(-6)), and BRCA2 (P = 5.70 × 10(-6)). Hierarchical modeling identified several pleiotropic associations with cancer risk in the base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination pathways. Only three susceptibility loci were identified, which had all been previously reported. In contrast, hierarchical modeling identified several pleiotropic cancer risk associations in key DNA repair pathways. Results suggest that many common variants in DNA repair genes are likely associated with cancer susceptibility through small effect sizes that do not meet stringent significance testing criteria. ©2015 American Association for Cancer Research.

Target capture enrichment of nuclear SNP markers for massively parallel sequencing of degraded and mixed samples.

PubMed

Bose, Nikhil; Carlberg, Katie; Sensabaugh, George; Erlich, Henry; Calloway, Cassandra

2018-05-01

DNA from biological forensic samples can be highly fragmented and present in limited quantity. When DNA is highly fragmented, conventional PCR based Short Tandem Repeat (STR) analysis may fail as primer binding sites may not be present on a single template molecule. Single Nucleotide Polymorphisms (SNPs) can serve as an alternative type of genetic marker for analysis of degraded samples because the targeted variation is a single base. However, conventional PCR based SNP analysis methods still require intact primer binding sites for target amplification. Recently, probe capture methods for targeted enrichment have shown success in recovering degraded DNA as well as DNA from ancient bone samples using next-generation sequencing (NGS) technologies. The goal of this study was to design and test a probe capture assay targeting forensically relevant nuclear SNP markers for clonal and massively parallel sequencing (MPS) of degraded and limited DNA samples as well as mixtures. A set of 411 polymorphic markers totaling 451 nuclear SNPs (375 SNPs and 36 microhaplotype markers) was selected for the custom probe capture panel. The SNP markers were selected for a broad range of forensic applications including human individual identification, kinship, and lineage analysis as well as for mixture analysis. Performance of the custom SNP probe capture NGS assay was characterized by analyzing read depth and heterozygote allele balance across 15 samples at 25 ng input DNA. Performance thresholds were established based on read depth ≥500X and heterozygote allele balance within ±10% deviation from 50:50, which was observed for 426 out of 451 SNPs. These 426 SNPs were analyzed in size selected samples (at ≤75 bp, ≤100 bp, ≤150 bp, ≤200 bp, and ≤250 bp) as well as mock degraded samples fragmented to an average of 150 bp. Samples selected for ≤75 bp exhibited 99-100% reportable SNPs across varied DNA amounts and as low as 0.5 ng. Mock degraded samples at 1 ng and 10 ng exhibited >90% reportable SNPs. Finally, two-person male-male mixtures were tested at 10 ng in contributor varying ratios. Overall, 85-100% of alleles unique to the minor contributor were observed at all mixture ratios. Results from these studies using the SNP probe capture NGS system demonstrates proof of concept for application to forensically relevant degraded and mixed DNA samples. Copyright © 2018 Elsevier B.V. All rights reserved.

The Science and Issues of Human DNA Polymorphisms: A Training Workshop for High School Biology Teachers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Micklos, David A.

2006-10-30

This project achieved its goal of implementing a nationwide training program to introduce high school biology teachers to the key uses and societal implications of human DNA polymorphisms. The 2.5-day workshop introduced high school biology faculty to a laboratory-based unit on human DNA polymorphisms â which provides a uniquely personal perspective on the science and Ethical, Legal and Social Implications (ELSI) of the Human Genome Project. As proposed, 12 workshops were conducted at venues across the United States. The workshops were attended by 256 high school faculty, exceeding proposed attendance of 240 by 7%. Each workshop mixed theoretical, laboratory, andmore » computer work with practical and ethical implications. Program participants learned simplified lab techniques for amplifying three types of chromosomal polymorphisms: an Alu insertion (PV92), a VNTR (pMCT118/D1S80), and single nucleotide polymorphisms (SNPs) in the mitochondrial control region. These polymorphisms illustrate the use of DNA variations in disease diagnosis, forensic biology, and identity testing - and provide a starting point for discussing the uses and potential abuses of genetic technology. Participants also learned how to use their Alu and mitochondrial data as an entrée to human population genetics and evolution. Our work to simplify lab techniques for amplifying human DNA polymorphisms in educational settings culminated with the release in 1998 of three Advanced Technology (AT) PCR kits by Carolina Biological Supply Company, the nationâÂÂs oldest educational science supplier. The kits use a simple 30-minute method to isolate template DNA from hair sheaths or buccal cells and streamlined PCR chemistry based on Pharmacia Ready-To-Go Beads, which incorporate Taq polymerase, deoxynucleotide triphosphates, and buffer in a freeze-dried pellet. These kits have greatly simplified teacher implementation of human PCR labs, and their use is growing at a rapid pace. Sales of human polymorphism kits by Carolina Biological rose from 700 units in 1999 to 1,132 in 2000 â a 62% increase. Competing kits using the Alu system, and based substantially on our earlier work, are also marketed by Biorad and Edvotek. In parallel with the lab experiments, we developed a suite of database/statistical applications and easy-to-use interfaces that allow students to use their own DNA data to explore human population genetics and to test theories of human evolution. Database searches and statistical analyses are launched from a centralized workspace. Workshop participants were introduced to these and other resources available at the DNALC WWW site (http://vector.cshl.org/bioserver/): 1) Allele Server tests Hardy-Weinberg equilibrium and statistically compares PV92 data from world populations. 2) Sequence Server uses DNA sequence data to search Genbank using BLASTN, compare sequences using CLUSTALW, and create phylogenetic trees using PHYLIP. 3) Simulation Server uses a Monte Carlo generator to model the long-term effects of drift, selection, and population bottlenecks. By targeting motivated and innovative biology faculty, we believe that this project offered a cost-effective means to bring high school biology education up-to-the-minute with genomic biology. The workshop reached a target audience of highly professional faculty who have already implemented hands-on labs in molecular genetics and many of whom offer laboratory electives in biotechnology. Many attend professional meetings, develop curriculum, collaborate with scientists, teach faculty workshops, and manage equipment-sharing programs. These individuals are life-long learners, anxious for deeper insight and additional training to further extend their leadership. This contention was supported by data from a mail survey, conducted in February-March 2000 and 2001, of 256 faculty who participated in workshops conducted during the current term of DOE support. Seventy percent of participants responded, providing direct reports on how their teaching behavior had changed since taking the DOE workshop. About nine of ten respondents said they had provided new classroom materials and first-hand accounts of DNA typing, sequencing, or PCR. Three-fourths had introduced new units on human molecular genetics. Most strikingly, half had students use PCR to amplify their own insertion polymorphisms (PV92), and better than one-fourth amplified a VNTR polymorphism and the mitochondrial control region. One in five had mitochondrial DNA sequenced by the DNALC Sequencing Service. A majority (58%) used online materials at the DNALC WWW site, and 28% analyzed student polymorphism data with Bioservers at the DNALC site. A majority (58%) assisted other faculty with student labs on polymorphisms, reaching an additional 786 teachers.« less

GrigoraSNPs: Optimized Analysis of SNPs for DNA Forensics.

PubMed

Ricke, Darrell O; Shcherbina, Anna; Michaleas, Adam; Fremont-Smith, Philip

2018-04-16

High-throughput sequencing (HTS) of single nucleotide polymorphisms (SNPs) enables additional DNA forensic capabilities not attainable using traditional STR panels. However, the inclusion of sets of loci selected for mixture analysis, extended kinship, phenotype, biogeographic ancestry prediction, etc., can result in large panel sizes that are difficult to analyze in a rapid fashion. GrigoraSNP was developed to address the allele-calling bottleneck that was encountered when analyzing SNP panels with more than 5000 loci using HTS. GrigoraSNPs uses a MapReduce parallel data processing on multiple computational threads plus a novel locus-identification hashing strategy leveraging target sequence tags. This tool optimizes the SNP calling module of the DNA analysis pipeline with runtimes that scale linearly with the number of HTS reads. Results are compared with SNP analysis pipelines implemented with SAMtools and GATK. GrigoraSNPs removes a computational bottleneck for processing forensic samples with large HTS SNP panels. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Evaluation of genetic diversity in Chinese kale (Brassica oleracea L. var. alboglabra Bailey) by using rapid amplified polymorphic DNA and sequence-related amplified polymorphism markers.

PubMed

Zhang, J; Zhang, L G

2014-02-14

Chinese kale is an original Chinese vegetable of the Cruciferae family. To select suitable parents for hybrid breeding, we thoroughly analyzed the genetic diversity of Chinese kale. Random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) molecular markers were used to evaluate the genetic diversity across 21 Chinese kale accessions from AVRDC and Guangzhou in China. A total of 104 bands were detected by 11 RAPD primers, of which 66 (63.5%) were polymorphic, and 229 polymorphic bands (68.4%) were observed in 335 bands amplified by 17 SRAP primer combinations. The dendrogram showed the grouping of the 21 accessions into 4 main clusters based on RAPD data, and into 6 clusters based on SRAP and combined data (RAPD + SRAP). The clustering of accessions based on SRAP data was consistent with petal colors. The Mantel test indicated a poor fit for the RAPD and SRAP data (r = 0.16). These results have an important implication for Chinese kale germplasm characterization and improvement.

Lack of association between ESR1 gene polymorphisms and premature ovarian failure in Serbian women.

PubMed

Li, J; Vujovic, S; Dalgleish, R; Thompson, J; Dragojevic-Dikic, S; Al-Azzawi, F

2014-06-01

It has previously been reported that estrogen receptor-alpha (ERα) gene (ESR1: estrogen receptor 1) polymorphisms are associated with premature ovarian failure (POF). The aim of this study was to investigate whether these genetic polymorphisms of ESR1 are associated with POF in Serbian women. A series of 197 POF cases matched with 547 fertile controls was recruited by the Institute for Endocrinology, Diabetes and Metabolic Disorders of Serbia between 2007 and 2010. Genomic DNA was extracted from saliva using Oragene® DNA sample collection kits. Two single-nucleotide polymorphisms (SNPs), PvuII and XbaI, in ESR1 were genotyped by dynamic allele-specific hybridization. Haplotype analyses were performed with the restriction fragment length polymorphism method. SNP and haplotype effects were analyzed by logistic regression models. No significant difference was found in the distribution of ESR1 PvuII and XbaI polymorphisms or haplotypes between the POF and control groups. The two ESR1 SNPs, PvuII and XbaI, are not commonly associated with POF in Serbian women and may not contribute to the genetic basis of the condition.

[Association of muscle segment homeobox gene 1 polymorphisms with nonsyndromic cleft lip with or without cleft palate].

PubMed

Zhang, Li; Tang, Jun-Ling; Liang, Shang-Zheng

2008-06-01

Muscle segment homeobox gene (MSX)1 has been proposed as a gene in which mutations may contribute to nonsyndromic cleft lip with or without cleft palate (NSCL/P). To study MSX1 polymorphisms in NSCL/ P by means of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), and investigate the association of MSX1 exons 1 polymorphisms with NSCL/P. DNA were extracted from blood samples from NSCL/P and unrelated normal subjects. Genome DNA from peripheral leukocyte with these blood samples were extracted, which was used as template to amplify desired gene fragment of MSX1 exons 1 by means of polymerase chain reaction (PCR). The PCR products were examined by single-strand conformation polymorphism (SSCP). The MSX1 exons 1 polymorphisms were examined by sequencing if mutations were found. MSX1 genes of exon 1 mutation was not been found in the NSCL/P and unrelated normal subjects by SSCP. No correlation between MSX1 exon 1 and NSCL/P was found. MSX1 exon 1 may not be a key gene (susceptibility gene) in NSCL/P.

Data from complete mtDNA sequencing of Tunisian centenarians: testing haplogroup association and the "golden mean" to longevity.

PubMed

Costa, Marta D; Cherni, Lotfi; Fernandes, Verónica; Freitas, Fernando; Ammar El Gaaied, Amel Ben; Pereira, Luísa

2009-04-01

Since the mitochondrial theory of ageing was proposed, mitochondrial DNA (mtDNA) diversity has been largely studied in old people, however complete genomes are still rare, being limited to Japanese and UK/US samples. In this work, we evaluated possible longevity associated polymorphisms/haplogroups in an African population, from Tunisia, by performing complete mtDNA sequencing. This population has a mixed Eurasian/sub-Saharan mtDNA gene pool, which could potentially facilitate the evaluation of association for sub-Saharan lineages. Sub-Saharan haplogroups were shown to be significantly less represented in centenarians (9.5%) than in controls (54.5%), but it is not possible to rule out an influence of population structure, which is high in these populations. No recurrent polymorphism were more frequent in centenarians than in controls, and although the Tunisian centenarians presented less synonymous and replacement polymorphisms than controls, this difference was not statistically significant. So far, it does not seem that centenarians have significantly less mildly deleterious substitutions, not only in Tunisia but also in Japanese and UK/US samples, as tested here, not favouring a "golden mean" to longevity.