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Sample records for nonsynonymous single nucleotide

  1. Predicting mendelian disease-causing non-synonymous single nucleotide variants in exome sequencing studies.

    PubMed

    Li, Miao-Xin; Kwan, Johnny S H; Bao, Su-Ying; Yang, Wanling; Ho, Shu-Leong; Song, Yong-Qiang; Sham, Pak C

    2013-01-01

    Exome sequencing is becoming a standard tool for mapping Mendelian disease-causing (or pathogenic) non-synonymous single nucleotide variants (nsSNVs). Minor allele frequency (MAF) filtering approach and functional prediction methods are commonly used to identify candidate pathogenic mutations in these studies. Combining multiple functional prediction methods may increase accuracy in prediction. Here, we propose to use a logit model to combine multiple prediction methods and compute an unbiased probability of a rare variant being pathogenic. Also, for the first time we assess the predictive power of seven prediction methods (including SIFT, PolyPhen2, CONDEL, and logit) in predicting pathogenic nsSNVs from other rare variants, which reflects the situation after MAF filtering is done in exome-sequencing studies. We found that a logit model combining all or some original prediction methods outperforms other methods examined, but is unable to discriminate between autosomal dominant and autosomal recessive disease mutations. Finally, based on the predictions of the logit model, we estimate that an individual has around 5% of rare nsSNVs that are pathogenic and carries ~22 pathogenic derived alleles at least, which if made homozygous by consanguineous marriages may lead to recessive diseases. PMID:23341771

  2. Phenotype Prediction of Pathogenic Nonsynonymous Single Nucleotide Polymorphisms in WFS1

    PubMed Central

    Qian, Xuli; Qin, Luyang; Xing, Guangqian; Cao, Xin

    2015-01-01

    Wolfram syndrome (WS) is a rare, progressive, neurodegenerative disorder that has an autosomal recessive pattern of inheritance. The gene for WS, wolfram syndrome 1 gene (WFS1), is located on human chromosome 4p16.1 and encodes a transmembrane protein. To date, approximately 230 mutations in WFS1 have been confirmed, in which nonsynonymous single nucleotide polymorphisms (nsSNPs) are the most common forms of genetic variation. Nonetheless, there is poor knowledge on the relationship between SNP genotype and phenotype in other nsSNPs of the WFS1 gene. Here, we analysed 395 nsSNPs associated with the WFS1 gene using different computational methods and identified 20 nsSNPs to be potentially pathogenic. Furthermore, to identify the amino acid distributions and significances of pathogenic nsSNPs in the protein of WFS1, its transmembrane domain was constructed by the TMHMM server, which suggested that mutations outside of the TMhelix could have more effects on protein function. The predicted pathogenic mutations for the nsSNPs of the WFS1 gene provide an excellent guide for screening pathogenic mutations. PMID:26435059

  3. Predicting Mendelian Disease-Causing Non-Synonymous Single Nucleotide Variants in Exome Sequencing Studies

    PubMed Central

    Bao, Su-Ying; Yang, Wanling; Ho, Shu-Leong; Song, Yong-Qiang; Sham, Pak C.

    2013-01-01

    Exome sequencing is becoming a standard tool for mapping Mendelian disease-causing (or pathogenic) non-synonymous single nucleotide variants (nsSNVs). Minor allele frequency (MAF) filtering approach and functional prediction methods are commonly used to identify candidate pathogenic mutations in these studies. Combining multiple functional prediction methods may increase accuracy in prediction. Here, we propose to use a logit model to combine multiple prediction methods and compute an unbiased probability of a rare variant being pathogenic. Also, for the first time we assess the predictive power of seven prediction methods (including SIFT, PolyPhen2, CONDEL, and logit) in predicting pathogenic nsSNVs from other rare variants, which reflects the situation after MAF filtering is done in exome-sequencing studies. We found that a logit model combining all or some original prediction methods outperforms other methods examined, but is unable to discriminate between autosomal dominant and autosomal recessive disease mutations. Finally, based on the predictions of the logit model, we estimate that an individual has around 5% of rare nsSNVs that are pathogenic and carries ∼22 pathogenic derived alleles at least, which if made homozygous by consanguineous marriages may lead to recessive diseases. PMID:23341771

  4. Functional analysis of non-synonymous single nucleotide polymorphisms in human SLC26A9

    PubMed Central

    Chen, An-Ping; Chang, Min-Hwang; Romero, Michael F.

    2012-01-01

    Slc26 anion transporters play crucial roles in transepithelial Cl− absorption and HCO3− secretion; Slc26 protein mutations lead to several diseases. Slc26a9 functions as a Cl− channel and electrogenic Cl−-HCO3− exchanger, and can interact with CFTR. Slc26a9(−/−) mice have reduced gastric acid secretion, yet no human disease is currently associated with SLC26A9 coding mutations. Therefore, we tested the function of non-synonymous, coding, single nucleotide polymorphisms (cSNPs) of SLC26A9. Presently, eight cSNPs are NCBI-documented: Y70N, T127N, I384T, R575W, P606L, V622L, V744M and H748R. Using two-electrode voltage-clamp and anion selective electrodes, we measured the biophysical consequences of these cSNPs. Y70N (cytoplasmic N-terminus) displays higher channel activity and enhanced Cl−-HCO3− exchange. T127N (transmembrane) results in smaller halide currents but not for SCN−. V622L (STAS domain) and V744M (STAS adjacent) decreased plasma membrane expression which partially accounts for decreased whole cell currents. Nevertheless, V622L transport is reduced to ~50%. SLC26A9 polymorphisms lead to several function modifications (increased activity, decreased activity, altered protein expression) which could lead to a spectrum of pathophysiologies. Thus, knowing an individual’s SLC26A9 genetics becomes important for understanding disease potentially caused by SLC26A9 mutations or modifying diseases, e.g., cystic fibrosis. Our results also provide a framework to understand SLC26A9 transport modalities and structure-function relationships. PMID:22544634

  5. Nonsynonymous single nucleotide polymorphisms of NHE3 differentially decrease NHE3 transporter activity

    PubMed Central

    Zhu, Xinjun Cindy; Sarker, Rafiquel; Horton, John R.; Chakraborty, Molee; Chen, Tian-E; Tse, C. Ming; Cha, Boyoung

    2015-01-01

    Genetic determinants appear to play a role in susceptibility to chronic diarrhea, but the genetic abnormalities involved have only been identified in a few conditions. The Na+/H+ exchanger 3 (NHE3) accounts for a large fraction of physiologic intestinal Na+ absorption. It is highly regulated through effects on its intracellular COOH-terminal regulatory domain. The impact of genetic variation in the NHE3 gene, such as single nucleotide polymorphisms (SNPs), on transporter activity remains unexplored. From a total of 458 SNPs identified in the entire NHE3 gene, we identified three nonsynonymous mutations (R474Q, V567M, and R799C), which were all in the protein's intracellular COOH-terminal domain. Here we evaluated whether these SNPs affect NHE3 activity by expressing them in a mammalian cell line that is null for all plasma membrane NHEs. These variants significantly reduced basal NHE3 transporter activity through a reduction in intrinsic NHE3 function in variant R474Q, abnormal trafficking in variant V567M, or defects in both intrinsic NHE3 function and trafficking in variant R799C. In addition, variants NHE3 R474Q and R799C failed to respond to acute dexamethasone stimulation, suggesting cells with these mutant proteins might be defective in NHE3 function during postprandial stimulation and perhaps under stressful conditions. Finally, variant R474Q was shown to exhibit an aberrant interaction with calcineurin B homologous protein (CHP), an NHE3 regulatory protein required for basal NHE3 activity. Taken together, these results demonstrate decreased transport activity in three SNPs of NHE3 and provide mechanistic insight into how these SNPs impact NHE3 function. PMID:25715704

  6. In silico Evaluation of Nonsynonymous Single Nucleotide Polymorphisms in the ADIPOQ Gene Associated with Diabetes, Obesity, and Inflammation

    PubMed Central

    Narayana Swamy, A; Valasala, Harika; Kamma, Sreenivasulu

    2015-01-01

    Background: The human ADIPOQ gene encodes adiponectin protein hormone, which is involved in regulating glucose levels as well as fatty acid breakdown. It is exclusively produced by adipose tissue and abundantly present in the circulation, with concentration of around 0.01% of total serum proteins, with important effect on metabolism. Methods: Most deleterious nonsynonymous single nucleotide polymorphisms in the coding region of the ADIPOQ gene were investigated using SNP databases, and detected nonsynonymous variants were analyzed in silico from the standpoint of relevant protein function and stability by using SIFT, PolyPhen-2, PROVEAN and MUpro, I-Mutant2.0 tools, respectively. Result: A total of 58 nonsynonymous SNPs consisting of 55 missense variations, 3 nonsense variations were found in the ADIPOQ gene. Next, 14 of the 55 missense variants were predicted to be damaging or deleterious by three different software programs (PolyPhen-2, SIFT, and PROVEAN), and 38 of them were predicted to be less stable (I-Mutant 2.0 and MUpro software). Totally, 10 variants out of 55 missense variants were predicted to be both deleterious and reduce protein stability. Additionally, 3 nonsense variants were predicted to produce a truncated ADIPOQ protein. RMSD and total energy were calculated for 4 nsSNPs out of 10 nsSNPs which were both deleterious and showed a decrease in protein stability. Conclusion: rs144526209 has high root-mean-square deviation (RMSD) and lower total energy value compared to the native modeled structure. It was concluded that this nsSNP, potentially functional and polymorphic in the ADIPOQ gene, might be associated with diabetes, obesity, and inflammation. PMID:26306152

  7. Prediction by Graph Theoretic Measures of Structural Effects in Proteins Arising from Non-Synonymous Single Nucleotide Polymorphisms

    PubMed Central

    Cheng, Tammy M. K.; Lu, Yu-En; Vendruscolo, Michele; Lio', Pietro; Blundell, Tom L.

    2008-01-01

    Recent analyses of human genome sequences have given rise to impressive advances in identifying non-synonymous single nucleotide polymorphisms (nsSNPs). By contrast, the annotation of nsSNPs and their links to diseases are progressing at a much slower pace. Many of the current approaches to analysing disease-associated nsSNPs use primarily sequence and evolutionary information, while structural information is relatively less exploited. In order to explore the potential of such information, we developed a structure-based approach, Bongo (Bonds ON Graph), to predict structural effects of nsSNPs. Bongo considers protein structures as residue–residue interaction networks and applies graph theoretical measures to identify the residues that are critical for maintaining structural stability by assessing the consequences on the interaction network of single point mutations. Our results show that Bongo is able to identify mutations that cause both local and global structural effects, with a remarkably low false positive rate. Application of the Bongo method to the prediction of 506 disease-associated nsSNPs resulted in a performance (positive predictive value, PPV, 78.5%) similar to that of PolyPhen (PPV, 77.2%) and PANTHER (PPV, 72.2%). As the Bongo method is solely structure-based, our results indicate that the structural changes resulting from nsSNPs are closely associated to their pathological consequences. PMID:18654622

  8. Construction and assessment of individualized proteogenomic databases for large-scale analysis of nonsynonymous single nucleotide variants.

    PubMed

    Krug, Karsten; Popic, Sasa; Carpy, Alejandro; Taumer, Christoph; Macek, Boris

    2014-12-01

    Next-generation sequencing projects focusing on genomes and transcriptomes identify millions of single nucleotide variants (SNVs), many of which result in single amino acid substitutions. These nonsynonymous (ns) SNVs are typically not incorporated into protein sequence databases used to identify MS/MS data. Here, we perform a comparative analysis of the assembly of nsSNV-containing proteogenomic databases. We use a comprehensive transcriptome and proteome dataset of HeLa cells from the literature to derive and to incorporate SNVs into databases applicable to proteomics search engines, and to assess their performance in the identification of nsSNVs. We assemble the databases by (1) translation of SNV-containing transcripts into all possible reading frames, (2) translation of predicted reading frame, (3) prediction of nsSNVs and subsequent incorporation into canonical protein sequences. We show substantial differences between generated databases in terms of represented nsSNVs and theoretical search space, affecting sensitivity and specificity of database search. We query the databases with >2.2M high-resolution MS/MS spectra using MaxQuant software and identify 451 variant peptides, containing 401 nsSNVs. We conclude that prediction of reading frame and, if applicable, SNV effect result in comprehensive yet compact databases necessary to retain sensitivity in large-scale analysis of nsSNVs called from transcriptomics data. PMID:25251379

  9. Prioritization Of Nonsynonymous Single Nucleotide Variants For Exome Sequencing Studies Via Integrative Learning On Multiple Genomic Data.

    PubMed

    Wu, Mengmeng; Wu, Jiaxin; Chen, Ting; Jiang, Rui

    2015-01-01

    The rapid advancement of next generation sequencing technology has greatly accelerated the progress for understanding human inherited diseases via such innovations as exome sequencing. Nevertheless, the identification of causative variants from sequencing data remains a great challenge. Traditional statistical genetics approaches such as linkage analysis and association studies have limited power in analyzing exome sequencing data, while relying on simply filtration strategies and predicted functional implications of mutations to pinpoint pathogenic variants are prone to produce false positives. To overcome these limitations, we herein propose a supervised learning approach, termed snvForest, to prioritize candidate nonsynonymous single nucleotide variants for a specific type of disease by integrating 11 functional scores at the variant level and 8 association scores at the gene level. We conduct a series of large-scale in silico validation experiments, demonstrating the effectiveness of snvForest across 2,511 diseases of different inheritance styles and the superiority of our approach over two state-of-the-art methods. We further apply snvForest to three real exome sequencing data sets of epileptic encephalophathies and intellectual disability to show the ability of our approach to identify causative de novo mutations for these complex diseases. The online service and standalone software of snvForest are found at http://bioinfo.au.tsinghua.edu.cn/jianglab/snvforest. PMID:26459872

  10. Prioritization Of Nonsynonymous Single Nucleotide Variants For Exome Sequencing Studies Via Integrative Learning On Multiple Genomic Data

    PubMed Central

    Wu, Mengmeng; Wu, Jiaxin; Chen, Ting; Jiang, Rui

    2015-01-01

    The rapid advancement of next generation sequencing technology has greatly accelerated the progress for understanding human inherited diseases via such innovations as exome sequencing. Nevertheless, the identification of causative variants from sequencing data remains a great challenge. Traditional statistical genetics approaches such as linkage analysis and association studies have limited power in analyzing exome sequencing data, while relying on simply filtration strategies and predicted functional implications of mutations to pinpoint pathogenic variants are prone to produce false positives. To overcome these limitations, we herein propose a supervised learning approach, termed snvForest, to prioritize candidate nonsynonymous single nucleotide variants for a specific type of disease by integrating 11 functional scores at the variant level and 8 association scores at the gene level. We conduct a series of large-scale in silico validation experiments, demonstrating the effectiveness of snvForest across 2,511 diseases of different inheritance styles and the superiority of our approach over two state-of-the-art methods. We further apply snvForest to three real exome sequencing data sets of epileptic encephalophathies and intellectual disability to show the ability of our approach to identify causative de novo mutations for these complex diseases. The online service and standalone software of snvForest are found at http://bioinfo.au.tsinghua.edu.cn/jianglab/snvforest. PMID:26459872

  11. Cellular signalling of non-synonymous single-nucleotide polymorphisms of the human μ-opioid receptor (OPRM1)

    PubMed Central

    Knapman, Alisa; Connor, Mark

    2015-01-01

    There is significant variability in individual responses to opioid drugs, which is likely to have a significant genetic component. A number of non-synonymous single-nucleotide polymorphisms (SNPs) in the coding regions of the μ-opioid receptor gene (OPRM1) have been postulated to contribute to this variability. Although many studies have investigated the clinical influences of these μ-opioid receptor variants, the outcomes are reported in the context of thousands of other genes and environmental factors, and we are no closer to being able to predict individual response to opioids based on genotype. Investigation of how μ-opioid receptor SNPs affect their expression, coupling to second messengers, desensitization and regulation is necessary to understand how subtle changes in receptor structure can impact individual responses to opioids. To date, the few functional studies that have investigated the consequences of SNPs on the signalling profile of the μ-opioid receptor in vitro have shown that the common N40D variant has altered functional responses to some opioids, while other, rarer, variants display altered signalling or agonist-dependent regulation. Here, we review the data available on the effects of μ-opioid receptor polymorphisms on receptor function, expression and regulation in vitro, and discuss the limitations of the studies to date. Whether or not μ-opioid receptor SNPs contribute to individual variability in opioid responses remains an open question, in large part because we have relatively little good data about how the amino acid changes affect μ-opioid receptor function. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24527749

  12. A Non-Synonymous Single Nucleotide Polymorphism in the HJURP Gene Associated with Susceptibility to Hepatocellular Carcinoma among Chinese

    PubMed Central

    Zhai, Yun; Wang, Shaoxia; Li, Yang; Ma, Fuchao; Li, Yuanfeng; Wang, Zhifu; Zhang, Yang; Zhang, Xiumei; Liang, Renxiang; Wei, Zhongliang; Cui, Ying; Li, Yongqiang; Yu, Xinsen; Ji, Hongzan; He, Fuchu; Xie, Weimin; Zhou, Gangqiao

    2016-01-01

    Objective HJURP (Holliday Junction-Recognizing Protein) plays dual roles in DNA repair and in accurate chromosome segregation during mitosis. We examined whether the single nucleotide polymorphisms (SNPs) of HJURP were associated with the risk of occurrence of hepatocellular carcinoma (HCC) among chronic hepatitis B virus (HBV) carriers from well-known high-risk regions for HCC in China. Methods Twenty-four haplotype-tagging SNPs across HJURP were selected from HapMap data using the Haploview software. We genotyped these 24 SNPs using the using Sequenom's iPLEX assay in the Fusui population, consisting of 348 patients with HCC and 359 cancer-free controls, and further investigated the significantly associated SNP using the TaqMan assay in the Haimen population, consisting of 100 cases and 103 controls. The genetic associations with the risk of HCC were analyzed by logistic regression. Results We observed an increased occurrence of HCC consistently associated with A/C or C/C genotypes of the non-synonymous SNP rs3771333 compared with the A/A genotype in both the Fusui and Haimen populations, with a pooled odds ratio 1.82 (95% confidence interval, 1.33–2.49; P = 1.9 × 10−4). Case-only analysis further indicated that carriers of the at-risk C allele were younger than those carrying the A/A genotype (P = 0.0016). In addition, the expression levels of HJURP in C allele carriers were lower than that in A/A genotype carriers (P = 0.0078 and 0.0010, for mRNA and protein levels, respectively). Conclusion Our findings suggest that rs3771333 in HJURP may play a role in mediating the susceptibility to HCC among Chinese. PMID:26863619

  13. Impacts of Nonsynonymous Single Nucleotide Polymorphisms of Adiponectin Receptor 1 Gene on Corresponding Protein Stability: A Computational Approach

    PubMed Central

    Saleh, Md. Abu; Solayman, Md.; Paul, Sudip; Saha, Moumoni; Khalil, Md. Ibrahim; Gan, Siew Hua

    2016-01-01

    Despite the reported association of adiponectin receptor 1 (ADIPOR1) gene mutations with vulnerability to several human metabolic diseases, there is lack of computational analysis on the functional and structural impacts of single nucleotide polymorphisms (SNPs) of the human ADIPOR1 at protein level. Therefore, sequence- and structure-based computational tools were employed in this study to functionally and structurally characterize the coding nsSNPs of ADIPOR1 gene listed in the dbSNP database. Our in silico analysis by SIFT, nsSNPAnalyzer, PolyPhen-2, Fathmm, I-Mutant 2.0, SNPs&GO, PhD-SNP, PANTHER, and SNPeffect tools identified the nsSNPs with distorting functional impacts, namely, rs765425383 (A348G), rs752071352 (H341Y), rs759555652 (R324L), rs200326086 (L224F), and rs766267373 (L143P) from 74 nsSNPs of ADIPOR1 gene. Finally the aforementioned five deleterious nsSNPs were introduced using Swiss-PDB Viewer package within the X-ray crystal structure of ADIPOR1 protein, and changes in free energy for these mutations were computed. Although increased free energy was observed for all the mutants, the nsSNP H341Y caused the highest energy increase amongst all. RMSD and TM scores predicted that mutants were structurally similar to wild type protein. Our analyses suggested that the aforementioned variants especially H341Y could directly or indirectly destabilize the amino acid interactions and hydrogen bonding networks of ADIPOR1. PMID:27294143

  14. Impacts of Nonsynonymous Single Nucleotide Polymorphisms of Adiponectin Receptor 1 Gene on Corresponding Protein Stability: A Computational Approach.

    PubMed

    Saleh, Md Abu; Solayman, Md; Paul, Sudip; Saha, Moumoni; Khalil, Md Ibrahim; Gan, Siew Hua

    2016-01-01

    Despite the reported association of adiponectin receptor 1 (ADIPOR1) gene mutations with vulnerability to several human metabolic diseases, there is lack of computational analysis on the functional and structural impacts of single nucleotide polymorphisms (SNPs) of the human ADIPOR1 at protein level. Therefore, sequence- and structure-based computational tools were employed in this study to functionally and structurally characterize the coding nsSNPs of ADIPOR1 gene listed in the dbSNP database. Our in silico analysis by SIFT, nsSNPAnalyzer, PolyPhen-2, Fathmm, I-Mutant 2.0, SNPs&GO, PhD-SNP, PANTHER, and SNPeffect tools identified the nsSNPs with distorting functional impacts, namely, rs765425383 (A348G), rs752071352 (H341Y), rs759555652 (R324L), rs200326086 (L224F), and rs766267373 (L143P) from 74 nsSNPs of ADIPOR1 gene. Finally the aforementioned five deleterious nsSNPs were introduced using Swiss-PDB Viewer package within the X-ray crystal structure of ADIPOR1 protein, and changes in free energy for these mutations were computed. Although increased free energy was observed for all the mutants, the nsSNP H341Y caused the highest energy increase amongst all. RMSD and TM scores predicted that mutants were structurally similar to wild type protein. Our analyses suggested that the aforementioned variants especially H341Y could directly or indirectly destabilize the amino acid interactions and hydrogen bonding networks of ADIPOR1. PMID:27294143

  15. Nonsynonymous single nucleotide polymorphisms in the complement component 3 gene are associated with risk of age-related macular degeneration: a meta-analysis.

    PubMed

    Qian-Qian, Yu; Yong, Yao; Jing, Zhu; Xin, Bao; Tian-Hua, Xie; Chao, Sun; Jia, Cao

    2015-05-01

    Nonsynonymous single nucleotide polymorphisms (SNPs) in complement component 3 (CC3) are associated with the risk of age-related macular degeneration (AMD), however, this association is not consistent among studies. To thoroughly address this issue, we performed an updated meta-analysis to evaluate the association between nine SNPs in the CC3 gene and AMD risk. A search was conducted of the PubMed database through 3rd Aug, 2014. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of associations. Based on the search criteria for manuscripts reporting AMD susceptibility related to CC3 in nine SNPs, 57 case-control studies from 22 different articles were retrieved. Significantly positive associations were found for the rs2230199 C/G SNP and AMD in the Caucasian population, as well as for the rs1047286 C/T SNP. Moreover, a relationship between the rs11569536 G/A SNP and AMD was detected. By contrast, a negative association was observed between rs2250656 A/G SNP and AMD risk. The present meta-analysis suggests that these four SNPs in the CC3 gene are potentially associated with the risk of AMD development. Further studies using larger sample sizes and accounting for gene-environment interactions should be conducted to elucidate the role of CC3 gene polymorphisms in AMD risk. PMID:25688879

  16. Effects of individually silenced N-glycosylation sites and non-synonymous single-nucleotide polymorphisms on the fusogenic function of human syncytin-2

    PubMed Central

    Cui, Lina; Wang, Huiying; Lu, Xiaoyin; Wang, Rui; Zheng, Ru; Li, Yue; Yang, Xiaokui; Jia, Wen-Tong; Zhao, Yangyu; Wang, Yongqing; Wang, Haibin; Wang, Yan-Ling; Zhu, Cheng; Lin, Hai-Yan; Wang, Hongmei

    2016-01-01

    ABSTRACT The placental syncytiotrophoblast, which is formed by the fusion of cytotrophoblast cells, is indispensable for the establishment and maintenance of normal pregnancy. The human endogenous retrovirus envelope glycoprotein syncytin-2 is the most important player in mediating trophoblast cell-cell fusion as a fusogen. We constructed expression plasmids of wild-type and 21 single-amino-acid substitution mutants of syncytin-2, including 10 N-glycosylation sites individually silenced by mutagenizing N to Q, 1 naturally occurring single-nucleotide polymorphism (SNP) N118S that introduced an N-glycosylation site, and another 10 non-synonymous SNPs located within important functional domains. We observed that syncytin-2 was highly fusogenic and that the mutants had different capacities in merging 293T cells. Of the 21 mutants, N133Q, N312Q, N443Q, C46R (in the CXXC motif) and R417H (in the heptad repeat region and immunosuppressive domain) lost their fusogenicity, whereas N332Q, N118S, T367M (in the fusion peptide), V483I (in the transmembrane domain) and T522M (in the cytoplasmic domain) enhanced the fusogenic activity. We also proved that N133, N146, N177, N220, N241, N247, N312, N332 and N443 were all glycosylated in 293T cells. A co-immunoprecipitation assay showed compromised interaction between mutants N443Q, C46R, T367M, R417H and the receptor MFSD2A, whereas N118S was associated with more receptors. We also sequenced the coding sequence of syncytin-2 in 125 severe pre-eclamptic patients and 272 normal pregnant Chinese women. Surprisingly, only 1 non-synonymous SNP T522M was found and the frequencies of heterozygous carriers were not significantly different. Taken together, our results suggest that N-glycans at residues 133, 312, 332 and 443 of syncytin-2 are required for optimal fusion induction, and that SNPs C46R, N118S, T367M, R417H, V483I and T522M can alter the fusogenic function of syncytin-2. PMID:26853155

  17. Structural modeling and in silico analysis of non-synonymous single nucleotide polymorphisms of human 3β-hydroxysteroid dehydrogenase type 2

    PubMed Central

    Goswami, Achintya Mohan

    2015-01-01

    Single-nucleotide polymorphisms (SNPs), a most common type of genetic mutations, result from single base pair alterations. Non-synonymous SNPs (nsSNP) occur in the coding regions of a gene and result in single amino acid substitution which might have the potential to affect the function as well as structure of the corresponding protein. In human the 3β-hydroxysteroid dehydrogenases/Δ4,5-isomerase type 2 (HSD3B2) is an important membrane-bound enzyme involved in the dehydrogenation and Δ4,5-isomerization of the Δ5-steroid precursors into their respective Δ4-ketosteroids in the biosynthesis of steroid hormones such as glucocorticoids, mineralocorticoids, progesterone, androgens, and estrogens in tissues such as adrenal gland, ovary, and testis. Most of the nsSNPs of HSD3B2 are still uncharacterized in terms of their disease causing potential. So, this study has been undertaken to explore and extend the knowledge related to the effect of nsSNPs on the stability and function of the HSD3B2. In this study sixteen nsSNP of HSD3B2 were subjected to in silico analysis using nine different algorithms: SIFT, PROVEAN, PolyPhen, MutPred, SNPeffect, nsSNP Analyzer, PhD SNP, stSNP, and I Mutant 2.0. The results obtained from the analysis revealed that the prioritization of diseases associated amino acid substitution as evident from possible alteration in structure–function relationship. Structural phylogenetic analysis using ConSurf revealed that the functional residues are highly conserved in human HSD3B2; and most of the disease associated nsSNPs are within these conserved residues. Structural theoritical models of HSD3B2 were created using HHPred, Phyre2 and RaptorX server. The predicted models were evaluated to get the best one for structural understanding of amino acid substitutions in three dimensional spaces. PMID:26288759

  18. A new role for the nonpathogenic nonsynonymous single-nucleotide polymorphisms of acetylcholinesterase in the treatment of Alzheimer's disease: a computational study.

    PubMed

    Saravanaraman, Ponne; Chinnadurai, Raj Kumar; Boopathy, Rathanam

    2014-08-01

    Single-nucleotide polymorphisms (SNPs) are implicated in the complexity of understanding the genetics of diseases and their therapeutics. Here we have attempted to determine the impact of nonsynonymous SNPs (nsSNPs) on structure, dynamics, and ligand-binding properties of the human acetylcholinesterase (hAChE) protein, which has been targeted in the treatment of Alzheimer's disease. Of the reported 153 SNPs, 4 nsSNPs, namely, A415G, P104A, V302E, and Y119H, were prioritized to be functionally unfavorable by SIFT and PolyPhen algorithms. Molecular dynamics simulation revealed these nsSNP forms to be structurally stable, and they are also considered functionally active as they lie away from the catalytic triad. However, the aromatic amino acids lining the active-site gorge exhibited altered degrees of side chain dihedral angles. Such conformational alterations were evaluated for their ability to interfere with binding of hAChE inhibitors. The inhibitors (donepezil, galantamine, rivastigmine, and tacrine) were oriented differently in comparison to the native because of the steric hindrance offered by the altered dihedral angles. Interestingly, huperzine A alone exhibited higher efficiency in its binding to the AChE and retained similar orientation irrespective of the polymorphisms since the orientation of Asp74 involved in its binding and trafficking remained unaltered in all protein forms. Therefore, we conclude that nsSNPs confer changes to the dynamicity of proteins, which in turn affects their ligand-binding properties rather than their stability. Considering the diverse polymorphic nature of hAChE, while contemplating any structure-based drug design, the common, nonpathogenic nsSNPs should be considered for the utmost efficacy of drugs. PMID:24611490

  19. Evaluation of All Nonsynonymous Single-Nucleotide Polymorphisms in the Gene Encoding Human Deoxyribonuclease I-Like 1, Possibly Implicated in the Blocking of Endocytosis-Mediated Foreign Gene Transfer

    PubMed Central

    Ueki, Misuzu; Kimura-Kataoka, Kaori; Fujihara, Junko; Iida, Reiko; Yasuda, Toshihiro

    2014-01-01

    Many nonsynonymous single-nucleotide polymorphisms (SNPs) in the human deoxyribonuclease I-like 1 (DNase 1L1) gene, possibly implicated in the blocking of endocytosis-mediated foreign gene transfer, have been identified, but only limited population data are available and no studies have evaluated whether such SNPs are functional. Genotyping of all 21 nonsynonymous human DNase 1L1 SNPs was performed in 16 different populations representing three ethnic groups using the PCR-restriction fragment length polymorphism technique. All of the nonsynonymous SNPs, except for SNP p.Val122Ile in Caucasian populations, exhibited a monoallelic distribution in all of the populations. On the basis of alterations in the activity levels resulting from the corresponding amino acid substitutions, two activity-abolishing and four activity-reducing SNPs were confirmed to be functional. Although all of the nonsynonymous SNPs that affected the catalytic activity showed extremely low genetic heterogeneity, it seems plausible that a minor allele of six SNPs producing a loss-of-function or extremely low-activity variant could serve directly as a genetic risk factor for diseases. Especially, the amino acid residues in activity-abolishing SNPs were conserved in animal DNases 1L1. Furthermore, results of phylogenetic analysis suggest that DNase 1L1 might have appeared latest among the DNase I family during the course of molecular evolution. PMID:24329527

  20. Evaluation of all nonsynonymous single-nucleotide polymorphisms in the gene encoding human deoxyribonuclease I-like 1, possibly implicated in the blocking of endocytosis-mediated foreign gene transfer.

    PubMed

    Ueki, Misuzu; Kimura-Kataoka, Kaori; Fujihara, Junko; Takeshita, Haruo; Iida, Reiko; Yasuda, Toshihiro

    2014-02-01

    Many nonsynonymous single-nucleotide polymorphisms (SNPs) in the human deoxyribonuclease I-like 1 (DNase 1L1) gene, possibly implicated in the blocking of endocytosis-mediated foreign gene transfer, have been identified, but only limited population data are available and no studies have evaluated whether such SNPs are functional. Genotyping of all 21 nonsynonymous human DNase 1L1 SNPs was performed in 16 different populations representing three ethnic groups using the PCR-restriction fragment length polymorphism technique. All of the nonsynonymous SNPs, except for SNP p.Val122Ile in Caucasian populations, exhibited a monoallelic distribution in all of the populations. On the basis of alterations in the activity levels resulting from the corresponding amino acid substitutions, two activity-abolishing and four activity-reducing SNPs were confirmed to be functional. Although all of the nonsynonymous SNPs that affected the catalytic activity showed extremely low genetic heterogeneity, it seems plausible that a minor allele of six SNPs producing a loss-of-function or extremely low-activity variant could serve directly as a genetic risk factor for diseases. Especially, the amino acid residues in activity-abolishing SNPs were conserved in animal DNases 1L1. Furthermore, results of phylogenetic analysis suggest that DNase 1L1 might have appeared latest among the DNase I family during the course of molecular evolution. PMID:24329527

  1. Non-synonymous single nucleotide polymorphisms in the watermelon eIF4E gene are closely associated with resistance to Zucchini yellow mosaic virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Zucchini yellow mosaic virus (ZYMV) is one of the most economically important potyviruses infecting cucurbit crops worldwide. Using a candidate gene approach, we cloned and sequenced eIF4E and eIF(iso)4E gene segments in watermelon. Analysis of the nucleotide sequences between the ZYMV-resistant wa...

  2. Large-scale mass spectrometric detection of variant peptides resulting from non-synonymous nucleotide differences

    PubMed Central

    Sheynkman, Gloria M.; Shortreed, Michael R.; Frey, Brian L.; Scalf, Mark; Smith, Lloyd M.

    2013-01-01

    Each individual carries thousands of non-synonymous single nucleotide variants (nsSNVs) in their genome, each corresponding to a single amino acid polymorphism (SAP) in the encoded proteins. It is important to be able to directly detect and quantify these variations at the protein level in order to study post-transcriptional regulation, differential allelic expression, and other important biological processes. However, such variant peptides are not generally detected in standard proteomic analyses, due to their absence from the generic databases that are employed for mass spectrometry searching. Here, we extend previous work that demonstrated the use of customized SAP databases constructed from sample-matched RNA-Seq data. We collected deep coverage RNA-Seq data from the Jurkat cell line, compiled the set of nsSNVs that are expressed, used this information to construct a customized SAP database, and searched it against deep coverage shotgun MS data obtained from the same sample. This approach enabled detection of 421 SAP peptides mapping to 395 nsSNVs. We compared these peptides to peptides identified from a large generic search database containing all known nsSNVs (dbSNP) and found that more than 70% of the SAP peptides from this dbSNP-derived search were not supported by the RNA-Seq data, and thus are likely false positives. Next, we increased the SAP coverage from the RNA-Seq derived database by utilizing multiple protease digestions, thereby increasing variant detection to 695 SAP peptides mapping to 504 nsSNV sites. These detected SAP peptides corresponded to moderate to high abundance transcripts (30+ transcripts per million, TPM). The SAP peptides included 192 allelic pairs; the relative expression levels of the two alleles were evaluated for 51 of those pairs, and found to be comparable in all cases. PMID:24175627

  3. The Single Nucleotide Polymorphism Consortium

    NASA Technical Reports Server (NTRS)

    Morgan, Michael

    2003-01-01

    I want to discuss both the Single Nucleotide Polymorphism (SNP) Consortium and the Human Genome Project. I am afraid most of my presentation will be thin on law and possibly too high on rhetoric. Having been engaged in a personal and direct way with these issues as a trained scientist, I find it quite difficult to be always as objective as I ought to be.

  4. Inter-individual variation in nucleotide excision repair pathway is modulated by non-synonymous polymorphisms in ERCC4 and MBD4 genes.

    PubMed

    Allione, Alessandra; Guarrera, Simonetta; Russo, Alessia; Ricceri, Fulvio; Purohit, Rituraj; Pagnani, Andrea; Rosa, Fabio; Polidoro, Silvia; Voglino, Floriana; Matullo, Giuseppe

    2013-01-01

    Inter-individual differences in DNA repair capacity (DRC) may lead to genome instability and, consequently, modulate individual cancer risk. Among the different DNA repair pathways, nucleotide excision repair (NER) is one of the most versatile, as it can eliminate a wide range of helix-distorting DNA lesions caused by ultraviolet light irradiation and chemical mutagens. We performed a genotype-phenotype correlation study in 122 healthy subjects in order to assess if any associations exist between phenotypic profiles of NER and DNA repair gene single nucleotide polymorphisms (SNPs). Individuals were genotyped for 768 SNPs with a custom Illumina Golden Gate Assay, and peripheral blood mononuclear cells (PBMCs) of the same subjects were tested for a NER comet assay to measure DRC after challenging cells by benzo(a)pyrene diolepoxide (BPDE). We observed a large inter-individual variability of NER capacity, with women showing a statistically significant lower DRC (mean ± SD: 6.68 ± 4.76; p = 0.004) than men (mean ± SD: 8.89 ± 5.20). Moreover, DRC was significantly lower in individuals carrying a variant allele for the ERCC4 rs1800124 non-synonymous SNP (nsSNP) (p = 0.006) and significantly higher in subjects with the variant allele of MBD4 rs2005618 SNP (p = 0.008), in linkage disequilibrium (r(2) = 0.908) with rs10342 nsSNP. Traditional in silico docking approaches on protein-DNA and protein-protein interaction showed that Gly875 variant in ERCC4 (rs1800124) decreases the DNA-protein interaction and that Ser273 and Thr273 variants in MBD4 (rs10342) indicate complete loss of protein-DNA interactions. Our results showed that NER inter-individual capacity can be modulated by cross-talk activity involving nsSNPs in ERCC4 and MBD4 genes, and they suggested to better investigate SNP effect on cancer risk and response to chemo- and radiotherapies. PMID:24004570

  5. High-Throughput Sequencing Reveals Single Nucleotide Variants in Longer-Kernel Bread Wheat

    PubMed Central

    Chen, Feng; Zhu, Zibo; Zhou, Xiaobian; Yan, Yan; Dong, Zhongdong; Cui, Dangqun

    2016-01-01

    The transcriptomes of bread wheat Yunong 201 and its ethyl methanesulfonate derivative Yunong 3114 were obtained by next-sequencing technology. Single nucleotide variants (SNVs) in the wheat strains were explored and compared. A total of 5907 and 6287 non-synonymous SNVs were acquired for Yunong 201 and 3114, respectively. A total of 4021 genes with SNVs were obtained. The genes that underwent non-synonymous SNVs were significantly involved in ATP binding, protein phosphorylation, and cellular protein metabolic process. The heat map analysis also indicated that most of these mutant genes were significantly differentially expressed at different developmental stages. The SNVs in these genes possibly contribute to the longer kernel length of Yunong 3114. Our data provide useful information on wheat transcriptome for future studies on wheat functional genomics. This study could also help in illustrating the gene functions of the non-synonymous SNVs of Yunong 201 and 3114. PMID:27551288

  6. High-Throughput Sequencing Reveals Single Nucleotide Variants in Longer-Kernel Bread Wheat.

    PubMed

    Chen, Feng; Zhu, Zibo; Zhou, Xiaobian; Yan, Yan; Dong, Zhongdong; Cui, Dangqun

    2016-01-01

    The transcriptomes of bread wheat Yunong 201 and its ethyl methanesulfonate derivative Yunong 3114 were obtained by next-sequencing technology. Single nucleotide variants (SNVs) in the wheat strains were explored and compared. A total of 5907 and 6287 non-synonymous SNVs were acquired for Yunong 201 and 3114, respectively. A total of 4021 genes with SNVs were obtained. The genes that underwent non-synonymous SNVs were significantly involved in ATP binding, protein phosphorylation, and cellular protein metabolic process. The heat map analysis also indicated that most of these mutant genes were significantly differentially expressed at different developmental stages. The SNVs in these genes possibly contribute to the longer kernel length of Yunong 3114. Our data provide useful information on wheat transcriptome for future studies on wheat functional genomics. This study could also help in illustrating the gene functions of the non-synonymous SNVs of Yunong 201 and 3114. PMID:27551288

  7. Single nucleotide variations: biological impact and theoretical interpretation.

    PubMed

    Katsonis, Panagiotis; Koire, Amanda; Wilson, Stephen Joseph; Hsu, Teng-Kuei; Lua, Rhonald C; Wilkins, Angela Dawn; Lichtarge, Olivier

    2014-12-01

    Genome-wide association studies (GWAS) and whole-exome sequencing (WES) generate massive amounts of genomic variant information, and a major challenge is to identify which variations drive disease or contribute to phenotypic traits. Because the majority of known disease-causing mutations are exonic non-synonymous single nucleotide variations (nsSNVs), most studies focus on whether these nsSNVs affect protein function. Computational studies show that the impact of nsSNVs on protein function reflects sequence homology and structural information and predict the impact through statistical methods, machine learning techniques, or models of protein evolution. Here, we review impact prediction methods and discuss their underlying principles, their advantages and limitations, and how they compare to and complement one another. Finally, we present current applications and future directions for these methods in biological research and medical genetics. PMID:25234433

  8. Single Nucleotide Polymorphisms and Osteoarthritis

    PubMed Central

    Wang, Ting; Liang, Yuting; Li, Hong; Li, Haibo; He, Quanze; Xue, Ying; Shen, Cong; Zhang, Chunhua; Xiang, Jingjing; Ding, Jie; Qiao, Longwei; Zheng, Qiping

    2016-01-01

    Abstract Osteoarthritis (OA) is a complex disorder characterized by degenerative articular cartilage and is largely attributed to genetic risk factors. Single nucleotide polymorphisms (SNPs) are common DNA variants that have shown promising and efficiency, compared with positional cloning, to map candidate genes of complex diseases, including OA. In this study, we aim to provide an overview of multiple SNPs from a number of genes that have recently been linked to OA susceptibility. We also performed a comprehensive meta-analysis to evaluate the association of SNP rs7639618 of double von Willebrand factor A domains (DVWA) gene with OA susceptibility. A systematic search of studies on the association of SNPs with susceptibility to OA was conducted in PubMed and Google scholar. Studies subjected to meta-analysis include human and case-control studies that met the Hardy–Weinberg equilibrium model and provide sufficient data to calculate an odds ratio (OR). A total of 9500 OA cases and 9365 controls in 7 case-control studies relating to SNP rs7639618 were included in this study and the ORs with 95% confidence intervals (CIs) were calculated. Over 50 SNPs from different genes have been shown to be associated with either hip (23), or knee (20), or both (13) OA. The ORs of these SNPs for OA and the subtypes are not consistent. As to SNP rs7639618 of DVWA, increased knee OA risk was observed in all genetic models analyzed. Specifically, people from Asian with G-allele showed significantly increased risk of knee OA (A versus G: OR = 1.28, 95% CI 1.13–1.46; AA versus GG: OR = 1.60, 95% CI 1.25–2.05; GA versus GG: OR = 1.31, 95% CI 1.18–1.44; AA versus GA+GG: OR = 1.34, 95% CI 1.12–1.61; AA+GA versus GG: OR = 1.40, 95% CI 1.19–1.64), but not in Caucasians or with hip OA. Our results suggest that multiple SNPs play different roles in the pathogenesis of OA and its subtypes; SNP rs7639618 of DVWA gene is associated with a significantly increased

  9. Genetic associations of nonsynonymous exonic variants with psychophysiological endophenotypes.

    PubMed

    Vrieze, Scott I; Malone, Stephen M; Pankratz, Nathan; Vaidyanathan, Uma; Miller, Michael B; Kang, Hyun Min; McGue, Matt; Abecasis, Gonçalo; Iacono, William G

    2014-12-01

    We mapped ∼85,000 rare nonsynonymous exonic single nucleotide polymorphisms (SNPs) to 17 psychophysiological endophenotypes in 4,905 individuals, including antisaccade eye movements, resting EEG, P300 amplitude, electrodermal activity, affect-modulated startle eye blink. Nonsynonymous SNPs are predicted to directly change or disrupt proteins encoded by genes and are expected to have significant biological consequences. Most such variants are rare, and new technologies can efficiently assay them on a large scale. We assayed 247,870 mostly rare SNPs on an Illumina exome array. Approximately 85,000 of the SNPs were polymorphic, rare (MAF < .05), and nonsynonymous. Single variant association tests identified a SNP in the PARD3 gene associated with theta resting EEG power. The sequence kernel association test, a gene-based test, identified a gene PNPLA7 associated with pleasant difference startle, the difference in startle magnitude between pleasant and neutral images. No other single nonsynonymous variant, or gene-based group of variants, was strongly associated with any endophenotype. PMID:25387709

  10. Monovar: single-nucleotide variant detection in single cells.

    PubMed

    Zafar, Hamim; Wang, Yong; Nakhleh, Luay; Navin, Nicholas; Chen, Ken

    2016-06-01

    Current variant callers are not suitable for single-cell DNA sequencing, as they do not account for allelic dropout, false-positive errors and coverage nonuniformity. We developed Monovar (https://bitbucket.org/hamimzafar/monovar), a statistical method for detecting and genotyping single-nucleotide variants in single-cell data. Monovar exhibited superior performance over standard algorithms on benchmarks and in identifying driver mutations and delineating clonal substructure in three different human tumor data sets. PMID:27088313

  11. From Single Nucleotide Polymorphism to Transcriptional Mechanism

    PubMed Central

    Martini, Sebastian; Nair, Viji; Patel, Sanjeevkumar R.; Eichinger, Felix; Nelson, Robert G.; Weil, E. Jennifer; Pezzolesi, Marcus G.; Krolewski, Andrzej S.; Randolph, Ann; Keller, Benjamin J.; Werner, Thomas; Kretzler, Matthias

    2013-01-01

    Genome-wide association studies have proven to be highly effective at defining relationships between single nucleotide polymorphisms (SNPs) and clinical phenotypes in complex diseases. Establishing a mechanistic link between a noncoding SNP and the clinical outcome is a significant hurdle in translating associations into biological insight. We demonstrate an approach to assess the functional context of a diabetic nephropathy (DN)-associated SNP located in the promoter region of the gene FRMD3. The approach integrates pathway analyses with transcriptional regulatory pattern-based promoter modeling and allows the identification of a transcriptional framework affected by the DN-associated SNP in the FRMD3 promoter. This framework provides a testable hypothesis for mechanisms of genomic variation and transcriptional regulation in the context of DN. Our model proposes a possible transcriptional link through which the polymorphism in the FRMD3 promoter could influence transcriptional regulation within the bone morphogenetic protein (BMP)-signaling pathway. These findings provide the rationale to interrogate the biological link between FRMD3 and the BMP pathway and serve as an example of functional genomics-based hypothesis generation. PMID:23434934

  12. Single Nucleotide Polymorphisms for Pig Identification and Parentage Exclusion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single nucleotide polymorphisms have become an important type of marker for commercial diagnostic and parentage genotyping applications as automated genotyping systems have been developed that yield accurate genotypes. Unfortunately, a large number of highly informative public SNP markers tested in ...

  13. A single-nucleotide exon found in Arabidopsis.

    PubMed

    Guo, Lei; Liu, Chun-Ming

    2015-01-01

    The presence of introns in gene-coding regions is one of the most mysterious evolutionary inventions in eukaryotic organisms. It has been proposed that, although sequences involved in intron recognition and splicing are mainly located in introns, exonic sequences also contribute to intron splicing. The smallest constitutively spliced exon known so far has 6 nucleotides, and the smallest alternatively spliced exon has 3 nucleotides. Here we report that the Anaphase Promoting Complex subunit 11 (APC11) gene in Arabidopsis thaliana carries a constitutive single-nucleotide exon. In vivo transcription and translation assays performed using APC11-Green Fluorescence Protein (GFP) fusion constructs revealed that intron splicing surrounding the single-nucleotide exon is effective in both Arabidopsis and rice. This discovery warrants attention to genome annotations in the future. PMID:26657562

  14. A single-nucleotide exon found in Arabidopsis

    PubMed Central

    Guo, Lei; Liu, Chun-Ming

    2015-01-01

    The presence of introns in gene-coding regions is one of the most mysterious evolutionary inventions in eukaryotic organisms. It has been proposed that, although sequences involved in intron recognition and splicing are mainly located in introns, exonic sequences also contribute to intron splicing. The smallest constitutively spliced exon known so far has 6 nucleotides, and the smallest alternatively spliced exon has 3 nucleotides. Here we report that the Anaphase Promoting Complex subunit 11 (APC11) gene in Arabidopsis thaliana carries a constitutive single-nucleotide exon. In vivo transcription and translation assays performed using APC11-Green Fluorescence Protein (GFP) fusion constructs revealed that intron splicing surrounding the single-nucleotide exon is effective in both Arabidopsis and rice. This discovery warrants attention to genome annotations in the future. PMID:26657562

  15. Characterization of single-nucleotide variation in Indian-origin rhesus macaques (Macaca mulatta)

    PubMed Central

    2011-01-01

    Background Rhesus macaques are the most widely utilized nonhuman primate model in biomedical research. Previous efforts have validated fewer than 900 single nucleotide polymorphisms (SNPs) in this species, which limits opportunities for genetic studies related to health and disease. Extensive information about SNPs and other genetic variation in rhesus macaques would facilitate valuable genetic analyses, as well as provide markers for genome-wide linkage analysis and the genetic management of captive breeding colonies. Results We used the available rhesus macaque draft genome sequence, new sequence data from unrelated individuals and existing published sequence data to create a genome-wide SNP resource for Indian-origin rhesus monkeys. The original reference animal and two additional Indian-origin individuals were resequenced to low coverage using SOLiD™ sequencing. We then used three strategies to validate SNPs: comparison of potential SNPs found in the same individual using two different sequencing chemistries, and comparison of potential SNPs in different individuals identified with either the same or different sequencing chemistries. Our approach validated approximately 3 million SNPs distributed across the genome. Preliminary analysis of SNP annotations suggests that a substantial number of these macaque SNPs may have functional effects. More than 700 non-synonymous SNPs were scored by Polyphen-2 as either possibly or probably damaging to protein function and these variants now constitute potential models for studying functional genetic variation relevant to human physiology and disease. Conclusions Resequencing of a small number of animals identified greater than 3 million SNPs. This provides a significant new information resource for rhesus macaques, an important research animal. The data also suggests that overall genetic variation is high in this species. We identified many potentially damaging non-synonymous coding SNPs, providing new opportunities to

  16. Single-nucleotide polymorphisms in porcine mannan-binding lectin A.

    PubMed

    Lillie, Brandon N; Keirstead, Natalie D; Squires, E James; Hayes, M Anthony

    2006-12-01

    The MBL1 and MBL2 genes encode mannan-binding lectins (MBL) A and C, respectively, that are collagenous lectins (collectins) produced mainly by the liver. Several single-nucleotide polymorphisms (SNPs) in the human MBL2 gene are responsible for various innate immune dysfunctions due to abnormal structure or expression of human MBL-C. The MBL1 gene encodes MBL-A, which has bacteria-binding properties in pigs and rodents but is mutated to a pseudogene in humans and chimpanzees. In these studies, we surveyed both porcine MBL genes for SNPs that might impair disease resistance. Single-strand conformational polymorphism (SSCP) analysis of MBL cDNAs from porcine liver revealed three SNPs within the coding region of MBL1 in various breeds of pigs. One nonsynonymous SNP that substituted cysteine for glycine in the collagen-like domain of pig MBL-A was found by a multiplex PCR test in all European pig breeds examined, with allele frequencies ranging from 1.4 to 46.4%. No SNPs were identified in the coding region of porcine MBL2 but the expression of MBL-C in the liver was widely variable in comparison to the expression of MBL-A, GAPDH, PigMAP, and haptoglobin. These results indicate that some pigs have a miscoding defect in MBL-A and a possible expression defect in MBL-C, which are analogous to coding and promoter polymorphisms that affect human MBL-C. PMID:17089118

  17. A Laboratory Exercise for Genotyping Two Human Single Nucleotide Polymorphisms

    ERIC Educational Resources Information Center

    Fernando, James; Carlson, Bradley; LeBard, Timothy; McCarthy, Michael; Umali, Finianne; Ashton, Bryce; Rose, Ferrill F., Jr.

    2016-01-01

    The dramatic decrease in the cost of sequencing a human genome is leading to an era in which a wide range of students will benefit from having an understanding of human genetic variation. Since over 90% of sequence variation between humans is in the form of single nucleotide polymorphisms (SNPs), a laboratory exercise has been devised in order to…

  18. Single Nucleotide Polymorphisms Predict Symptom Severity of Autism Spectrum Disorder

    ERIC Educational Resources Information Center

    Jiao, Yun; Chen, Rong; Ke, Xiaoyan; Cheng, Lu; Chu, Kangkang; Lu, Zuhong; Herskovits, Edward H.

    2012-01-01

    Autism is widely believed to be a heterogeneous disorder; diagnosis is currently based solely on clinical criteria, although genetic, as well as environmental, influences are thought to be prominent factors in the etiology of most forms of autism. Our goal is to determine whether a predictive model based on single-nucleotide polymorphisms (SNPs)…

  19. Discovery, Validation and Characterization of 1039 Cattle Single Nucleotide Polymorphisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We identified approximately 13000 putative single nucleotide polymorphisms (SNPs) by comparison of repeat-masked BAC-end sequences from the cattle RPCI-42 BAC library with whole-genome shotgun contigs of cattle genome assembly Btau 1.0. Genotyping of a subset of these SNPs was performed on a panel ...

  20. A Bioinformatics Approach to Prioritize Single Nucleotide Polymorphisms in TLRs Signaling Pathway Genes.

    PubMed

    Alipoor, Behnam; Ghaedi, Hamid; Omrani, Mir Davood; Bastami, Milad; Meshkani, Reza; Golmohammadi, Taghi

    2016-01-01

    It has been suggested that single nucleotide polymorphisms (SNPs) in genes involved in Toll-like receptors (TLRs) pathway may exhibit broad effects on function of this network and might contribute to a range of human diseases. However, the extent to which these variations affect TLR signaling is not well understood. In this study, we adopted a bioinformatics approach to predict the consequences of SNPs in TLRs network. The consequences of non-synonymous coding SNPs (nsSNPs) were predicted by SIFT, PolyPhen, PANTHER, SNPs&GO, I-Mutant, ConSurf and NetSurf tools. Structural visualization of wild type and mutant protein was performed using the project HOPE and Swiss PDB viewer. The influence of 5'-UTR and 3'- UTR SNPs were analyzed by appropriate computational approaches. Nineteen nsSNPs in TLRs pathway genes were found to have deleterious consequences as predicted by the combination of different algorithms. Moreover, our results suggested that SNPs located at UTRs of TLRs pathway genes may potentially influence binding of transcription factors or microRNAs. By applying a pathway-based bioinformatics analysis of genetic variations, we provided a prioritized list of potentially deleterious variants. These findings may facilitate the selection of proper variants for future functional and/or association studies. PMID:27478803

  1. Development of 101 novel EST-derived single nucleotide polymorphism markers for Zhikong scallop ( Chlamys farreri)

    NASA Astrophysics Data System (ADS)

    Li, Jiqin; Bao, Zhenmin; Li, Ling; Wang, Xiaojian; Wang, Shi; Hu, Xiaoli

    2013-09-01

    Zhikong scallop ( Chlamys farreri) is an important maricultured species in China. Many researches on this species, such as population genetics and QTL fine-mapping, need a large number of molecular markers. In this study, based on the expressed sequence tags (EST), a total of 300 putative single nucleotide polymorphisms (SNPs) were selected and validated using high resolution melting (HRM) technology with unlabeled probe. Of them, 101 (33.7%) were found to be polymorphic in 48 individuals from 4 populations. Further evaluation with 48 individuals from Qingdao population showed that all the polymorphic loci had two alleles with the minor allele frequency ranged from 0.046 to 0.500. The observed and expected heterozygosities ranged from 0.000 to 0.925 and from 0.089 to 0.505, respectively. Fifteen loci deviated significantly from Hardy-Weinberg equilibrium and significant linkage disequilibrate was detected in one pair of markers. BLASTx gave significant hits for 72 of the 101 polymorphic SNP-containing ESTs. Thirty four polymorphic SNP loci were predicted to be non-synonymous substitutions as they caused either the change of codons (33 SNPs) or pretermination of translation (1 SNP). The markers developed can be used for the population studies and genetic improvement on Zhikong scallop.

  2. StructMAn: annotation of single-nucleotide polymorphisms in the structural context.

    PubMed

    Gress, Alexander; Ramensky, Vasily; Büch, Joachim; Keller, Andreas; Kalinina, Olga V

    2016-07-01

    The next generation sequencing technologies produce unprecedented amounts of data on the genetic sequence of individual organisms. These sequences carry a substantial amount of variation that may or may be not related to a phenotype. Phenotypically important part of this variation often comes in form of protein-sequence altering (non-synonymous) single nucleotide variants (nsSNVs). Here we present StructMAn, a Web-based tool for annotation of human and non-human nsSNVs in the structural context. StructMAn analyzes the spatial location of the amino acid residue corresponding to nsSNVs in the three-dimensional (3D) protein structure relative to other proteins, nucleic acids and low molecular-weight ligands. We make use of all experimentally available 3D structures of query proteins, and also, unlike other tools in the field, of structures of proteins with detectable sequence identity to them. This allows us to provide a structural context for around 20% of all nsSNVs in a typical human sequencing sample, for up to 60% of nsSNVs in genes related to human diseases and for around 35% of nsSNVs in a typical bacterial sample. Each nsSNV can be visualized and inspected by the user in the corresponding 3D structure of a protein or protein complex. The StructMAn server is available at http://structman.mpi-inf.mpg.de. PMID:27150811

  3. Investigating single nucleotide polymorphism (SNP) density in the human genome and its implications for molecular evolution.

    PubMed

    Zhao, Zhongming; Fu, Yun-Xin; Hewett-Emmett, David; Boerwinkle, Eric

    2003-07-17

    We investigated the single nucleotide polymorphism (SNP) density across the human genome and in different genic categories using two SNP databases: Celera's CgsSNP, which includes SNPs identified by comparing genomic sequences, and Celera's RefSNP, which includes SNPs from a variety of sources and is biased toward disease-associated genes. Based on CgsSNP, the average numbers of SNPs per 10 kb was 8.33, 8.44, and 8.09 in the human genome, in intergenic regions, and in genic regions, respectively. In genic regions, the SNP density in intronic, exonic and adjoining untranslated regions was 8.21, 5.28, and 7.51 SNPs per 10 kb, respectively. The pattern of SNP density based on RefSNP was different from that based on CgsSNP, emphasizing its utility for genotype-phenotype association studies but not for most population genetic studies. The number of SNPs per chromosome was correlated with chromosome length, but the density of SNPs estimated by CgsSNP was not significantly correlated with the GC content of the chromosome. Based on CgsSNP, the ratio of nonsense to missense mutations (0.027), the ratio of missense to silent mutations (1.15), and the ratio of non-synonymous to synonymous mutations (1.18) was less than half of that expected in a human protein coding sequence under the neutral mutation theory, reflecting a role for natural selection, especially purifying selection. PMID:12909357

  4. Alteration of Antiviral Signalling by Single Nucleotide Polymorphisms (SNPs) of Mitochondrial Antiviral Signalling Protein (MAVS)

    PubMed Central

    Xing, Fei; Matsumiya, Tomoh; Hayakari, Ryo; Yoshida, Hidemi; Kawaguchi, Shogo; Takahashi, Ippei; Nakaji, Shigeyuki; Imaizumi, Tadaatsu

    2016-01-01

    Genetic variation is associated with diseases. As a type of genetic variation occurring with certain regularity and frequency, the single nucleotide polymorphism (SNP) is attracting more and more attention because of its great value for research and real-life application. Mitochondrial antiviral signalling protein (MAVS) acts as a common adaptor molecule for retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), which can recognize foreign RNA, including viral RNA, leading to the induction of type I interferons (IFNs). Therefore, MAVS is thought to be a crucial molecule in antiviral innate immunity. We speculated that genetic variation of MAVS may result in susceptibility to infectious diseases. To assess the risk of viral infection based on MAVS variation, we tested the effects of twelve non-synonymous MAVS coding-region SNPs from the National Center for Biotechnology Information (NCBI) database that result in amino acid substitutions. We found that five of these SNPs exhibited functional alterations. Additionally, four resulted in an inhibitory immune response, and one had the opposite effect. In total, 1,032 human genomic samples obtained from a mass examination were genotyped at these five SNPs. However, no homozygous or heterozygous variation was detected. We hypothesized that these five SNPs are not present in the Japanese population and that such MAVS variations may result in serious immune diseases. PMID:26954674

  5. StructMAn: annotation of single-nucleotide polymorphisms in the structural context

    PubMed Central

    Gress, Alexander; Ramensky, Vasily; Büch, Joachim; Keller, Andreas; Kalinina, Olga V.

    2016-01-01

    The next generation sequencing technologies produce unprecedented amounts of data on the genetic sequence of individual organisms. These sequences carry a substantial amount of variation that may or may be not related to a phenotype. Phenotypically important part of this variation often comes in form of protein-sequence altering (non-synonymous) single nucleotide variants (nsSNVs). Here we present StructMAn, a Web-based tool for annotation of human and non-human nsSNVs in the structural context. StructMAn analyzes the spatial location of the amino acid residue corresponding to nsSNVs in the three-dimensional (3D) protein structure relative to other proteins, nucleic acids and low molecular-weight ligands. We make use of all experimentally available 3D structures of query proteins, and also, unlike other tools in the field, of structures of proteins with detectable sequence identity to them. This allows us to provide a structural context for around 20% of all nsSNVs in a typical human sequencing sample, for up to 60% of nsSNVs in genes related to human diseases and for around 35% of nsSNVs in a typical bacterial sample. Each nsSNV can be visualized and inspected by the user in the corresponding 3D structure of a protein or protein complex. The StructMAn server is available at http://structman.mpi-inf.mpg.de. PMID:27150811

  6. Single-Nucleotide Variations in Cardiac Arrhythmias: Prospects for Genomics and Proteomics Based Biomarker Discovery and Diagnostics

    PubMed Central

    Abunimer, Ayman; Smith, Krista; Wu, Tsung-Jung; Lam, Phuc; Simonyan, Vahan; Mazumder, Raja

    2014-01-01

    Cardiovascular diseases are a large contributor to causes of early death in developed countries. Some of these conditions, such as sudden cardiac death and atrial fibrillation, stem from arrhythmias—a spectrum of conditions with abnormal electrical activity in the heart. Genome-wide association studies can identify single nucleotide variations (SNVs) that may predispose individuals to developing acquired forms of arrhythmias. Through manual curation of published genome-wide association studies, we have collected a comprehensive list of 75 SNVs associated with cardiac arrhythmias. Ten of the SNVs result in amino acid changes and can be used in proteomic-based detection methods. In an effort to identify additional non-synonymous mutations that affect the proteome, we analyzed the post-translational modification S-nitrosylation, which is known to affect cardiac arrhythmias. We identified loss of seven known S-nitrosylation sites due to non-synonymous single nucleotide variations (nsSNVs). For predicted nitrosylation sites we found 1429 proteins where the sites are modified due to nsSNV. Analysis of the predicted S-nitrosylation dataset for over- or under-representation (compared to the complete human proteome) of pathways and functional elements shows significant statistical over-representation of the blood coagulation pathway. Gene Ontology (GO) analysis displays statistically over-represented terms related to muscle contraction, receptor activity, motor activity, cystoskeleton components, and microtubule activity. Through the genomic and proteomic context of SNVs and S-nitrosylation sites presented in this study, researchers can look for variation that can predispose individuals to cardiac arrhythmias. Such attempts to elucidate mechanisms of arrhythmia thereby add yet another useful parameter in predicting susceptibility for cardiac diseases. PMID:24705329

  7. Discovery and validation of genic single nucleotide polymorphisms in the Pacific oyster Crassostrea gigas.

    PubMed

    Wang, Jiafeng; Qi, Haigang; Li, Li; Que, Huayong; Wang, Di; Zhang, Guofan

    2015-01-01

    The economic and ecological importance of the oyster necessitates further research on the molecular mechanisms, which both regulate the commercially important traits of the oyster and help it to survive in the variable marine environment. Single nucleotide polymorphisms (SNPs) have been widely used to assess genetic variation and identify genes underlying target traits. In addition, high-resolution melting (HRM) analysis is a potentially powerful method for validating candidate SNPs. In this study, we adopted a rapid and efficient pipeline for the screening and validation of SNPs in the genic region of Crassostrea gigas based on transcriptome sequencing and HRM analysis. Transcriptomes of three wild oyster populations were sequenced using Illumina sequencing technology. In total, 50-60 million short reads, corresponding to 4.5-5.4 Gbp, from each population were aligned to the oyster genome, and 5.8 × 10(5) SNPs were putatively identified, resulting in a predicted SNP every 47 nucleotides on average. The putative SNPs were unevenly distributed in the genome and high-density (≥2%), nonsynonymous coding SNPs were enriched in genes related to apoptosis and responses to biotic stimuli. Subsequently, 1,671 loci were detected by HRM analysis, accounting for 64.7% of the total selected candidate primers, and finally, 1,301 polymorphic SNP markers were developed based on HRM analysis. All of the validated SNPs were distributed into 897 genes and located in 672 scaffolds, and 275 of these genes were stress inducible under unfavourable salinity, temperature, and exposure to air and heavy metals. The validated SNPs in this study provide valuable molecular markers for genetic mapping and characterization of important traits in oysters. PMID:24823694

  8. 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.

  9. Establishment of a pipeline to analyse non-synonymous SNPs in Bos taurus

    PubMed Central

    Lee, Michael A; Keane, Orla M; Glass, Belinda C; Manley, Tim R; Cullen, Neil G; Dodds, Ken G; McCulloch, Alan F; Morris, Chris A; Schreiber, Mark; Warren, Jonathan; Zadissa, Amonida; Wilson, Theresa; McEwan, John C

    2006-01-01

    Background Single nucleotide polymorphisms (SNPs) are an abundant form of genetic variation in the genome of every species and are useful for gene mapping and association studies. Of particular interest are non-synonymous SNPs, which may alter protein function and phenotype. We therefore examined bovine expressed sequences for non-synonymous SNPs and validated and tested selected SNPs for their association with measured traits. Results Over 500,000 public bovine expressed sequence tagged (EST) sequences were used to search for coding SNPs (cSNPs). A total of 15,353 SNPs were detected in the transcribed sequences studied, of which 6,325 were predicted to be coding SNPs with the remaining 9,028 SNPs presumed to be in untranslated regions. Of the cSNPs detected, 2,868 were predicted to result in a change in the amino acid encoded. In order to determine the actual number of non-synonymous polymorphic SNPs we designed assays for 920 of the putative SNPs. These SNPs were then genotyped through a panel of cattle DNA pools using chip-based MALDI-TOF mass spectrometry. Of the SNPs tested, 29% were found to be polymorphic with a minor allele frequency >10%. A subset of the SNPs was genotyped through animal resources in order to look for association with age of puberty, facial eczema resistance or meat yield. Three SNPs were nominally associated with resistance to the disease facial eczema (P < 0.01). Conclusion We have identified 15,353 putative SNPs in or close to bovine genes and 2,868 of these SNPs were predicted to be non-synonymous. Approximately 29% of the non-synonymous SNPs were polymorphic and common with a minor allele frequency >10%. Of the SNPs detected in this study, 99% have not been previously reported. These novel SNPs will be useful for association studies or gene mapping. PMID:17125523

  10. Pathway analysis of single-nucleotide polymorphisms potentially associated with glioblastoma multiforme susceptibility using random forests.

    PubMed

    Chang, Jeffrey S; Yeh, Ru-Fang; Wiencke, John K; Wiemels, Joseph L; Smirnov, Ivan; Pico, Alexander R; Tihan, Tarik; Patoka, Joe; Miike, Rei; Sison, Jennette D; Rice, Terri; Wrensch, Margaret R

    2008-06-01

    Glioma is a complex disease that is unlikely to result from the effect of a single gene. Genetic analysis at the pathway level involving multiple genes may be more likely to capture gene-disease associations than analyzing genes one at a time. The current pilot study included 112 Caucasians with glioblastoma multiforme and 112 Caucasian healthy controls frequency matched to cases by age and gender. Subjects were genotyped using a commercially available (ParAllele/Affymetrix) assay panel of 10,177 nonsynonymous coding single-nucleotide polymorphisms (SNP) spanning the genome known at the time the panel was constructed. For this analysis, we selected 10 pathways potentially involved in gliomagenesis that had SNPs represented on the panel. We performed random forests (RF) analyses of SNPs within each pathway group and logistic regression to assess interaction among genes in the one pathway for which the RF prediction error was better than chance and the permutation P < 0.10. Only the DNA repair pathway had a better than chance classification of case-control status with a prediction error of 45.5% and P = 0.09. Three SNPs (rs1047840 of EXO1, rs12450550 of EME1, and rs799917 of BRCA1) of the DNA repair pathway were identified as promising candidates for further replication. In addition, statistically significant interactions (P < 0.05) between rs1047840 of EXO1 and rs799917 or rs1799966 of BRCA1 were observed. Despite less than complete inclusion of genes and SNPs relevant to glioma and a small sample size, RF analysis identified one important biological pathway and several SNPs potentially associated with the development of glioblastoma. PMID:18559551

  11. Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms.

    PubMed

    Fakhrai-Rad, Hossein; Pourmand, Nader; Ronaghi, Mostafa

    2002-05-01

    Pyrosequencing, a non-electrophoretic method for DNA sequencing, is emerging as a popular platform for analysis of single nucleotide polymorphisms (SNPs). This technology has the advantage of accuracy, ease-of-use, and high flexibility for different applications. Here, we review the methodology and the use of this technique for SNP genotyping, SNP discovery, haplotyping, and allelic frequency studies. In addition, we describe new schemes for template preparation and multiplexing as an effort for cost reduction in large-scale studies. PMID:11968080

  12. Single nucleotide polymorphisms in the MATP gene are associated with normal human pigmentation variation.

    PubMed

    Graf, Justin; Hodgson, Richard; van Daal, Angela

    2005-03-01

    Human physical pigmentation is determined by the type and amount of melanin and the process of pigmentation production probably involves more than 100 genes. A failure to synthesize melanin results in oculocutaneous albinism (OCA). A recently identified form of OCA results from mutations in the Membrane Associated Transporter Protein (MATP) gene. The role of MATP in human pigmentation is not clear. We investigated the role of two nonpathogenic nonsynonymous single nucleotide polymorphisms (SNPs) in the MATP gene to determine if they are associated with normal human skin, hair, and eye color variation. A total of 608 individuals from four different population groups (456 Caucasians, 31 Asians, 70 African-Americans, and 51 Australian Aborigines) were genotyped for c.814G>A (p.Glu272Lys) and c.1122C>G (p.Phe374Leu). Results indicate that the allele frequencies of both polymorphisms are significantly different between population groups. The two alleles, 374Leu and 272Lys, are significantly associated with dark hair, skin, and eye color in Caucasians. The odds ratios (ORs) of the LeuLeu genotype for black hair and olive skin are 25.63 and 28.65, respectively, and for the LysLys genotype are 43.23 and 8.27, respectively. The OR for eye color is lower at 3.48 for the LeuLeu and 6.57 for LysLys genotypes. This is the first report of this highly significant association of MATP polymorphisms with normal human pigmentation variation. PMID:15714523

  13. Postzygotic single-nucleotide mosaicisms in whole-genome sequences of clinically unremarkable individuals

    PubMed Central

    Huang, August Y; Xu, Xiaojing; Ye, Adam Y; Wu, Qixi; Yan, Linlin; Zhao, Boxun; Yang, Xiaoxu; He, Yao; Wang, Sheng; Zhang, Zheng; Gu, Bowen; Zhao, Han-Qing; Wang, Meng; Gao, Hua; Gao, Ge; Zhang, Zhichao; Yang, Xiaoling; Wu, Xiru; Zhang, Yuehua; Wei, Liping

    2014-01-01

    Postzygotic single-nucleotide mutations (pSNMs) have been studied in cancer and a few other overgrowth human disorders at whole-genome scale and found to play critical roles. However, in clinically unremarkable individuals, pSNMs have never been identified at whole-genome scale largely due to technical difficulties and lack of matched control tissue samples, and thus the genome-wide characteristics of pSNMs remain unknown. We developed a new Bayesian-based mosaic genotyper and a series of effective error filters, using which we were able to identify 17 SNM sites from ∼80× whole-genome sequencing of peripheral blood DNAs from three clinically unremarkable adults. The pSNMs were thoroughly validated using pyrosequencing, Sanger sequencing of individual cloned fragments, and multiplex ligation-dependent probe amplification. The mutant allele fraction ranged from 5%-31%. We found that C→T and C→A were the predominant types of postzygotic mutations, similar to the somatic mutation profile in tumor tissues. Simulation data showed that the overall mutation rate was an order of magnitude lower than that in cancer. We detected varied allele fractions of the pSNMs among multiple samples obtained from the same individuals, including blood, saliva, hair follicle, buccal mucosa, urine, and semen samples, indicating that pSNMs could affect multiple sources of somatic cells as well as germ cells. Two of the adults have children who were diagnosed with Dravet syndrome. We identified two non-synonymous pSNMs in SCN1A, a causal gene for Dravet syndrome, from these two unrelated adults and found that the mutant alleles were transmitted to their children, highlighting the clinical importance of detecting pSNMs in genetic counseling. PMID:25312340

  14. Nonrandom Distribution of miRNAs Genes and Single Nucleotide Variants in Keratoconus Loci

    PubMed Central

    Nowak, Dorota M.; Gajecka, Marzena

    2015-01-01

    Despite numerous studies, the causes of both development and progression of keratoconus remain elusive. Previous studies of this disorder focused mainly on one or two genetic factors only. However, in the analysis of such complex diseases all potential factors should be taken into consideration. The purpose of this study was a comprehensive analysis of known keratoconus loci to uncover genetic factors involved in this disease causation in the general population, which could be omitted in the original studies. In this investigation genomic data available in various databases and experimental own data were assessed. The lists of single nucleotide variants and miRNA genes localized in reported keratoconus loci were obtained from Ensembl and miRBase, respectively. The potential impact of nonsynonymous amino acid substitutions on protein structure and function was assessed with PolyPhen-2 and SIFT. For selected protein genes the ranking was made to choose those most promising for keratoconus development. Ranking results were based on topological features in the protein-protein interaction network. High specificity for the populations in which the causative sequence variants have been identified was found. In addition, the possibility of links between previously analyzed keratoconus loci was confirmed including miRNA-gene interactions. Identified number of genes associated with oxidative stress and inflammatory agents corroborated the hypothesis of their effect on the disease etiology. Distribution of the numerous sequences variants within both exons and mature miRNA which forces you to search for a broader look at the determinants of keratoconus. Our findings highlight the complexity of the keratoconus genetics. PMID:26176855

  15. Functional and Structural Consequences of Damaging Single Nucleotide Polymorphisms in Human Prostate Cancer Predisposition Gene RNASEL

    PubMed Central

    Datta, Amit; Mazumder, Md. Habibul Hasan; Chowdhury, Afrin Sultana; Hasan, Md. Anayet

    2015-01-01

    A commonly diagnosed cancer, prostate cancer (PrCa), is being regulated by the gene RNASEL previously known as PRCA1 codes for ribonuclease L which is an integral part of interferon regulated system that mediates antiviral and antiproliferative role of the interferons. Both somatic and germline mutations have been implicated to cause prostate cancer. With an array of available Single Nucleotide Polymorphism data on dbSNP this study is designed to sort out functional SNPs in RNASEL by implementing different authentic computational tools such as SIFT, PolyPhen, SNPs&GO, Fathmm, ConSurf, UTRScan, PDBsum, Tm-Align, I-Mutant, and Project HOPE for functional and structural assessment, solvent accessibility, molecular dynamics, and energy minimization study. Among 794 RNASEL SNP entries 124 SNPs were found nonsynonymous from which SIFT predicted 13 nsSNPs as nontolerable whereas PolyPhen-2 predicted 28. SNPs found on the 3′ and 5′ UTR were also assessed. By analyzing six tools having different perspectives an aggregate result was produced where nine nsSNPs were found to be most likely to exert deleterious effect. 3D models of mutated proteins were generated to determine the functional and structural effect of the mutations on ribonuclease L. The initial findings were reinforced by the results from I-Mutant and Project HOPE as these tools predicted significant structural and functional instability of the mutated proteins. Expasy-ProSit tool defined the mutations to be situated in the functional domains of the protein. Considering previous analysis this study revealed a conclusive result deducing the available SNP data on the database by identifying the most damaging three nsSNP rs151296858 (G59S), rs145415894 (A276V), and rs35896902 (R592H). As such studies involving polymorphisms of RNASEL were none to be found, the results of the current study would certainly be helpful in future prospects concerning prostate cancer in males. PMID:26236721

  16. Association of loblolly pine xylem development gene expression with single-nucleotide polymorphisms.

    PubMed

    Palle, Sreenath R; Seeve, Candace M; Eckert, Andrew J; Wegrzyn, Jill L; Neale, David B; Loopstra, Carol A

    2013-07-01

    Variation in the expression of genes with putative roles in wood development was associated with single-nucleotide polymorphisms (SNPs) using a population of loblolly pine (Pinus taeda L.) that included individuals from much of the native range. Association studies were performed using 3938 SNPs and expression data obtained using quantitative real-time polymerase chain reaction (PCR) (qRT-PCR) for 106 xylem development genes in 400 clonally replicated loblolly pine individuals. A general linear model (GLM) approach, which takes the underlying population structure into consideration, was used to discover significant associations. After adjustment for multiple testing using a false discovery rate correction, 88 statistically significant associations (Q<0.05) were observed for 80 SNPs with the expression data of 33 xylem development genes. Thirty SNPs caused nonsynonymous mutations, 18 resulted in synonymous mutations, 11 were in 3' untranslated regions (UTRs), 1 was in a 5' UTR and 20 were in introns. Using AraNet, we found that Arabidopsis genes with high similarity to the loblolly pine genes involved in 21 of the 88 statistically significant associations are connected in functional gene networks. Comparisons of gene expression values revealed that in most cases the average expression in plants homozygous for the rare SNP allele was lower than that of plants that were heterozygous or homozygous for the abundant allele. Although there are association studies of SNPs and expression profiles for humans, Arabidopsis and white spruce, to the best of our knowledge, this is the first example of such an association genetic study in pines. Functional validation of these associations will lead to a deeper understanding of the molecular basis of phenotypic differences in wood development among individuals in conifer populations. PMID:23933831

  17. Functional and Structural Consequences of Damaging Single Nucleotide Polymorphisms in Human Prostate Cancer Predisposition Gene RNASEL.

    PubMed

    Datta, Amit; Mazumder, Md Habibul Hasan; Chowdhury, Afrin Sultana; Hasan, Md Anayet

    2015-01-01

    A commonly diagnosed cancer, prostate cancer (PrCa), is being regulated by the gene RNASEL previously known as PRCA1 codes for ribonuclease L which is an integral part of interferon regulated system that mediates antiviral and antiproliferative role of the interferons. Both somatic and germline mutations have been implicated to cause prostate cancer. With an array of available Single Nucleotide Polymorphism data on dbSNP this study is designed to sort out functional SNPs in RNASEL by implementing different authentic computational tools such as SIFT, PolyPhen, SNPs&GO, Fathmm, ConSurf, UTRScan, PDBsum, Tm-Align, I-Mutant, and Project HOPE for functional and structural assessment, solvent accessibility, molecular dynamics, and energy minimization study. Among 794 RNASEL SNP entries 124 SNPs were found nonsynonymous from which SIFT predicted 13 nsSNPs as nontolerable whereas PolyPhen-2 predicted 28. SNPs found on the 3' and 5' UTR were also assessed. By analyzing six tools having different perspectives an aggregate result was produced where nine nsSNPs were found to be most likely to exert deleterious effect. 3D models of mutated proteins were generated to determine the functional and structural effect of the mutations on ribonuclease L. The initial findings were reinforced by the results from I-Mutant and Project HOPE as these tools predicted significant structural and functional instability of the mutated proteins. Expasy-ProSit tool defined the mutations to be situated in the functional domains of the protein. Considering previous analysis this study revealed a conclusive result deducing the available SNP data on the database by identifying the most damaging three nsSNP rs151296858 (G59S), rs145415894 (A276V), and rs35896902 (R592H). As such studies involving polymorphisms of RNASEL were none to be found, the results of the current study would certainly be helpful in future prospects concerning prostate cancer in males. PMID:26236721

  18. Discovery and verification of functional single nucleotide polymorphisms in regulatory genomic regions: Current and developing technologies

    PubMed Central

    Chorley, Brian N.; Wang, Xuting; Campbell, Michelle R.; Pittman, Gary S.; Noureddine, Maher A.; Bell, Douglas A.

    2008-01-01

    The most common form of genetic variation, single nucleotide polymorphisms or SNPs, can affect the way an individual responds to the environment and modify disease risk. Although most of the millions of SNPs have little or no effect on gene regulation and protein activity, there are many circumstances where base changes can have deleterious effects. Non-synonymous SNPs that result in amino acid changes in proteins have been studied because of their obvious impact on protein activity. It is well known that SNPs within regulatory regions of the genome can result in disregulation of gene transcription. However, the impact of SNPs located in putative regulatory regions, or rSNPs, is harder to predict for two primary reasons. First, the mechanistic roles of non-coding genomic sequence remain poorly defined. Second, experimental validation of the functional consequences of rSNPs is often slow and laborious. In this review, we summarize traditional and novel methodologies for candidate rSNPs selection, in particular in silico techniques that aid in candidate rSNP selection. Additionally we will discuss molecular biological techniques that assess the impact of rSNPs on binding of regulatory machinery, as well as functional consequences on transcription. Standard techniques such as EMSA and luciferase reporter constructs are still widely used to assess effects of rSNPs on binding and gene transcription; however, these protocols are often bottlenecks in the discovery process. Therefore, we highlight novel and developing high-throughput protocols that promise to aid in shortening the process of rSNP validation. Given the large amount of genomic information generated from a multitude of re-sequencing and genome-wide SNP array efforts, future focus should be to develop validation techniques that will allow greater understanding of the impact these polymorphisms have on human health and disease. PMID:18565787

  19. Shifting Paradigm of Association Studies: Value of Rare Single-Nucleotide Polymorphisms

    PubMed Central

    Gorlov, Ivan P.; Gorlova, Olga Y.; Sunyaev, Shamil R.; Spitz, Margaret R.; Amos, Christopher I.

    2008-01-01

    Summary Currently, single-nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) of >5% are preferentially used in case-control association studies of common human diseases. Recent technological developments enable inexpensive and accurate genotyping of a large number of SNPs in thousands of cases and controls, which can provide adequate statistical power to analyze SNPs with MAF <5%. Our purpose was to determine whether evaluating rare SNPs in case-control association studies could help identify causal SNPs for common diseases. We suggest that slightly deleterious SNPs (sdSNPs) subjected to weak purifying selection are major players in genetic control of susceptibility to common diseases. We compared the distribution of MAFs of synonymous SNPs with that of nonsynonymous SNPs (1) predicted to be benign, (2) predicted to be possibly damaging, and (3) predicted to be probably damaging by PolyPhen. Our sources of data were the International HapMap Project, ENCODE, and the SeattleSNPs project. We found that the MAF distribution of possibly and probably damaging SNPs was shifted toward rare SNPs compared with the MAF distribution of benign and synonymous SNPs that are not likely to be functional. We also found an inverse relationship between MAF and the proportion of nsSNPs predicted to be protein disturbing. On the basis of this relationship, we estimated the joint probability that a SNP is functional and would be detected as significant in a case-control study. Our analysis suggests that including rare SNPs in genotyping platforms will advance identification of causal SNPs in case-control association studies, particularly as sample sizes increase. PMID:18179889

  20. 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

  1. Identification of single nucleotides in MoS2 nanopores.

    PubMed

    Feng, Jiandong; Liu, Ke; Bulushev, Roman D; Khlybov, Sergey; Dumcenco, Dumitru; Kis, Andras; Radenovic, Aleksandra

    2015-12-01

    The size of the sensing region in solid-state nanopores is determined by the size of the pore and the thickness of the pore membrane, so ultrathin membranes such as graphene and single-layer molybdenum disulphide could potentially offer the necessary spatial resolution for nanopore DNA sequencing. However, the fast translocation speeds (3,000-50,000 nt ms(-1)) of DNA molecules moving across such membranes limit their usability. Here, we show that a viscosity gradient system based on room-temperature ionic liquids can be used to control the dynamics of DNA translocation through MoS2 nanopores. The approach can be used to statistically detect all four types of nucleotide, which are identified according to current signatures recorded during their transient residence in the narrow orifice of the atomically thin MoS2 nanopore. Our technique, which exploits the high viscosity of room-temperature ionic liquids, provides optimal single nucleotide translocation speeds for DNA sequencing, while maintaining a signal-to-noise ratio higher than 10. PMID:26389660

  2. Evaluation of published single nucleotide polymorphisms associated with acute GVHD.

    PubMed

    Chien, Jason W; Zhang, Xinyi Cindy; Fan, Wenhong; Wang, Hongwei; Zhao, Lue Ping; Martin, Paul J; Storer, Barry E; Boeckh, Michael; Warren, Edus H; Hansen, John A

    2012-05-31

    Candidate genetic associations with acute GVHD (aGVHD) were evaluated with the use of genotyped and imputed single-nucleotide polymorphism data from genome-wide scans of 1298 allogeneic hematopoietic cell transplantation (HCT) donors and recipients. Of 40 previously reported candidate SNPs, 6 were successfully genotyped, and 10 were imputed and passed criteria for analysis. Patient and donor genotypes were assessed for association with grades IIb-IV and III-IV aGVHD, stratified by donor type, in univariate and multivariate allelic, recessive and dominant models. Use of imputed genotypes to replicate previous IL10 associations was validated. Similar to previous publications, the IL6 donor genotype for rs1800795 was associated with a 20%-50% increased risk for grade IIb-IV aGVHD after unrelated HCT in the allelic (adjusted P = .011) and recessive (adjusted P = .0013) models. The donor genotype was associated with a 60% increase in risk for grade III-IV aGVHD after related HCT (adjusted P = .028). Other associations were found for IL2, CTLA4, HPSE, and MTHFR but were inconsistent with original publications. These results illustrate the advantages of using imputed single-nucleotide polymorphism data in genetic analyses and demonstrate the importance of validation in genetic association studies. PMID:22282500

  3. Syndrome-based discrimination of single nucleotide polymorphism.

    PubMed

    May, E E; Dolan, P; Crozier, P; Brozik, S

    2006-01-01

    The ability to discriminate nucleic acid sequences is necessary for a wide variety of applications: high throughput screening, distinguishing genetically modified organisms (GMOs), molecular computing, differentiating biological markers, fingerprinting a specific sensor response for complex systems, etc. Hybridization-based target recognition and discrimination is central to the operation of nucleic acid sensor systems. Therefore developing a quantitative correlation between mishybridization events and sensor out put is critical to the accurate interpretation of results. In this work, using experimental data produced by introducing single mutations (single nucleotide polymorphisms, SNPs) in the probe sequence of computational catalytic molecular beacons (deoxyribozyme gates) [1], we investigate coding theory algorithms for uniquely categorizing SNPs based on the calculation of syndromes. PMID:17947098

  4. A single natural nucleotide mutation alters bacterial pathogen host tropism.

    PubMed

    Viana, David; Comos, María; McAdam, Paul R; Ward, Melissa J; Selva, Laura; Guinane, Caitriona M; González-Muñoz, Beatriz M; Tristan, Anne; Foster, Simon J; Fitzgerald, J Ross; Penadés, José R

    2015-04-01

    The capacity of microbial pathogens to alter their host tropism leading to epidemics in distinct host species populations is a global public and veterinary health concern. To investigate the molecular basis of a bacterial host-switching event in a tractable host species, we traced the evolutionary trajectory of the common rabbit clone of Staphylococcus aureus. We report that it evolved through a likely human-to-rabbit host jump over 40 years ago and that only a single naturally occurring nucleotide mutation was required and sufficient to convert a human-specific S. aureus strain into one that could infect rabbits. Related mutations were identified at the same locus in other rabbit strains of distinct clonal origin, consistent with convergent evolution. This first report of a single mutation that was sufficient to alter the host tropism of a microorganism during its evolution highlights the capacity of some pathogens to readily expand into new host species populations. PMID:25685890

  5. Single nucleotide variations in CLCN6 identified in patients with benign partial epilepsies in infancy and/or febrile seizures.

    PubMed

    Yamamoto, Toshiyuki; Shimojima, Keiko; Sangu, Noriko; Komoike, Yuta; Ishii, Atsushi; Abe, Shinpei; Yamashita, Shintaro; Imai, Katsumi; Kubota, Tetsuo; Fukasawa, Tatsuya; Okanishi, Tohru; Enoki, Hideo; Tanabe, Takuya; Saito, Akira; Furukawa, Toru; Shimizu, Toshiaki; Milligan, Carol J; Petrou, Steven; Heron, Sarah E; Dibbens, Leanne M; Hirose, Shinichi; Okumura, Akihisa

    2015-01-01

    Nucleotide alterations in the gene encoding proline-rich transmembrane protein 2 (PRRT2) have been identified in most patients with benign partial epilepsies in infancy (BPEI)/benign familial infantile epilepsy (BFIE). However, not all patients harbor these PRRT2 mutations, indicating the involvement of genes other than PRRT2. In this study, we performed whole exome sequencing analysis for a large family affected with PRRT2-unrelated BPEI. We identified a non-synonymous single nucleotide variation (SNV) in the voltage-sensitive chloride channel 6 gene (CLCN6). A cohort study of 48 BPEI patients without PRRT2 mutations revealed a different CLCN6 SNV in a patient, his sibling and his father who had a history of febrile seizures (FS) but not BPEI. Another study of 48 patients with FS identified an additional SNV in CLCN6. Chloride channels (CLCs) are involved in a multitude of physiologic processes and some members of the CLC family have been linked to inherited diseases. However, a phenotypic correlation has not been confirmed for CLCN6. Although we could not detect significant biological effects linked to the identified CLCN6 SNVs, further studies should investigate potential CLCN6 variants that may underlie the genetic susceptibility to convulsive disorders. PMID:25794116

  6. Single Nucleotide Variations in CLCN6 Identified in Patients with Benign Partial Epilepsies in Infancy and/or Febrile Seizures

    PubMed Central

    Yamamoto, Toshiyuki; Shimojima, Keiko; Sangu, Noriko; Komoike, Yuta; Ishii, Atsushi; Abe, Shinpei; Yamashita, Shintaro; Imai, Katsumi; Kubota, Tetsuo; Fukasawa, Tatsuya; Okanishi, Tohru; Enoki, Hideo; Tanabe, Takuya; Saito, Akira; Furukawa, Toru; Shimizu, Toshiaki; Milligan, Carol J.; Petrou, Steven; Heron, Sarah E.; Dibbens, Leanne M.; Hirose, Shinichi; Okumura, Akihisa

    2015-01-01

    Nucleotide alterations in the gene encoding proline-rich transmembrane protein 2 (PRRT2) have been identified in most patients with benign partial epilepsies in infancy (BPEI)/benign familial infantile epilepsy (BFIE). However, not all patients harbor these PRRT2 mutations, indicating the involvement of genes other than PRRT2. In this study, we performed whole exome sequencing analysis for a large family affected with PRRT2-unrelated BPEI. We identified a non-synonymous single nucleotide variation (SNV) in the voltage-sensitive chloride channel 6 gene (CLCN6). A cohort study of 48 BPEI patients without PRRT2 mutations revealed a different CLCN6 SNV in a patient, his sibling and his father who had a history of febrile seizures (FS) but not BPEI. Another study of 48 patients with FS identified an additional SNV in CLCN6. Chloride channels (CLCs) are involved in a multitude of physiologic processes and some members of the CLC family have been linked to inherited diseases. However, a phenotypic correlation has not been confirmed for CLCN6. Although we could not detect significant biological effects linked to the identified CLCN6 SNVs, further studies should investigate potential CLCN6 variants that may underlie the genetic susceptibility to convulsive disorders. PMID:25794116

  7. Y-Single Nucleotide Polymorphisms Diversity in Chinese Indigenous Horse

    PubMed Central

    Han, Haoyuan; Zhang, Qin; Gao, Kexin; Yue, Xiangpeng; Zhang, Tao; Dang, Ruihua; Lan, Xianyong; Chen, Hong; Lei, Chuzhao

    2015-01-01

    In contrast to high genetic diversity of mitochondrial DNA (mtDNA), equine Y chromosome shows extremely low variability, implying limited patrilines in the domesticated horse. In this study, we applied direct sequencing and restriction fragment length polymorphism (RFLP) methods to investigate the polymorphisms of 33 Y chromosome specific loci in 304 Chinese indigenous horses from 13 breeds. Consequently, two Y-single nucleotide polymorphisms (SNPs) (Y-45701/997 and Y-50869) and one Y-indel (Y-45288) were identified. Of those, the Y-50869 (T>A) revealed the highest variation frequency (24.67%), whereas it was only 3.29% and 1.97% in Y-45288 (T/-) and Y-45701/997 (G>T) locus, respectively. These three mutations accounted for 27.96% of the total samples and identified five Y-SNP haplotypes, demonstrating genetic diversity of Y chromosome in Chinese horses. In addition, all the five Y-SNP haplotypes were shared by different breeds. Among 13 horse breeds analyzed, Balikun horse displayed the highest nucleotide diversity (π = 5.6×10−4) and haplotype diversity (h = 0.527), while Ningqiang horse showed the lowest nucleotide diversity (π = 0.00000) and haplotype diversity (h = 0.000). The results also revealed that Chinese horses had a different polymorphic pattern of Y chromosome from European and American horses. In conclusion, Chinese horses revealed genetic diversity of Y chromosome, however more efforts should be made to better understand the domestication and paternal origin of Chinese indigenous horses. PMID:26104513

  8. A MEMS-Based Approach to Single Nucleotide Polymorphism Genotyping

    PubMed Central

    Zhu, Jing; Palla, Mirkó; Ronca, Stefano; Warpner, Ronald; Ju, Jingyue; Lin, Qiao

    2014-01-01

    Genotyping of single nucleotide polymorphisms (SNPs) allows diagnosis of human genetic disorders associated with single base mutations. Conventional SNP genotyping methods are capable of providing either accurate or high-throughput detection, but are still labor-, time-, and resource-intensive. Microfluidics has been applied to SNP detection to provide fast, low-cost, and automated alternatives, although these applications are still limited by either accuracy or throughput issues. To address this challenge, we present a MEMS-based SNP genotyping approach that uses solid-phase-based reactions in a single microchamber on a temperature control chip. Polymerase chain reaction (PCR), allele specific single base extension (SBE), and desalting on microbeads are performed in the microchamber, which is coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze the SBE product. Experimental results from genotyping of the SNP on exon 1 of the HBB gene, which causes sickle cell anemia, demonstrate the potential of the device for rapid, accurate, multiplexed and high-throughput detection of SNPs. PMID:24729659

  9. [Application of single nucleotide polymorphism in crop genetics and improvement].

    PubMed

    Du, Chun-Fang; Liu, Hui-Min; Li, Run-Zhi; Li, Peng-Bo; Ren, Zhi-Qiang

    2003-11-01

    Single nucleotide polymorphism(SNP) is the most common type of sequence difference between alleles, which can be used as a kind of high-throughput genetic marker. Several different routes have been developed to discover and identify SNP. These include the direct sequencing of PCR amplicons, electronic SNP(eSNP) and so on. SNP assays have been made in many crop species such as maize and soybean. The elite germplasm of some crops have been narrowed in genetic diversity, increasing the amount of linkage disequilibrium (LD) present and facilitating the association of SNP haplotypes at candidate gene loci with phenotypes. SNP analysis has been broadly used in the field of plant gene mapping, integration of genetic and physical maps, DNA marker-assisted breeding and functional genomics. PMID:15639972

  10. Single Nucleotide Polymorphism Mapping Using Genome-Wide Unique Sequences

    PubMed Central

    Chen, Leslie Y.Y.; Lu, Szu-Hsien; Shih, Edward S.C.; Hwang, Ming-Jing

    2002-01-01

    As more and more genomic DNAs are sequenced to characterize human genetic variations, the demand for a very fast and accurate method to genomically position these DNA sequences is high. We have developed a new mapping method that does not require sequence alignment. In this method, we first identified DNA fragments of 15 bp in length that are unique in the human genome and then used them to position single nucleotide polymorphism (SNP) sequences. By use of four desktop personal computers with AMD K7 (1 GHz) processors, our new method mapped more than 1.6 million SNP sequences in 20 hr and achieved a very good agreement with mapping results from alignment-based methods. PMID:12097348

  11. Current research status, databases and application of single nucleotide polymorphism.

    PubMed

    Javed, R; Mukesh

    2010-07-01

    Single Nucleotide Polymorphisms (SNPs) are the most frequent form of DNA variation in the genome. SNPs are genetic markers which are bi-allelic in nature and grow at a very fast rate. Current genomic databases contain information on several million SNPs. More than 6 million SNPs have been identified and the information is publicly available through the efforts of the SNP Consortium and others data bases. The NCBI plays a major role in facillating the identification and cataloging of SNPs through creation and maintenance of the public SNP database (dbSNP) by the biomedical community worldwide and stimulate many areas of biological research including the identification of the genetic components of disease. In this review article, we are compiling the existing SNP databases, research status and their application. PMID:21717869

  12. ENGINES: exploring single nucleotide variation in entire human genomes

    PubMed Central

    2011-01-01

    Background Next generation ultra-sequencing technologies are starting to produce extensive quantities of data from entire human genome or exome sequences, and therefore new software is needed to present and analyse this vast amount of information. The 1000 Genomes project has recently released raw data for 629 complete genomes representing several human populations through their Phase I interim analysis and, although there are certain public tools available that allow exploration of these genomes, to date there is no tool that permits comprehensive population analysis of the variation catalogued by such data. Description We have developed a genetic variant site explorer able to retrieve data for Single Nucleotide Variation (SNVs), population by population, from entire genomes without compromising future scalability and agility. ENGINES (ENtire Genome INterface for Exploring SNVs) uses data from the 1000 Genomes Phase I to demonstrate its capacity to handle large amounts of genetic variation (>7.3 billion genotypes and 28 million SNVs), as well as deriving summary statistics of interest for medical and population genetics applications. The whole dataset is pre-processed and summarized into a data mart accessible through a web interface. The query system allows the combination and comparison of each available population sample, while searching by rs-number list, chromosome region, or genes of interest. Frequency and FST filters are available to further refine queries, while results can be visually compared with other large-scale Single Nucleotide Polymorphism (SNP) repositories such as HapMap or Perlegen. Conclusions ENGINES is capable of accessing large-scale variation data repositories in a fast and comprehensive manner. It allows quick browsing of whole genome variation, while providing statistical information for each variant site such as allele frequency, heterozygosity or FST values for genetic differentiation. Access to the data mart generating scripts and to

  13. Electrophoretic Transport of Single DNA Nucleotides through Nanoslits: A Molecular Dynamics Simulation Study.

    PubMed

    Xia, Kai; Novak, Brian R; Weerakoon-Ratnayake, Kumuditha M; Soper, Steven A; Nikitopoulos, Dimitris E; Moldovan, Dorel

    2015-09-01

    There is potential for flight time based DNA sequencing involving disassembly into individual nucleotides which would pass through a nanochannel with two or more detectors. We performed molecular dynamics simulations of electrophoretic motion of single DNA nucleotides through 3 nm wide hydrophobic slits with both smooth and rough walls. The electric field (E) varied from 0.0 to 0.6 V/nm. The nucleotides adsorb and desorb from walls multiple times during their transit through the slit. The nucleotide-wall interactions differed due to nucleotide hydrophobicities and wall roughness which determined duration and frequency of nucleotide adsorptions and their velocities while adsorbed. Transient association of nucleotides with one, two, or three sodium ions occurred, but the mean association numbers (ANs) were weak functions of nucleotide type. Nucleotide-wall interactions contributed more to separation of nucleotide flight time distributions than ion association and thus indicate that nucleotide-wall interactions play a defining role in successfully discriminating between nucleotides on the basis of their flight times through nanochannels/slits. With smooth walls, smaller nucleotides moved faster, but with rough walls larger nucleotides moved faster due to fewer favorable wall adsorption sites. This indicates that roughness, or surface patterning, might be exploited to achieve better time-of-flight based discrimination between nucleotides. PMID:26237155

  14. Differential Single Nucleotide Polymorphism-Based Analysis of an Outbreak Caused by Salmonella enterica Serovar Manhattan Reveals Epidemiological Details Missed by Standard Pulsed-Field Gel Electrophoresis

    PubMed Central

    Scaltriti, Erika; Sassera, Davide; Comandatore, Francesco; Morganti, Marina; Mandalari, Carmen; Gaiarsa, Stefano; Bandi, Claudio; Zehender, Gianguglielmo; Bolzoni, Luca; Casadei, Gabriele

    2015-01-01

    We retrospectively analyzed a rare Salmonella enterica serovar Manhattan outbreak that occurred in Italy in 2009 to evaluate the potential of new genomic tools based on differential single nucleotide polymorphism (SNP) analysis in comparison with the gold standard genotyping method, pulsed-field gel electrophoresis. A total of 39 isolates were analyzed from patients (n = 15) and food, feed, animal, and environmental sources (n = 24), resulting in five different pulsed-field gel electrophoresis (PFGE) profiles. Isolates epidemiologically related to the outbreak clustered within the same pulsotype, SXB_BS.0003, without any further differentiation. Thirty-three isolates were considered for genomic analysis based on different sets of SNPs, core, synonymous, nonsynonymous, as well as SNPs in different codon positions, by Bayesian and maximum likelihood algorithms. Trees generated from core and nonsynonymous SNPs, as well as SNPs at the second and first plus second codon positions detailed four distinct groups of isolates within the outbreak pulsotype, discriminating outbreak-related isolates of human and food origins. Conversely, the trees derived from synonymous and third-codon-position SNPs clustered food and human isolates together, indicating that all outbreak-related isolates constituted a single clone, which was in line with the epidemiological evidence. Further experiments are in place to extend this approach within our regional enteropathogen surveillance system. PMID:25653407

  15. A Microfluidic Device for Multiplex Single-Nucleotide Polymorphism Genotyping

    PubMed Central

    Zhu, Jing; Qiu, Chunmei; Palla, Mirkó; Nguyen, ThaiHuu; Russo, James J.; Ju, Jingyue; Lin, Qiao

    2015-01-01

    Single-nucleotide polymorphisms (SNPs) are the most abundant type of genetic variations; they provide the genetic fingerprint of individuals and are essential for genetic biomarker discoveries. Accurate detection of SNPs is of great significance for disease prevention, diagnosis and prognosis, and for prediction of drug response and clinical outcomes in patients. Nevertheless, conventional SNP genotyping methods are still limited by insufficient accuracy or labor-, time-, and resource-intensive procedures. Microfluidics has been increasingly utilized to improve efficiency; however, the currently available microfluidic genotyping systems still have shortcomings in accuracy, sensitivity, throughput and multiplexing capability. To address these challenges, we developed a multi-step SNP genotyping microfluidic device, which performs single-base extension of SNP specific primers and solid-phase purification of the extension products on a temperature-controlled chip. The products are ready for immediate detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), providing identification of the alleles at the target loci. The integrated device enables efficient and automated operation, while maintaining the high accuracy and sensitivity provided by MS. The multiplex genotyping capability was validated by performing rapid, accurate and simultaneous detection of 4 loci on a synthetic template. The microfluidic device has the potential to perform automatic, accurate, quantitative and high-throughput assays covering a broad spectrum of applications in biological and clinical research, drug development and forensics. PMID:26594354

  16. Single nucleotide polymorphism markers for genetic mapping in Drosophila melanogaster

    SciTech Connect

    Hoskins, Roger A.; Phan, Alexander C.; Naeemuddin, Mohammed; Mapa, Felipa A.; Ruddy, David A.; Ryan, Jessica J.; Young, Lynn M.; Wells, Trent; Kopczynski, Casey; Ellis, Michael C.

    2001-04-16

    For nearly a century, genetic analysis in Drosophila melanogaster has been a powerful tool for analyzing gene function, yet Drosophila lacks the molecular genetic mapping tools that have recently revolutionized human, mouse and plant genetics. Here, we describe the systematic characterization of a dense set of molecular markers in Drosophila using an STS-based physical map of the genome. We identify 474 biallelic markers in standard laboratory strains of Drosophila that the genome. The majority of these markers are single nucleotide polymorphisms (SNPs) and sequences for these variants are provided in an accessible format. The average density of the new markers is 1 marker per 225 kb on the autosomes and 1 marker per 1 Mb on the X chromosome. We include in this survey a set of P-element strains that provide additional utility for high-resolution mapping. We demonstrate one application of the new markers in a simple set of crosses to map a mutation in the hedgehog gene to an interval of <1 Mb. This new map resource significantly increases the efficiency and resolution of recombination mapping and will be of immediate value to the Drosophila research community.

  17. Single nucleotide polymorphism genotyping using BeadChip microarrays.

    PubMed

    Lambert, Gilliam; Tsinajinnie, Darwin; Duggan, David

    2013-07-01

    The genotyping of single nucleotide polymorphisms (SNPs) has successfully contributed to the study of complex diseases more than any other technology to date. Genome-wide association studies (GWAS) using 10,000s to >1,000,000 SNPs have identified 1000s of statistically significant SNPs pertaining to 17 different human disease and trait categories. Post-GWAS fine-mapping studies using 10,000s to 100,000s SNPs on a microarray have narrowed the region of interest for many of these GWAS findings; in addition, independent signals within the original GWAS region have been identified. Focused content, SNP-based microarrays such as the human exome, for example, have too been used successfully to identify novel disease associations. Success has come to studies where 100s to 10,000s (mostly) to >100,000 samples were genotyped. For the time being, SNP-based microarrays remain cost-effective especially when studying large numbers of samples compared to other "genotyping" technologies including next generation sequencing. In this unit, protocols for manual (LIMS-free), semi-manual, and automated processing of BeadChip microarrays are presented. Lower throughput studies will find value in the manual and semi-manual protocols, while all types of studies--low-, medium-, and high-throughput--will find value in the semi-manual and automated protocols. PMID:23853082

  18. Research on Single Nucleotide Polymorphisms Interaction Detection from Network Perspective

    PubMed Central

    Su, Lingtao; Liu, Guixia; Wang, Han; Tian, Yuan; Zhou, Zhihui; Han, Liang; Yan, Lun

    2015-01-01

    Single Nucleotide Polymorphisms (SNPs) found in Genome-Wide Association Study (GWAS) mainly influence the susceptibility of complex diseases, but they still could not comprehensively explain the relationships between mutations and diseases. Interactions between SNPs are considered so important for deeply understanding of those relationships that several strategies have been proposed to explore such interactions. However, part of those methods perform poorly when marginal effects of disease loci are weak or absent, others may lack of considering high-order SNPs interactions, few methods have achieved the requirements in both performance and accuracy. Considering the above reasons, not only low-order, but also high-order SNP interactions as well as main-effect SNPs, should be taken into account in detection methods under an acceptable computational complexity. In this paper, a new pairwise (or low-order) interaction detection method IG (Interaction Gain) is introduced, in which disease models are not required and parallel computing is utilized. Furthermore, high-order SNP interactions were proposed to be detected by finding closely connected function modules of the network constructed from IG detection results. Tested by a wide range of simulated datasets and four WTCCC real datasets, the proposed methods accurately detected both low-order and high-order SNP interactions as well as disease-associated main-effect SNPS and it surpasses all competitors in performances. The research will advance complex diseases research by providing more reliable SNP interactions. PMID:25763929

  19. Research on single nucleotide polymorphisms interaction detection from network perspective.

    PubMed

    Su, Lingtao; Liu, Guixia; Wang, Han; Tian, Yuan; Zhou, Zhihui; Han, Liang; Yan, Lun

    2015-01-01

    Single Nucleotide Polymorphisms (SNPs) found in Genome-Wide Association Study (GWAS) mainly influence the susceptibility of complex diseases, but they still could not comprehensively explain the relationships between mutations and diseases. Interactions between SNPs are considered so important for deeply understanding of those relationships that several strategies have been proposed to explore such interactions. However, part of those methods perform poorly when marginal effects of disease loci are weak or absent, others may lack of considering high-order SNPs interactions, few methods have achieved the requirements in both performance and accuracy. Considering the above reasons, not only low-order, but also high-order SNP interactions as well as main-effect SNPs, should be taken into account in detection methods under an acceptable computational complexity. In this paper, a new pairwise (or low-order) interaction detection method IG (Interaction Gain) is introduced, in which disease models are not required and parallel computing is utilized. Furthermore, high-order SNP interactions were proposed to be detected by finding closely connected function modules of the network constructed from IG detection results. Tested by a wide range of simulated datasets and four WTCCC real datasets, the proposed methods accurately detected both low-order and high-order SNP interactions as well as disease-associated main-effect SNPS and it surpasses all competitors in performances. The research will advance complex diseases research by providing more reliable SNP interactions. PMID:25763929

  20. Single nucleotide polymorphisms in clinics: Fantasy or reality for cancer?

    PubMed

    Srinivasan, Srilakshmi; Clements, Judith A; Batra, Jyotsna

    2016-01-01

    Single nucleotide polymorphisms (SNPs) have been classically used for dissecting various human complex disorders using candidate gene studies. During the last decade, large scale SNP analysis, i.e. genome-wide association studies (GWAS) have provided an agnostic approach to identify possible genetic loci associated with heterogeneous disease such as cancer susceptibility, prognosis of survival or drug response. Further, the advent of new technologies, including microarray-based genotyping as well as high throughput next generation sequencing has opened new avenues for SNPs to be used in clinical practice. It is speculated that the utility of SNPs to understand the mechanisms, biology of variable drug response and ultimately treatment individualization based on the individual's genome composition will be indispensable in the near future. In the current review, we discuss the advantages and disadvantages of the clinical utility of genetic variants in disease risk-prediction, prognosis, clinical outcome and pharmacogenomics. The lessons and challenges for the utility of SNP-based biomarkers are also discussed, including the need for additional functional validation studies. PMID:26398894

  1. Single nucleotide polymorphisms of Kit gene in Chinese indigenous horses.

    PubMed

    Han, Haoyuan; Mao, Chunchun; Chen, Ningbo; Lan, Xianyong; Chen, Hong; Lei, Chuzhao; Dang, Ruihua

    2016-02-01

    Kit gene is a genetic determinant of horse white coat color which has been a highly valued trait in horses for at least 2,000 years. Single nucleotide polymorphisms (SNPs) in Kit are of importance due to their strong associations with melanoblast survival during embryonic development. In this study, a mutation analysis of all 21 Kit exons in 14 Chinese domestic horse breeds revealed six SNPs (g.91214T>G, g.143245T>G, g.164297C>T, g.170189C>T, g.171356C>G, and g.171471G>A), which located in 5'-UTR region, intron 6, exon 15, exon 20, intron 20, and exon 21 of the equine Kit gene, respectively. Subsequently, these six SNPs loci were genotyped in 632 Chinese horses by PCR-RFLP or direct sequencing. The six SNPs together defined 18 haplotypes, demonstrating abundant haplotype diversities in Chinese horses. All the mutant alleles and haplotypes were shared among different breeds. But fewer mutations were detected in horses from China than that from abroad, indicating that Chinese horses belong to a more ancient genetic pool. This study will provide fundamental genetic information for evaluating the genetic diversity of Kit gene in Chinese indigenous horse breeds. PMID:27348891

  2. Single nucleotide polymorphism-based dispersal estimates using noninvasive sampling.

    PubMed

    Norman, Anita J; Spong, Göran

    2015-08-01

    Quantifying dispersal within wild populations is an important but challenging task. Here we present a method to estimate contemporary, individual-based dispersal distance from noninvasively collected samples using a specialized panel of 96 SNPs (single nucleotide polymorphisms). One main issue in conducting dispersal studies is the requirement for a high sampling resolution at a geographic scale appropriate for capturing the majority of dispersal events. In this study, fecal samples of brown bear (Ursus arctos) were collected by volunteer citizens, resulting in a high sampling resolution spanning over 45,000 km(2) in Gävleborg and Dalarna counties in Sweden. SNP genotypes were obtained for unique individuals sampled (n = 433) and subsequently used to reconstruct pedigrees. A Mantel test for isolation by distance suggests that the sampling scale was appropriate for females but not for males, which are known to disperse long distances. Euclidean distance was estimated between mother and offspring pairs identified through the reconstructed pedigrees. The mean dispersal distance was 12.9 km (SE 3.2) and 33.8 km (SE 6.8) for females and males, respectively. These results were significantly different (Wilcoxon's rank-sum test: P-value = 0.02) and are in agreement with the previously identified pattern of male-biased dispersal. Our results illustrate the potential of using a combination of noninvasively collected samples at high resolution and specialized SNPs for pedigree-based dispersal models. PMID:26357536

  3. Single nucleotide polymorphisms predict symptom severity of autism spectrum disorder

    PubMed Central

    Jiao, Yun; Chen, Rong; Ke, Xiaoyan; Cheng, Lu; Chu, Kangkang; Lu, Zuhong; Herskovits, Edward H

    2011-01-01

    Autism is widely believed to be a heterogeneous disorder; diagnosis is currently based solely on clinical criteria, although genetic, as well as environmental, influences are thought to be prominent factors in the etiology of most forms of autism. Our goal is to determine whether a predictive model based on single-nucleotide polymorphisms (SNPs) can predict symptom severity of autism spectrum disorder (ASD). We divided 118 ASD children into a mild/moderate autism group (n = 65) and a severe autism group (n = 53), based on the Childhood Autism Rating Scale (CARS). For each child, we obtained 29 SNPs of 9 ASD-related genes. To generate predictive models, we employed three machine-learning techniques: decision stumps (DSs), alternating decision trees (ADTrees), and FlexTrees. DS and FlexTree generated modestly better classifiers, with accuracy = 67%, sensitivity = 0.88 and specificity = 0.42. The SNP rs878960 in GABRB3 was selected by all models, and was related associated with CARS assessment. Our results suggest that SNPs have the potential to offer accurate classification of ASD symptom severity. PMID:21786105

  4. Single nucleotide polymorphism-based dispersal estimates using noninvasive sampling

    PubMed Central

    Norman, Anita J; Spong, Göran

    2015-01-01

    Quantifying dispersal within wild populations is an important but challenging task. Here we present a method to estimate contemporary, individual-based dispersal distance from noninvasively collected samples using a specialized panel of 96 SNPs (single nucleotide polymorphisms). One main issue in conducting dispersal studies is the requirement for a high sampling resolution at a geographic scale appropriate for capturing the majority of dispersal events. In this study, fecal samples of brown bear (Ursus arctos) were collected by volunteer citizens, resulting in a high sampling resolution spanning over 45,000 km2 in Gävleborg and Dalarna counties in Sweden. SNP genotypes were obtained for unique individuals sampled (n = 433) and subsequently used to reconstruct pedigrees. A Mantel test for isolation by distance suggests that the sampling scale was appropriate for females but not for males, which are known to disperse long distances. Euclidean distance was estimated between mother and offspring pairs identified through the reconstructed pedigrees. The mean dispersal distance was 12.9 km (SE 3.2) and 33.8 km (SE 6.8) for females and males, respectively. These results were significantly different (Wilcoxon’s rank-sum test: P-value = 0.02) and are in agreement with the previously identified pattern of male-biased dispersal. Our results illustrate the potential of using a combination of noninvasively collected samples at high resolution and specialized SNPs for pedigree-based dispersal models. PMID:26357536

  5. Single Nucleotide Polymorphism in Patients with Moyamoya Disease

    PubMed Central

    2015-01-01

    Moyamoya disease (MMD) is a chronic, progressive, cerebrovascular occlusive disorder that displays various clinical features and results in cerebral infarct or hemorrhagic stroke. Specific genes associated with the disease have not yet been identified, making identification of at-risk patients difficult before clinical manifestation. Familial MMD is not uncommon, with as many as 15% of MMD patients having a family history of the disease, suggesting a genetic etiology. Studies of single nucleotide polymorphisms (SNPs) in MMD have mostly focused on mechanical stress on vessels, endothelium, and the relationship to atherosclerosis. In this review, we discuss SNPs studies targeting the genetic etiology of MMD. Genetic analyses in familial MMD and genome-wide association studies represent promising strategies for elucidating the pathophysiology of this condition. This review also discusses future research directions, not only to offer new insights into the origin of MMD, but also to enhance our understanding of the genetic aspects of MMD. There have been several SNP studies of MMD. Current SNP studies suggest a genetic contribution to MMD, but further reliable and replicable data are needed. A large cohort or family-based design would be important. Modern SNP studies of MMD depend on novel genetic, experimental, and database methods that will hopefully hasten the arrival of a consensus conclusion. PMID:26180609

  6. Single Nucleotide Polymorphism Clustering in Systemic Autoimmune Diseases

    PubMed Central

    Charlon, Thomas; Bossini-Castillo, Lara; Carmona, F. David; Di Cara, Alessandro; Wojcik, Jérôme; Voloshynovskiy, Sviatoslav

    2016-01-01

    Systemic Autoimmune Diseases, a group of chronic inflammatory conditions, have variable symptoms and difficult diagnosis. In order to reclassify them based on genetic markers rather than clinical criteria, we performed clustering of Single Nucleotide Polymorphisms. However naive approaches tend to group patients primarily by their geographic origin. To reduce this “ancestry signal”, we developed SNPClust, a method to select large sources of ancestry-independent genetic variations from all variations detected by Principal Component Analysis. Applied to a Systemic Lupus Erythematosus case control dataset, SNPClust successfully reduced the ancestry signal. Results were compared with association studies between the cases and controls without or with reference population stratification correction methods. SNPClust amplified the disease discriminating signal and the ratio of significant associations outside the HLA locus was greater compared to population stratification correction methods. SNPClust will enable the use of ancestry-independent genetic information in the reclassification of Systemic Autoimmune Diseases. SNPClust is available as an R package and demonstrated on the public Human Genome Diversity Project dataset at https://github.com/ThomasChln/snpclust. PMID:27490238

  7. Single Nucleotide Polymorphism Clustering in Systemic Autoimmune Diseases.

    PubMed

    Charlon, Thomas; Martínez-Bueno, Manuel; Bossini-Castillo, Lara; Carmona, F David; Di Cara, Alessandro; Wojcik, Jérôme; Voloshynovskiy, Sviatoslav; Martín, Javier; Alarcón-Riquelme, Marta E

    2016-01-01

    Systemic Autoimmune Diseases, a group of chronic inflammatory conditions, have variable symptoms and difficult diagnosis. In order to reclassify them based on genetic markers rather than clinical criteria, we performed clustering of Single Nucleotide Polymorphisms. However naive approaches tend to group patients primarily by their geographic origin. To reduce this "ancestry signal", we developed SNPClust, a method to select large sources of ancestry-independent genetic variations from all variations detected by Principal Component Analysis. Applied to a Systemic Lupus Erythematosus case control dataset, SNPClust successfully reduced the ancestry signal. Results were compared with association studies between the cases and controls without or with reference population stratification correction methods. SNPClust amplified the disease discriminating signal and the ratio of significant associations outside the HLA locus was greater compared to population stratification correction methods. SNPClust will enable the use of ancestry-independent genetic information in the reclassification of Systemic Autoimmune Diseases. SNPClust is available as an R package and demonstrated on the public Human Genome Diversity Project dataset at https://github.com/ThomasChln/snpclust. PMID:27490238

  8. A survey of genome-wide single nucleotide polymorphisms through genome resequencing in the Périgord black truffle (Tuber melanosporum Vittad.).

    PubMed

    Payen, Thibaut; Murat, Claude; Gigant, Anaïs; Morin, Emmanuelle; De Mita, Stéphane; Martin, Francis

    2015-09-01

    The Périgord black truffle (Tuber melanosporum Vittad.), considered a gastronomic delicacy worldwide, is an ectomycorrhizal filamentous fungus that is ecologically important in Mediterranean French, Italian and Spanish woodlands. In this study, we developed a novel resource of single nucleotide polymorphisms (SNPs) for T. melanosporum using Illumina high-throughput resequencing. The genome from six T. melanosporum geographical accessions was sequenced to a depth of approximately 20×. These geographical accessions were selected from different populations within the northern and southern regions of the geographical species distribution. Approximately 80% of the reads for each of the six resequenced geographical accessions mapped against the reference T. melanosporum genome assembly, estimating the core genome size of this organism to be approximately 110 Mbp. A total of 442 326 SNPs corresponding to 3540 SNPs/Mbps were identified as being included in all seven genomes. The SNPs occurred more frequently in repeated sequences (85%), although 4501 SNPs were also identified in the coding regions of 2587 genes. Using the ratio of nonsynonymous mutations per nonsynonymous site (pN) to synonymous mutations per synonymous site (pS) and Tajima's D index scanning the whole genome, we were able to identify genomic regions and genes potentially subjected to positive or purifying selection. The SNPs identified represent a valuable resource for future population genetics and genomics studies. PMID:25703414

  9. Systematic Dissection of Coding Exons at Single Nucleotide Resolution Supports an Additional Role in Cell-Specific Transcriptional Regulation

    PubMed Central

    Kim, Mee J.; Findlay, Gregory M.; Martin, Beth; Zhao, Jingjing; Bell, Robert J. A.; Smith, Robin P.; Ku, Angel A.; Shendure, Jay; Ahituv, Nadav

    2014-01-01

    In addition to their protein coding function, exons can also serve as transcriptional enhancers. Mutations in these exonic-enhancers (eExons) could alter both protein function and transcription. However, the functional consequence of eExon mutations is not well known. Here, using massively parallel reporter assays, we dissect the enhancer activity of three liver eExons (SORL1 exon 17, TRAF3IP2 exon 2, PPARG exon 6) at single nucleotide resolution in the mouse liver. We find that both synonymous and non-synonymous mutations have similar effects on enhancer activity and many of the deleterious mutation clusters overlap known liver-associated transcription factor binding sites. Carrying a similar massively parallel reporter assay in HeLa cells with these three eExons found differences in their mutation profiles compared to the liver, suggesting that enhancers could have distinct operating profiles in different tissues. Our results demonstrate that eExon mutations could lead to multiple phenotypes by disrupting both the protein sequence and enhancer activity and that enhancers can have distinct mutation profiles in different cell types. PMID:25340400

  10. Are Immune Modulating Single Nucleotide Polymorphisms Associated with Necrotizing Enterocolitis?

    PubMed Central

    Franklin, Ashanti L.; Said, Mariam; Cappiello, Clint D.; Gordish-Dressman, Heather; Tatari-Calderone, Zohreh; Vukmanovic, Stanislav; Rais-Bahrami, Khodayar; Luban, Naomi L. C.; Devaney, Joseph M.; Sandler, Anthony D.

    2015-01-01

    Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency. The purpose of this study is to determine if functional single nucleotide polymorphisms (SNPs) in immune-modulating genes pre-dispose infants to NEC. After Institutional Review Board approval and parental consent, buccal swabs were collected for DNA extraction. TaqMan allelic discrimination assays and BglII endonuclease digestion were used to genotype specific inflammatory cytokines and TRIM21. Statistical analysis was completed using logistic regression. 184 neonates were analyzed in the study. Caucasian neonates with IL-6 (rs1800795) were over 6 times more likely to have NEC (p = 0.013; OR = 6.61, 95% CI 1.48–29.39), and over 7 times more likely to have Stage III disease (p = 0.011; OR = 7.13, (95% CI 1.56–32.52). Neonates with TGFβ-1 (rs2241712) had a decreased incidence of NEC-related perforation (p = 0.044; OR = 0.28, 95% CI: 0.08–0.97) and an increased incidence of mortality (p = 0.049; OR = 2.99, 95% CI: 1.01 – 8.86). TRIM21 (rs660) was associated with NEC-related intestinal perforation (p = 0.038; OR = 4.65, 95% CI 1.09–19.78). In premature Caucasian neonates, the functional SNP IL-6 (rs1800795) is associated with both the development and increased severity of NEC. TRIM21 (rs660) and TGFβ-1 (rs2241712) were associated with NEC- related perforation in all neonates in the cohort. These findings suggest a possible genetic role in the development of NEC. PMID:26670709

  11. A three-single-nucleotide polymorphism haplotype in intron 1 of OCA2 explains most human eye-color variation.

    PubMed

    Duffy, David L; Montgomery, Grant W; Chen, Wei; Zhao, Zhen Zhen; Le, Lien; James, Michael R; Hayward, Nicholas K; Martin, Nicholas G; Sturm, Richard A

    2007-02-01

    We have previously shown that a quantitative-trait locus linked to the OCA2 region of 15q accounts for 74% of variation in human eye color. We conducted additional genotyping to clarify the role of the OCA2 locus in the inheritance of eye color and other pigmentary traits associated with skin-cancer risk in white populations. Fifty-eight synonymous and nonsynonymous exonic single-nucleotide polymorphisms (SNPs) and tagging SNPs were typed in a collection of 3,839 adolescent twins, their siblings, and their parents. The highest association for blue/nonblue eye color was found with three OCA2 SNPs: rs7495174 T/C, rs6497268 G/T, and rs11855019 T/C (P values of 1.02x10(-61), 1.57x10(-96), and 4.45x10(-54), respectively) in intron 1. These three SNPs are in one major haplotype block, with TGT representing 78.4% of alleles. The TGT/TGT diplotype found in 62.2% of samples was the major genotype seen to modify eye color, with a frequency of 0.905 in blue or green compared with only 0.095 in brown eye color. This genotype was also at highest frequency in subjects with light brown hair and was more frequent in fair and medium skin types, consistent with the TGT haplotype acting as a recessive modifier of lighter pigmentary phenotypes. Homozygotes for rs11855019 C/C were predominantly without freckles and had lower mole counts. The minor population impact of the nonsynonymous coding-region polymorphisms Arg305Trp and Arg419Gln associated with nonblue eyes and the tight linkage of the major TGT haplotype within the intron 1 of OCA2 with blue eye color and lighter hair and skin tones suggest that differences within the 5' proximal regulatory control region of the OCA2 gene alter expression or messenger RNA-transcript levels and may be responsible for these associations. PMID:17236130

  12. The application and performance of single nucleotide polymorphism markers for population genetic analyses of Lepidoptera

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single nucleotide polymorphisms (SNPs) are nucleotide substitution mutations that tend to be at high densities within eukaryotic genomes. The development of assays that detect allelic variation at SNP loci is attractive for genome mapping, population genetics, and phylogeographic applications. A p...

  13. Identification and validation of single nucleotide polymorphisms in growth- and maturation-related candidate genes in sole (Solea solea L.).

    PubMed

    Diopere, Eveline; Hellemans, Bart; Volckaert, Filip A M; Maes, Gregory E

    2013-03-01

    Genomic methodologies applied in evolutionary and fisheries research have been of great benefit to understand the marine ecosystem and the management of natural resources. Although single nucleotide polymorphisms (SNPs) are attractive for the study of local adaptation, spatial stock management and traceability, and investigating the effects of fisheries-induced selection, they have rarely been exploited in non-model organisms. This is partly due to difficulties in finding and validating SNPs in species with limited or no genomic resources. Complementary to random genome-scan approaches, a targeted candidate gene approach has the potential to unveil pre-selected functional diversity and provides more in depth information on the action of selection at specific genes. For example genes can be under selective pressure due to climate change and sustained periods of heavy fishing pressure. In this study, we applied a candidate gene approach in sole (Solea solea L.), an important member of the demersal ecosystem. As consumption flatfish it is heavy exploited and has experienced associated life-history changes over the last 60years. To discover novel genetic polymorphisms in or around genes linked to important life history traits in sole, we screened a total of 76 candidate genes related to growth and maturation using a targeted resequencing approach. We identified in total 86 putative SNPs in 22 genes and validated 29 SNPs using a multiplex single-base extension genotyping assay. We found 22 informative SNPs, of which two represent non-synonymous mutations, potentially of functional relevance. These novel markers should be rapidly and broadly applicable in analyses of natural sole populations, as a measure of the evolutionary signature of overfishing and for initiatives on marker assisted selection. PMID:23067785

  14. Single nucleotide polymorphism discovery in rainbow trout using reduced representation libraries

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single Nucleotide Polymorphisms (SNPs) are highly abundant, widespread and evenly distributed markers, which can be easily genotyped using high-throughput assays. These characteristics explain their increasing popularity in genome analyses such as quantitative trait loci mapping, linkage disequilibr...

  15. Detection of single-nucleotide polymorphisms using gold nanoparticles and single-strand-specific nucleases.

    PubMed

    Chen, Yen-Ting; Hsu, Chiao-Ling; Hou, Shao-Yi

    2008-04-15

    The current study reports an assay approach that can detect single-nucleotide polymorphisms (SNPs) and identify the position of the point mutation through a single-strand-specific nuclease reaction and a gold nanoparticle assembly. The assay can be implemented via three steps: a single-strand-specific nuclease reaction that allows the enzyme to truncate the mutant DNA; a purification step that uses capture probe-gold nanoparticles and centrifugation; and a hybridization reaction that induces detector probe-gold nanoparticles, capture probe-gold nanoparticles, and the target DNA to form large DNA-linked three-dimensional aggregates of gold nanoparticles. At high temperature (63 degrees C in the current case), the purple color of the perfect match solution would not change to red, whereas a mismatched solution becomes red as the assembled gold nanoparticles separate. Using melting analysis, the position of the point mutation could be identified. This assay provides a convenient colorimetric detection that enables point mutation identification without the need for expensive mass spectrometry. To our knowledge, this is the first report concerning SNP detection based on a single-strand-specific nuclease reaction and a gold nanoparticle assembly. PMID:18211817

  16. A novel MALDI-TOF based methodology for genotyping single nucleotide polymorphisms.

    PubMed

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

    2003-12-15

    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

  17. Two single nucleotide polymorphisms in the human nescient helix-loop-helix 2 (NHLH2) gene reduce mRNA stability and DNA binding.

    PubMed

    Al Rayyan, Numan; Wankhade, Umesh D; Bush, Korie; Good, Deborah J

    2013-01-01

    Nescient helix-loop-helix-2 (NHLH2) is a basic helix-loop-helix transcription factor, which has been implicated, using mouse knockouts, in adult body weight regulation and fertility. A scan of the known single nucleotide polymorphisms (SNPs) in the NHLH2 gene revealed one in the 3' untranslated region (3'UTR), which lies within an AUUUA RNA stability motif. A second SNP is nonsynonymous within the coding region of NHLH2, and was found in a genome-wide association study for obesity. Both of these SNPs were examined for their effect on NLHL2 by creating mouse mimics and examining mRNA stability, and protein function in mouse hypothalamic cell lines. The 3'UTR SNP causes increased instability and, when the SNP-containing Nhlh2 3'UTR is attached to luciferase mRNA, reduced protein levels in cells. The nonsynonymous SNP at position 83 in the protein changes an alanine residue, conserved in NHLH2 orthologs through the Drosophila sp. to a proline residue. This change affects migration of the protein on an SDS-PAGE gel, and appears to alter secondary structure of the protein, as predicted using in silico methods. These results provide functional information on two rare human SNPs in the NHLH2 gene. One of these has been linked to human obese phenotypes, while the other is present in a relatively high proportion of individuals. Given their effects on NHLH2 protein levels, both SNPs deserve further analysis in whether they are causative and/or additive for human body weight and fertility phenotypes. PMID:23026212

  18. Bulk segregant analysis using single nucleotide polymorphism microarrays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bulk segregant analysis using microarrays, and extreme array mapping have recently been used to rapidly identify genomic regions associated with phenotypes in multiple species. These experiments, however require the identification of single feature polymorphisms between the cross parents for each ne...

  19. Whole genome sequencing of a single Bos taurus animal for single nucleotide polymorphism discovery

    PubMed Central

    Eck, Sebastian H; Benet-Pagès, Anna; Flisikowski, Krzysztof; Meitinger, Thomas; Fries, Ruedi; Strom, Tim M

    2009-01-01

    Background The majority of the 2 million bovine single nucleotide polymorphisms (SNPs) currently available in dbSNP have been identified in a single breed, Hereford cattle, during the bovine genome project. In an attempt to evaluate the variance of a second breed, we have produced a whole genome sequence at low coverage of a single Fleckvieh bull. Results We generated 24 gigabases of sequence, mainly using 36-bp paired-end reads, resulting in an average 7.4-fold sequence depth. This coverage was sufficient to identify 2.44 million SNPs, 82% of which were previously unknown, and 115,000 small indels. A comparison with the genotypes of the same animal, generated on a 50 k oligonucleotide chip, revealed a detection rate of 74% and 30% for homozygous and heterozygous SNPs, respectively. The false positive rate, as determined by comparison with genotypes determined for 196 randomly selected SNPs, was approximately 1.1%. We further determined the allele frequencies of the 196 SNPs in 48 Fleckvieh and 48 Braunvieh bulls. 95% of the SNPs were polymorphic with an average minor allele frequency of 24.5% and with 83% of the SNPs having a minor allele frequency larger than 5%. Conclusions This work provides the first single cattle genome by next-generation sequencing. The chosen approach - low to medium coverage re-sequencing - added more than 2 million novel SNPs to the currently publicly available SNP resource, providing a valuable resource for the construction of high density oligonucleotide arrays in the context of genome-wide association studies. PMID:19660108

  20. Lineage and genogroup-defining single nucleotide polymorphisms of Escherichia coli 0157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O157:H7 is a zoonotic human pathogen for which cattle are an important reservoir host. Using both previously published and new sequencing data, a 48-locus single nucleotide polymorphism (SNP) based typing panel was developed that redundantly identified eleven genogroups that span ...

  1. Short communication: Relationship of call rate and accuracy of single nucleotide polymorphism genotypes in dairy cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Call rate has been used as a measure of quality on both a single nucleotide polymorphism (SNP) and animal basis since SNP genotypes were first used in genomic evaluation of dairy cattle. The genotyping laboratories perform initial quality control screening and genotypes that fail are usually exclude...

  2. Using 90,113 single nucleotide polymorphisms in genomic evaluation of dairy cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Accuracy of genomic evaluation is expected to increase when more markers are used because of better tracking of causative genetic variants. However, Illumina BovineHD genotypes based on 777,962 single nucleotide polymorphisms (SNP) have not been used for US genomic evaluation because the small relia...

  3. Subtyping of Salmonella enterica subspecies I using single nucleotide polymorphisms in adenylate cyclase (cyaA)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single nucleotide polymorphisms (SNPs) were characterized within adenylate cyclas...

  4. ASSOCIATION OF RESISTANCE TO AVIAN COCCIDIOSIS WITH SINGLE NUCLEOTIDE POLYMORPHISMS IN THE ZYXIN GENE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our previous genetic studies demonstrated that resistance to avian coccidiosis was linked with microsatellite markers LEI0071 and LEI0101 on chromosome 1. In this study, the associations between parameters of resistance to coccidiosis and single nucleotide polymorphisms (SNPs) in 3 candidate genes ...

  5. The effects of single nucleotide polymorphisms (SNPs) of calpastatin (CAST) gene on meat tenderness of yak.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The association of single nucleotide polymorphisms (SNPs) of calpastatin (CAST) gene with shear force of 2.54 cm steaks from M. longissimus dorsi from Gannan yaks (Bos grunniens, n=181) was studied. Yaks were harvested at 2, 3, and 4 yr of age (n=51, 59, and 71, respectively), and samples of each ya...

  6. Development of a web services based system for dissemination of single nucleotide polymorphism data

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single nucleotide polymorphisms (SNPs) can be used to generate DNA-based fingerprints for individual identification. The efficiency of DNA fingerprinting is greatest when the frequency of both SNP alleles is near 0.50. A number of SNPs have been identified in cattle populations with minor allele f...

  7. Relationships among calpastatin single nucleotide polymorphisms, calpastatin expression and tenderness in pork longissimus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome scans in the pig have identified a region on chromosome 2 (SSC2) associated with tenderness. Calpastatin is a likely positional candidate gene in this region because of its inhibitory role in the calpain system that is involved in postmortem tenderization. Novel single nucleotide polymorphism...

  8. Performance of single nucleotide polymorphisms versus haplotypes for genome-wide association analysis in barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome-wide association studies (GWAS) may benefit from using haplotype information for making marker-phenotype associations. Several rationales for grouping single nucleotide polymorphisms (SNPs) into haplotype blocks exist, but any advantage may depend on the genetic architecture of traits, patter...

  9. Single nucleotide polymorphism discovery in rainbow trout by deep sequencing of a reduced representation library

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: To enhance capabilities for genomic analyses in rainbow trout, such as genomic selection, a large suite of polymorphic markers that are amenable to high-throughput genotyping protocols must be identified. Expressed Sequence Tags (ESTs) have been used for single nucleotide polymorphism (...

  10. Increasing the number of single nucleotide polymorphisms used in genomic evaluation of dairy cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    GeneSeek designed a new version of the GeneSeek Genomic Profiler HD BeadChip for Dairy Cattle, which had >77,000 single nucleotide polymorphisms (SNPs). A set of >140,000 SNPs was selected that included all SNPs on the existing GeneSeek chip, all SNPs used in U.S. national genomic evaluations, SNPs ...

  11. Association of a single nucleotide polymorphism of calpain 1 gene with meat tenderness of the yak

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The association of a single nucleotide polymorphism (SNP) of calpain 1 (CAPN1) gene with shear force of 2.54 cm steaks from M. longissimus dorsi from Gannan yaks (Bos grunniens, n = 181) was studied. The experimental design was a repeated measures with the main unit in a completely randomized design...

  12. Development of Single Nucleotide Polymorphism Markers via Sequence-based Genotyping in Cotton (Gossypium spp)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High-throughput single nucleotide polymorphism (SNP) genotyping has become the dominant approach to genomic analysis and genetic manipulation in many crop plants. In cotton (Gossypium spp), however, only a very limited number of loci and a dearth of information have been generated from SNP genotypi...

  13. Association of Single Nucleotide Polymorphisms in the CAST Gene Associated with Longissimus Tenderness in Beef Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective was to assess the association of single nucleotide polymorphisms (SNP) developed on the CAST gene, with longissimus tenderness. Forty one SNP were identified in the CAST gene and assays were developed. Markers were scattered throughout the gene. These markers, in conjunction with a com...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  15. Association of single nucleotide polymorphisms in candidate genes residing under quantitative trait loci in beef cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective was to assess the association of single nucleotide polymorphisms (SNP) developed on candidate genes residing under previously identified quantitative trait loci for marbling score and meat tenderness. Two hundred five SNP were identified on twenty candidate genes. Genes selected under ...

  16. Development of Single Nucleotide Polymorphism (SNP) Markers for Use in Commercial Maize (Zea Mays L.) Germplasm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The development of single nucleotide polymorphism (SNP) markers in maize offer the opportunity to utilize DNA markers in many new areas of population genetics, gene discovery, plant breeding, and germplasm identification. However, the steps from sequencing and SNP discovery to SNP marker design and ...

  17. Single nucleotide polymorphisms in sheep varying in tolerance to elevated dietary nitrate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Discovery of single nucleotide polymorphisms (SNPs) may lead to development of marker panels predictive of tolerance to high dietary nitrate (NO3-). The aims of this research were to identify SNPs in Arginiosuccinate Lyase (ASL), determine the relationship of ASL SNP genotypes on NO3- tolerance, an...

  18. Single nucleotide polymorphism in wheat chromosome region harboring Fhb1 for Fusarium head blight resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fusarium head blight (FHB) is a destructive disease that reduces wheat grain yield and quality. To date, the quantitative trait locus on 3BS (Fhb1) from Sumai 3 has shown the largest effect on FHB resistance. Single nucleotide polymorphism (SNP) is the most common form of genetic variation and suita...

  19. Characterization of 22 novel single nucleotide polymorphism markers in steelhead and rainbow trout

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thirty-two individuals representing coastal and inland populations of steelhead and rainbow trout (Oncorhynchus mykiss) were sequenced at 15 ESTs and 9 microsatellite loci to identify single nucleotide polymorphisms (SNPs). Sixty-two polymorphisms were discovered during the screen and 13 were devel...

  20. Rapid and label-free single-nucleotide discrimination via an integrative nanoparticle-nanopore approach.

    PubMed

    Ang, Yan Shan; Yung, Lin-Yue Lanry

    2012-10-23

    Single-nucleotide polymorphism (SNP) is an important biomarker for disease diagnosis, treatment monitoring, and development of personalized medicine. Recent works focused primarily on ultrasensitive detection, while the need for rapid and label-free single-nucleotide discrimination techniques, which are crucial criteria for translation into clinical applications, remains relatively unexplored. In this work, we developed a novel SNP detection assay that integrates two complementary nanotechnology systems, namely, a highly selective nanoparticle-DNA detection system and a single-particle sensitive nanopore readout platform, for rapid detection of single-site mutations. Discrete nanoparticle-DNA structures formed in the presence of perfectly matched (PM) or single-mismatched (SM) targets exhibited distinct size differences, which were resolved on a size-tunable nanopore platform to generate corresponding "yes/no" readout signals. Leveraging the in situ reaction monitoring capability of the nanopore platform, we demonstrated that real-time single-nucleotide discrimination of a model G487A mutation, responsible for glucose-6-phosphate dehydrogenase deficiency, can be achieved within 30 min with no false positives. Semiquantification of DNA samples down to picomolar concentration was carried out using a simple parameter of particle count without the need for sample labeling or signal amplification. The unique combination of nanoparticle-based detection and nanopore readout presented in this work brings forth a rapid, specific, yet simple biosensing strategy that can potentially be developed for point-of-care application. PMID:22994459

  1. Insights into the Molecular Mechanisms Underlying Mammalian P2X7 Receptor Functions and Contributions in Diseases, Revealed by Structural Modeling and Single Nucleotide Polymorphisms

    PubMed Central

    Jiang, Lin-Hua; Baldwin, Jocelyn M.; Roger, Sebastien; Baldwin, Stephen A.

    2013-01-01

    The mammalian P2X7 receptors (P2X7Rs), a member of the ionotropic P2X receptor family with distinctive functional properties, play an important part in mediating extracellular ATP signaling in health and disease. A clear delineation of the molecular mechanisms underlying the key receptor properties, such as ATP-binding, ion permeation, and large pore formation of the mammalian P2X7Rs, is still lacking, but such knowledge is crucial for a better understanding of their physiological functions and contributions in diseases and for development of therapeutics. The recent breakthroughs in determining the atomic structures of the zebrafish P2X4.1R in the closed and ATP-bound open states have provided the long-awaited structural information. The human P2RX7 gene is abundant with non-synonymous single nucleotide polymorphisms (NS-SNPs), which generate a repertoire of human P2X7Rs with point mutations. Characterizations of the NS-SNPs identified in patients of various disease conditions and the resulting mutations have informed previously unknown molecular mechanisms determining the mammalian P2X7R functions and diseases. In this review, we will discuss the new insights into such mechanisms provided by structural modeling and recent functional and genetic linkage studies of NS-SNPs. PMID:23675347

  2. Single nucleotide polymorphism mining and nucleotide sequence analysis of Mx1 gene in exonic regions of Japanese quail

    PubMed Central

    Niraj, Diwesh Kumar; Kumar, Pushpendra; Mishra, Chinmoy; Narayan, Raj; Bhattacharya, Tarun Kumar; Shrivastava, Kush; Bhushan, Bharat; Tiwari, Ashok Kumar; Saxena, Vishesh; Sahoo, Nihar Ranjan; Sharma, Deepak

    2015-01-01

    Aim: An attempt has been made to study the Myxovirus resistant (Mx1) gene polymorphism in Japanese quail. Materials and Methods: In the present, investigation four fragments viz. Fragment I of 185 bp (Exon 3 region), Fragment II of 148 bp (Exon 5 region), Fragment III of 161 bp (Exon 7 region), and Fragment IV of 176 bp (Exon 13 region) of Mx1 gene were amplified and screened for polymorphism by polymerase chain reaction-single-strand conformation polymorphism technique in 170 Japanese quail birds. Results: Out of the four fragments, one fragment (Fragment II) was found to be polymorphic. Remaining three fragments (Fragment I, III, and IV) were found to be monomorphic which was confirmed by custom sequencing. Overall nucleotide sequence analysis of Mx1 gene of Japanese quail showed 100% homology with common quail and more than 80% homology with reported sequence of chicken breeds. Conclusion: The Mx1 gene is mostly conserved in Japanese quail. There is an urgent need of comprehensive analysis of other regions of Mx1 gene along with its possible association with the traits of economic importance in Japanese quail. PMID:27047057

  3. OmpF, a nucleotide-sensing nanoprobe, computational evaluation of single channel activities

    NASA Astrophysics Data System (ADS)

    Abdolvahab, R. H.; Mobasheri, H.; Nikouee, A.; Ejtehadi, M. R.

    2016-09-01

    The results of highthroughput practical single channel experiments should be formulated and validated by signal analysis approaches to increase the recognition precision of translocating molecules. For this purpose, the activities of the single nano-pore forming protein, OmpF, in the presence of nucleotides were recorded in real time by the voltage clamp technique and used as a means for nucleotide recognition. The results were analyzed based on the permutation entropy of current Time Series (TS), fractality, autocorrelation, structure function, spectral density, and peak fraction to recognize each nucleotide, based on its signature effect on the conductance, gating frequency and voltage sensitivity of channel at different concentrations and membrane potentials. The amplitude and frequency of ion current fluctuation increased in the presence of Adenine more than Cytosine and Thymine in milli-molar (0.5 mM) concentrations. The variance of the current TS at various applied voltages showed a non-monotonic trend whose initial increasing slope in the presence of Thymine changed to a decreasing one in the second phase and was different from that of Adenine and Cytosine; e.g., by increasing the voltage from 40 to 140 mV in the 0.5 mM concentration of Adenine or Cytosine, the variance decreased by one third while for the case of Thymine it was doubled. Moreover, according to the structure function of TS, the fractality of current TS differed as a function of varying membrane potentials (pd) and nucleotide concentrations. Accordingly, the calculated permutation entropy of the TS, validated the biophysical approach defined for the recognition of different nucleotides at various concentrations, pd's and polarities. Thus, the promising outcomes of the combined experimental and theoretical methodologies presented here can be implemented as a complementary means in pore-based nucleotide recognition approaches.

  4. An Engineered Kinetic Amplification Mechanism for Single Nucleotide Variant Discrimination by DNA Hybridization Probes.

    PubMed

    Chen, Sherry Xi; Seelig, Georg

    2016-04-20

    Even a single-nucleotide difference between the sequences of two otherwise identical biological nucleic acids can have dramatic functional consequences. Here, we use model-guided reaction pathway engineering to quantitatively improve the performance of selective hybridization probes in recognizing single nucleotide variants (SNVs). Specifically, we build a detection system that combines discrimination by competition with DNA strand displacement-based catalytic amplification. We show, both mathematically and experimentally, that the single nucleotide selectivity of such a system in binding to single-stranded DNA and RNA is quadratically better than discrimination due to competitive hybridization alone. As an additional benefit the integrated circuit inherits the property of amplification and provides at least 10-fold better sensitivity than standard hybridization probes. Moreover, we demonstrate how the detection mechanism can be tuned such that the detection reaction is agnostic to the position of the SNV within the target sequence. in contrast, prior strand displacement-based probes designed for kinetic discrimination are highly sensitive to position effects. We apply our system to reliably discriminate between different members of the let-7 microRNA family that differ in only a single base position. Our results demonstrate the power of systematic reaction network design to quantitatively improve biotechnology. PMID:27010123

  5. Agonist-dependence of functional properties for common nonsynonymous variants of human transient receptor potential vanilloid 1.

    PubMed

    Wang, Sen; Joseph, John; Diatchenko, Luda; Ro, Jin Y; Chung, Man-Kyo

    2016-07-01

    Transient receptor potential vanilloid 1 (TRPV1) is a polymodal receptor activated by capsaicin, heat, and acid, which plays critical roles in thermosensation and pain. In addition, TRPV1 also contributes to multiple pathophysiological states in respiratory, cardiovascular, metabolic, and renal systems. These contributions are further supported by evidence that variations in the human TRPV1 (hTRPV1) gene are associated with various physiological and pathological phenotypes. However, it is not well understood how the variations in hTRPV1 affect channel functions. In this study, we examined functional consequences of amino acid variations of hTRPV1 induced by 5 nonsynonymous single-nucleotide polymorphisms (SNPs) that most commonly exist in the human population. Using electrophysiological assays in HEK293 cells, we examined 9 parameters: activation, Ca permeation, and desensitization after activation by capsaicin, acid, and heat. Our results demonstrated that the 5 SNPs differentially affected functional properties of hTRPV1 in an agonist-dependent manner. Based upon the directionality of change of each phenotype and cumulative changes in each SNP, we classified the 5 SNPs into 3 presumptive functional categories: gain of function (hTRPV1 Q85R, P91S, and T469I), loss of function (I585V), and mixed (M315I). These results reveal a spectrum of functional variation among common hTRPV1 polymorphisms in humans and may aid mechanistic interpretation of phenotypes associated with nonsynonymous hTRPV1 SNPs under pathophysiological conditions. PMID:26967694

  6. Association of Nitric Oxide Synthase and Matrix Metalloprotease Single Nucleotide Polymorphisms with Preeclampsia and Its Complications

    PubMed Central

    Leonardo, Daniela P.; Albuquerque, Dulcinéia M.; Lanaro, Carolina; Baptista, Letícia C.; Cecatti, José G.; Surita, Fernanda G.; Parpinelli, Mary A.; Costa, Fernando F.; Franco-Penteado, Carla F.; Fertrin, Kleber Y.; Costa, Maria Laura

    2015-01-01

    Background Preeclampsia is one of the leading causes of maternal and neonatal morbidity and mortality in the world, but its appearance is still unpredictable and its pathophysiology has not been entirely elucidated. Genetic studies have associated single nucleotide polymorphisms in genes encoding nitric oxide synthase and matrix metalloproteases with preeclampsia, but the results are largely inconclusive across different populations. Objectives To investigate the association of single nucleotide polymorphisms (SNPs) in NOS3 (G894T, T-786C, and a variable number of tandem repetitions VNTR in intron 4), MMP2 (C-1306T), and MMP9 (C-1562T) genes with preeclampsia in patients from Southeastern Brazil. Methods This prospective case-control study enrolled 77 women with preeclampsia and 266 control pregnant women. Clinical data were collected to assess risk factors and the presence of severe complications, such as eclampsia and HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome. Results We found a significant association between the single nucleotide polymorphism NOS3 T-786C and preeclampsia, independently from age, height, weight, or the other SNPs studied, and no association was found with the other polymorphisms. Age and history of preeclampsia were also identified as risk factors. The presence of at least one polymorphic allele for NOS3 T-786C was also associated with the occurrence of eclampsia or HELLP syndrome among preeclamptic women. Conclusions Our data support that the NOS3 T-786C SNP is associated with preeclampsia and the severity of its complications. PMID:26317342

  7. New single nucleotide variation in the promoter region of androgen receptor (AR) gene in hypospadic patients

    PubMed Central

    Borhani, Nasim; Ghaffari Novin, Marefat; Manoochehri, Mehdi; Rouzrokh, Mohsen; Kazemi, Bahram; Koochaki, Ameneh; Hosseini, Ahmad; Masteri Farahani, Reza; Omrani, Mir Davood

    2014-01-01

    Background: Hypospadias is one of the most common congenital abnormalities in the male which is characterized by altered development of urethra, foreskin and ventral surface of the penis. Androgen receptor gene plays a critical role in the development of the male genital system by mediating the androgens effects. Objective: In present study, we looked for new variations in androgen receptor promoter and screened its exon 1 for five single nucleotide polymorphisms (SNP) in healthy and hypospadias Iranian men. Materials and Methods: In our study, at first DNA was extracted from patients (n=100) and controls (n=100) blood samples. Desired fragments of promoter and exon 1 were amplified using polymerase chain reaction. The promoter region was sequenced for the new variation and exone 1 screened for five SNPs (rs139767835, rs78686797, rs62636528, rs62636529, rs145326748) using restriction fragment length polymorphism technique. Results: The results showed a new single nucleotide variation (C→T) at -480 of two patients’ promoter region (2%). None of the mentioned SNPs were detected in patients and controls groups (0%). Conclusion: This finding indicates that new single nucleotide polymorphism in androgen receptor promoter may have role in etiology of hypospadias and development of this anomaly. This article extracted from Ph.D. thesis. (Nasim Borhani) PMID:24799883

  8. A two-layer assay for single-nucleotide variants utilizing strand displacement and selective digestion.

    PubMed

    Yu, Yingjie; Wu, Tongbo; Johnson-Buck, Alexander; Li, Lidan; Su, Xin

    2016-08-15

    Point mutations have emerged as prominent biomarkers for disease diagnosis, particularly in the case of cancer. Discovering single-nucleotide variants (SNVs) is also of great importance for the identification of single-nucleotide polymorphisms within the population. The competing requirements of thermodynamic stability and specificity in conventional nucleic acid hybridization probes make it challenging to achieve highly precise detection of point mutants. Here, we present a fluorescence-based assay for low-abundance mutation detection based on toehold-mediated strand displacement and nuclease-mediated strand digestion that enables highly precise detection of point mutations. We demonstrate that this combined assay provides 50-1000-fold discrimination (mean value: 255) between all possible single-nucleotide mutations and their corresponding wild-type sequence for a model DNA target. Using experiments and kinetic modeling, we investigate probe properties that obtain additive benefits from both strand displacement and nucleolytic digestion, thus providing guidance for the design of enzyme-mediated nucleic acid assays in the future. PMID:27100949

  9. Single nucleotide polymorphism analysis reveals heterogeneity within a seedling tree population of a polyembryonic mango cultivar.

    PubMed

    Winterhagen, Patrick; Wünsche, Jens-Norbert

    2016-05-01

    Within a polyembryonic mango seedling tree population, the genetic background of individuals should be identical because vigorous plants for cultivation are expected to develop from nucellar embryos representing maternal clones. Due to the fact that the mango cultivar 'Hôi' is assigned to the polyembryonic ecotype, an intra-cultivar variability of ethylene receptor genes was unexpected. Ethylene receptors in plants are conserved, but the number of receptors or receptor isoforms is variable regarding different plant species. However, it is shown here that the ethylene receptor MiETR1 is present in various isoforms within the mango cultivar 'Hôi'. The investigation of single nucleotide polymorphisms revealed that different MiETR1 isoforms can not be discriminated simply by individual single nucleotide exchanges but by the specific arrangement of single nucleotide polymorphisms at certain positions in the exons of MiETR1. Furthermore, an MiETR1 isoform devoid of introns in the genomic sequence was identified. The investigation demonstrates some limitations of high resolution melting and ScreenClust analysis and points out the necessity of sequencing to identify individual isoforms and to determine the variability within the tree population. PMID:27093244

  10. High-throughput profiling of influenza A virus hemagglutinin gene at single-nucleotide resolution

    PubMed Central

    Wu, Nicholas C.; Young, Arthur P.; Al-Mawsawi, Laith Q.; Olson, C. Anders; Feng, Jun; Qi, Hangfei; Chen, Shu-Hwa; Lu, I.-Hsuan; Lin, Chung-Yen; Chin, Robert G.; Luan, Harding H.; Nguyen, Nguyen; Nelson, Stanley F.; Li, Xinmin; Wu, Ting-Ting; Sun, Ren

    2014-01-01

    Genetic research on influenza virus biology has been informed in large part by nucleotide variants present in seasonal or pandemic samples, or individual mutants generated in the laboratory, leaving a substantial part of the genome uncharacterized. Here, we have developed a single-nucleotide resolution genetic approach to interrogate the fitness effect of point mutations in 98% of the amino acid positions in the influenza A virus hemagglutinin (HA) gene. Our HA fitness map provides a reference to identify indispensable regions to aid in drug and vaccine design as targeting these regions will increase the genetic barrier for the emergence of escape mutations. This study offers a new platform for studying genome dynamics, structure-function relationships, virus-host interactions, and can further rational drug and vaccine design. Our approach can also be applied to any virus that can be genetically manipulated. PMID:24820965

  11. A genetic variation map for chicken with 2.8 million single nucleotide polymorphisms

    SciTech Connect

    Wong, G K; Hillier, L; Brandstrom, M; Croojmans, R; Ovcharenko, I; Gordon, L; Stubbs, L; Lucas, S; Glavina, T; Kaiser, P; Gunnarsson, U; Webber, C; Overton, I

    2005-02-20

    We describe a genetic variation map for the chicken genome containing 2.8 million single nucleotide polymorphisms (SNPs), based on a comparison of the sequences of 3 domestic chickens (broiler, layer, Silkie) to their wild ancestor Red Jungle Fowl (RJF). Subsequent experiments indicate that at least 90% are true SNPs, and at least 70% are common SNPs that segregate in many domestic breeds. Mean nucleotide diversity is about 5 SNP/kb for almost every possible comparison between RJF and domestic lines, between two different domestic lines, and within domestic lines--contrary to the idea that domestic animals are highly inbred relative to their wild ancestors. In fact, most of the SNPs originated prior to domestication, and there is little to no evidence of selective sweeps for adaptive alleles on length scales of greater than 100 kb.

  12. Developing Single Nucleotide Polymorphism (SNP) markers from transcriptome sequences for the identification of longan (Dimocarpus longan) germplasm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Longan (Dimocarpus longan Lour.) is an important tropical fruit tree crop. Accurate varietal identification is essential for germplasm management and breeding. Using longan transcriptome sequences from public databases, we developed single nucleotide polymorphism (SNP) markers; validated 60 SNPs in...

  13. Prediction of Unobserved Single Nucleotide Polymorphism Genotypes of Jersey Cattle Using Reference Panels and Population-Based Imputation Algorithms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The availability of dense single nucleotide polymorphism (SNP) genotypes for dairy cattle has created exciting research opportunities and revolutionized practical breeding programs. Broader application of this technology will lead to situations in which genotypes from different low-, medium-, or hig...

  14. Regulatory single nucleotide polymorphisms at the beginning of intron 2 of the human KRAS gene.

    PubMed

    Antontseva, Elena V; Matveeva, Marina Yu; Bondar, Natalia P; Kashina, Elena V; Leberfarb, Elena Yu; Bryzgalov, Leonid O; Gervas, Polina A; Ponomareva, Anastasia A; Cherdyntseva, Nadezhda V; Orlov, Yury L; Merkulova, Tatiana I

    2015-12-01

    There are two regulatory single nucleotide polymorphisms (rSNPs) at the beginning of the second intron of the mouse K-ras gene that are strongly associated with lung cancer susceptibility. We performed functional analysis of three SNPs (rs12228277: T greater than A, rs12226937: G greater than A, and rs61761074: T greater than G) located in the same region of human KRAS. We found that rs12228277 and rs61761074 result in differential binding patterns of lung nuclear proteins to oligonucleotide probes corresponding two alternative alleles; in both cases, the transcription factor NF-Y is involved. G greater than A substitution (rs12226937) had no effect on the binding of lung nuclear proteins. However, all the nucleotide substitutions under study showed functional effects in a luciferase reporter assay. Among them, rs61761074 demonstrated a significant correlation with allele frequency in non-small-cell lung cancer (NSCLC). Taken together, the results of our study suggest that a T greater than G substitution at nucleotide position 615 in the second intron of the KRAS gene (rs61761074) may represent a promising genetic marker of NSCLC. PMID:26648033

  15. Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome.

    PubMed

    Linder, Bastian; Grozhik, Anya V; Olarerin-George, Anthony O; Meydan, Cem; Mason, Christopher E; Jaffrey, Samie R

    2015-08-01

    N(6)-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate. Current mapping approaches localize m6A residues to transcript regions 100-200 nt long but cannot identify precise m6A positions on a transcriptome-wide level. Here we developed m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) and used it to demonstrate that antibodies to m6A can induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA cross-linking and reverse transcription. We found that these antibodies similarly induced mutational signatures at N(6),2'-O-dimethyladenosine (m6Am), a modification found at the first nucleotide of certain mRNAs. Using these signatures, we mapped m6A and m6Am at single-nucleotide resolution in human and mouse mRNA and identified small nucleolar RNAs (snoRNAs) as a new class of m6A-containing non-coding RNAs (ncRNAs). PMID:26121403

  16. Genotyping single nucleotide polymorphisms in barley by tetra-primer ARMS-PCR.

    PubMed

    Chiapparino, E; Lee, D; Donini, P

    2004-04-01

    Single nucleotide polymorphisms (SNPs) are the most abundant form of DNA polymorphism. These polymorphisms can be used in plants as simple genetic markers for many breeding applications, for population studies, and for germplasm fingerprinting. The great increase in the available DNA sequences in the databases has made it possible to identify SNPs by "database mining", and the single most important factor preventing their widespread use appears to be the genotyping cost. Many genotyping platforms rely on the use of sophisticated, automated equipment coupled to costly chemistry and detection systems. A simple and economical method involving a single PCR is reported here for barley SNP genotyping. Using the tetra-primer ARMS-PCR procedure, we have been able to assay unambiguously five SNPs in a set of 132 varieties of cultivated barley. The results show the reliability of this technique and its potential for use in low- to moderate-throughput situations; the association of agronomically important traits is discussed. PMID:15060595

  17. Morpholino-functionalized nanochannel array for label-free single nucleotide polymorphisms detection.

    PubMed

    Gao, Hong-Li; Wang, Min; Wu, Zeng-Qiang; Wang, Chen; Wang, Kang; Xia, Xing-Hua

    2015-04-01

    The sensitive identification of single nucleotide polymorphisms becomes increasingly important for disease diagnosis, prevention, and practical applicability of pharmacogenomics. Herein, we propose a simple, highly selective, label-free single nucleotide polymorphisms (SNPs) sensing device by electrochemically monitoring the diffusion flux of ferricyanide probe across probe DNA/morpholino duplex functionalized nanochannels of porous anodic alumina. When perfectly matched or mismatched target DNA flows through the nanochannels modified with probe DNA/morpholino duplex, it competes for the probe DNA from morpholino, resulting in a change of the surface charges. Thus, the diffusion flux of negatively charged electroactive probe ferricyanide is modulated since it is sensitive to the surface charge due to the electrostatic interactions in electric double layer-merged nanochannels. Monitoring of the change in diffusion flux of probe enables us to detect not only a single base or two base mismatched sequence but also the specific location of the mismatched base. As is demonstrated, SNPs in the PML/RARα fusion gene, known as a biomarker of acute promyelocytic leukemia (APL), have been successfully detected. PMID:25734499

  18. Using Digital Polymerase Chain Reaction to Detect Single-Nucleotide Substitutions Induced by Genome Editing.

    PubMed

    Miyaoka, Yuichiro; Chan, Amanda H; Conklin, Bruce R

    2016-01-01

    This protocol is designed to detect single-nucleotide substitutions generated by genome editing in a highly sensitive and quantitative manner. It uses a combination of allele-specific hydrolysis probes and a new digital polymerase chain reaction (dPCR) technology called droplet digital PCR (ddPCR). ddPCR partitions a reaction into more than 10,000 nanoliter-scale water-in-oil droplets. As a result, each droplet contains only a few copies of the genome so that ddPCR is able to detect rare genome-editing events without missing them. PMID:27250210

  19. Steady-State Kinetic Analysis of DNA Polymerase Single-Nucleotide Incorporation Products

    PubMed Central

    O'Flaherty, Derek K.

    2014-01-01

    This unit describes the experimental procedures for the steady-state kinetic analysis of DNA synthesis across DNA nucleotides (native or modified) by DNA polymerases. In vitro primer extension experiments with a single nucleoside triphosphate species followed by denaturing polyacrylamide gel electrophoresis of the extended products is described. Data analysis procedures and fitting to steady-state kinetic models is presented to highlight the kinetic differences involved in the bypass of damaged versus undamaged DNA. Moreover, explanations concerning problems encountered in these experiments are addressed. This approach provides useful quantitative parameters for the processing of damaged DNA by DNA polymerases. PMID:25501593

  20. Investigation of bacterial nucleotide excision repair using single-molecule techniques.

    PubMed

    Van Houten, Bennett; Kad, Neil

    2014-08-01

    Despite three decades of biochemical and structural analysis of the prokaryotic nucleotide excision repair (NER) system, many intriguing questions remain with regard to how the UvrA, UvrB, and UvrC proteins detect, verify and remove a wide range of DNA lesions. Single-molecule techniques have begun to allow more detailed understanding of the kinetics and action mechanism of this complex process. This article reviews how atomic force microscopy and fluorescence microscopy have captured new glimpses of how these proteins work together to mediate NER. PMID:24472181

  1. VarMod: modelling the functional effects of non-synonymous variants.

    PubMed

    Pappalardo, Morena; Wass, Mark N

    2014-07-01

    Unravelling the genotype-phenotype relationship in humans remains a challenging task in genomics studies. Recent advances in sequencing technologies mean there are now thousands of sequenced human genomes, revealing millions of single nucleotide variants (SNVs). For non-synonymous SNVs present in proteins the difficulties of the problem lie in first identifying those nsSNVs that result in a functional change in the protein among the many non-functional variants and in turn linking this functional change to phenotype. Here we present VarMod (Variant Modeller) a method that utilises both protein sequence and structural features to predict nsSNVs that alter protein function. VarMod develops recent observations that functional nsSNVs are enriched at protein-protein interfaces and protein-ligand binding sites and uses these characteristics to make predictions. In benchmarking on a set of nearly 3000 nsSNVs VarMod performance is comparable to an existing state of the art method. The VarMod web server provides extensive resources to investigate the sequence and structural features associated with the predictions including visualisation of protein models and complexes via an interactive JSmol molecular viewer. VarMod is available for use at http://www.wasslab.org/varmod. PMID:24906884

  2. DNA sequencing by a single molecule detection of labeled nucleotides sequentially cleaved from a single strand of DNA

    SciTech Connect

    Goodwin, P.M.; Schecker, J.A.; Wilkerson, C.W.; Hammond, M.L.; Ambrose, W.P.; Jett, J.H.; Martin, J.C.; Marrone, B.L.; Keller, R.A. ); Haces, A.; Shih, P.J.; Harding, J.D. )

    1993-01-01

    We are developing a laser-based technique for the rapid sequencing of large DNA fragments (several kb in size) at a rate of 100 to 1000 bases per second. Our approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA into a flowing sample stream, sequential cleavage of the end nucleotide from the DNA fragment with an exonuclease, and detection of the individual fluorescently labeled bases by laser-induced fluorescence.

  3. DNA sequencing by a single molecule detection of labeled nucleotides sequentially cleaved from a single strand of DNA

    SciTech Connect

    Goodwin, P.M.; Schecker, J.A.; Wilkerson, C.W.; Hammond, M.L.; Ambrose, W.P.; Jett, J.H.; Martin, J.C.; Marrone, B.L.; Keller, R.A.; Haces, A.; Shih, P.J.; Harding, J.D.

    1993-02-01

    We are developing a laser-based technique for the rapid sequencing of large DNA fragments (several kb in size) at a rate of 100 to 1000 bases per second. Our approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA into a flowing sample stream, sequential cleavage of the end nucleotide from the DNA fragment with an exonuclease, and detection of the individual fluorescently labeled bases by laser-induced fluorescence.

  4. Single Nucleotide Polymorphism–Single Nucleotide Polymorphism Interactions Among Inflammation Genes in the Genetic Architecture of Blood Pressure in the Framingham Heart Study

    PubMed Central

    de las Fuentes, Lisa; Rao, Dabeeru C.

    2015-01-01

    BACKGROUND Hypertension is a major global health burden, but, although systolic and diastolic blood pressure (BP) each have estimated heritability of at least 30%, <3% of their variance has been attributed to particular genetic variants. Few studies have shown interactions between pairs of single nucleotide polymorphisms (SNPs) to be associated with BP. Although many studies use a Bonferroni correction for multiple testing to control type I error, thereby potentially reducing power, false discovery rate (FDR) approaches are also used in genome-wide studies. Renal ion balance genes have been associated with BP regulation, but, although inflammation has been studied in connection with BP, few studies have reported associations between inflammation genes and BP. METHODS We analyzed SNP-SNP interactions among 31 SNPs from genes involved in renal ion balance and 30 SNPs from genes involved in inflammation using data from the Framingham Heart Study. RESULTS No evidence of association was found for interactions among renal ion balance SNPs for either systolic or diastolic BP. A group of 3 interactions involving 6 inflammation genes (IKBKB–NFKBIA, IKBKE–CHUK, and ADIPOR2–RETN) showed evidence of association with diastolic BP with an FDR of 4.2%; no single interaction reached experiment-wide significance. CONCLUSIONS This study identified promising and biologically plausible candidates for interactions between inflammation genes that may be associated with DBP. Analysis using the FDR may allow detection of signals in the presence of modest noise (false positives) that a stringent approach based on Bonferroni-corrected P value thresholds may miss. PMID:25063733

  5. Computational Analysis of Single Nucleotide Polymorphisms Associated with Altered Drug Responsiveness in Type 2 Diabetes

    PubMed Central

    Costa, Valerio; Federico, Antonio; Pollastro, Carla; Ziviello, Carmela; Cataldi, Simona; Formisano, Pietro; Ciccodicola, Alfredo

    2016-01-01

    Type 2 diabetes (T2D) is one of the most frequent mortality causes in western countries, with rapidly increasing prevalence. Anti-diabetic drugs are the first therapeutic approach, although many patients develop drug resistance. Most drug responsiveness variability can be explained by genetic causes. Inter-individual variability is principally due to single nucleotide polymorphisms, and differential drug responsiveness has been correlated to alteration in genes involved in drug metabolism (CYP2C9) or insulin signaling (IRS1, ABCC8, KCNJ11 and PPARG). However, most genome-wide association studies did not provide clues about the contribution of DNA variations to impaired drug responsiveness. Thus, characterizing T2D drug responsiveness variants is needed to guide clinicians toward tailored therapeutic approaches. Here, we extensively investigated polymorphisms associated with altered drug response in T2D, predicting their effects in silico. Combining different computational approaches, we focused on the expression pattern of genes correlated to drug resistance and inferred evolutionary conservation of polymorphic residues, computationally predicting the biochemical properties of polymorphic proteins. Using RNA-Sequencing followed by targeted validation, we identified and experimentally confirmed that two nucleotide variations in the CAPN10 gene—currently annotated as intronic—fall within two new transcripts in this locus. Additionally, we found that a Single Nucleotide Polymorphism (SNP), currently reported as intergenic, maps to the intron of a new transcript, harboring CAPN10 and GPR35 genes, which undergoes non-sense mediated decay. Finally, we analyzed variants that fall into non-coding regulatory regions of yet underestimated functional significance, predicting that some of them can potentially affect gene expression and/or post-transcriptional regulation of mRNAs affecting the splicing. PMID:27347941

  6. Associations between novel single nucleotide polymorphisms in the Bos taurus growth hormone gene and performance traits in Holstein-Friesian dairy cattle.

    PubMed

    Mullen, M P; Berry, D P; Howard, D J; Diskin, M G; Lynch, C O; Berkowicz, E W; Magee, D A; MacHugh, D E; Waters, S M

    2010-12-01

    Growth hormone, produced in the anterior pituitary gland, stimulates the release of insulin-like growth factor-I from the liver and is of critical importance in the control of nutrient utilization and partitioning for lactogenesis, fertility, growth, and development in cattle. The aim of this study was to discover novel polymorphisms in the bovine growth hormone gene (GH1) and to quantify their association with performance using estimates of genetic merit on 848 Holstein-Friesian AI (artificial insemination) dairy sires. Associations with previously reported polymorphisms in the bovine GH1 gene were also undertaken. A total of 38 novel single nucleotide polymorphisms (SNP) were identified across a panel of 22 beef and dairy cattle by sequence analysis of the 5' promoter, intronic, exonic, and 3' regulatory regions, encompassing approximately 7 kb of the GH1 gene. Following multiple regression analysis on all SNP, associations were identified between 11 SNP (2 novel and 9 previously identified) and milk fat and protein yield, milk composition, somatic cell score, survival, body condition score, and body size. The G allele of a previously identified SNP in exon 5 at position 2141 of the GH1 sequence, resulting in a nonsynonymous substitution, was associated with decreased milk protein yield. The C allele of a novel SNP, GH32, was associated with inferior carcass conformation. In addition, the T allele of a previously characterized SNP, GH35, was associated with decreased survival. Both GH24 (novel) and GH35 were independently associated with somatic cell count, and 3 SNP, GH21, 2291, and GH35, were independently associated with body depth. Furthermore, 2 SNP, GH24 and GH63, were independently associated with carcass fat. Results of this study further demonstrate the multifaceted influences of GH1 on milk production, fertility, and growth-related traits in cattle. PMID:21094770

  7. In silico Analysis Revealed High-risk Single Nucleotide Polymorphisms in Human Pentraxin-3 Gene and their Impact on Innate Immune Response against Microbial Pathogens

    PubMed Central

    Thakur, Raman; Shankar, Jata

    2016-01-01

    Pentraxin-3 (PTX-3) protein is an evolutionary conserved protein that acts as a soluble pattern-recognition receptor for pathogens and plays important role in innate immune response. It recognizes various pathogens by interacting with extracellular moieties such as glactomannan of conidia (Aspergillus fumigatus), lipopolysaccharide of Pseudomonas aeruginosa, Streptococcus pneumonia and Salmonella typhimurium. Thus, PTX-3 protein helps to clear these pathogens by activating downstream innate immune process. In this study, computational methods were used to analyze various non-synonymous single nucleotide polymorphisms (nsSNPs) in PTX-3 gene. Three different databases were used to retrieve SNP data sets followed by seven different in silico algorithms to screen nsSNPs in PTX-3 gene. Sequence homology based approach was used to identify nsSNPs. Conservation profile of PTX-3 protein amino acid residues were predicted by ConSurf web server. In total, 10 high-risk nsSNPs were identified in pentraxin-domain of PTX-3 gene. Out of these 10 high-risk nsSNPs, 4 were present in the conserved structural and functional residues of the pentraxin-domain, hence, selected for structural analyses. The results showed alteration in the putative structure of pentraxin-domain. Prediction of protein–protein interactions analysis showed association of PTX-3 protein with C1q component of complement pathway. Different functional and structural residues along with various putative phosphorylation sites and evolutionary relationship were also predicted for PTX-3 protein. This is the first extensive computational analyses of pentraxin protein family with nsSNPs and will serve as a valuable resource for future population based studies. PMID:26941719

  8. Single nucleotide polymorphisms upstream from the β-carotene 15,15'-monoxygenase gene influence provitamin A conversion efficiency in female volunteers.

    PubMed

    Lietz, Georg; Oxley, Anthony; Leung, Wing; Hesketh, John

    2012-01-01

    β-Carotene, the most abundant provitamin A carotenoid in the diet, is converted to retinal by β-carotene 15,15'-monoxygenase (BCMO1). However, β-carotene absorption and conversion into retinal is extremely variable among individuals, with proportions of low responders to dietary β-carotene as high as 45%. Recently, 2 common nonsynonymous single nucleotide polymorphisms (SNPs) within the BCMO1 coding region (R267S; rs12934922 and A379V; rs7501331) revealed reduced catalytic activity, confirming that genetic variations contribute to the low responder phenotype. Because 4 SNPs 5' upstream from the BCMO1 gene were recently shown to affect circulating carotenoid concentrations, the current study aimed to investigate the effects of these SNPs on β-carotene conversion efficiency. Three of the 4 polymorphisms (rs6420424, rs11645428, and rs6564851) reduced the catalytic activity of BCMO1 in female volunteers by 59, 51, and 48%, respectively. The TG-rich lipoprotein fraction retinyl palmitate:β-carotene ratio was negatively correlated with the G allele of rs11645428 (r = -0.44; P = 0.018), whereas it was positively correlated with the G allele of rs6420424 (r = 0.53; P = 0.004) and the T allele of rs6564851 (r = 0.41; P = 0.028). Furthermore, large inter-ethnic variations in frequency of affected alleles were detected, with frequencies varying from 43 to 84% (rs6420424), 52 to 100% (rs11645428), and 19 to 67% (rs6564851). In summary, a range of SNPs can influence the effectiveness of using plant-based provitamin A carotenoids to increase vitamin A status in at-risk population groups and this effect may vary depending on ethnic origin. PMID:22113863

  9. Two common single nucleotide polymorphisms in the gene encoding beta-carotene 15,15'-monoxygenase alter beta-carotene metabolism in female volunteers.

    PubMed

    Leung, W C; Hessel, S; Méplan, C; Flint, J; Oberhauser, V; Tourniaire, F; Hesketh, J E; von Lintig, J; Lietz, G

    2009-04-01

    The key enzyme responsible for beta-carotene conversion into retinal is beta-carotene 15,15'-monoxygenase (BCMO1). Since it has been reported that the conversion of beta-carotene into vitamin A is highly variable in up to 45% of healthy individuals, we hypothesized that genetic polymorphisms in the BCMO1 gene could contribute to the occurrence of the poor converter phenotype. Here we describe the screening of the total open reading frame of the BCMO1 coding region that led to the identification of two common nonsynonymous single nucleotide polymorphisms (R267S: rs12934922; A379V: rs7501331) with variant allele frequencies of 42 and 24%, respectively. In vitro biochemical characterization of the recombinant 267S + 379V double mutant revealed a reduced catalytic activity of BCMO1 by 57% (P<0.001). Assessment of the responsiveness to a pharmacological dose of beta-carotene in female volunteers confirmed that carriers of both the 379V and 267S + 379V variant alleles had a reduced ability to convert beta-carotene, as indicated through reduced retinyl palmitate:beta-carotene ratios in the triglyceride-rich lipoprotein fraction [-32% (P=0.005) and -69% (P=0.001), respectively] and increased fasting beta-carotene concentrations [+160% (P=0.025) and +240% (P=0.041), respectively]. Our data show that there is genetic variability in beta-carotene metabolism and may provide an explanation for the molecular basis of the poor converter phenotype within the population. PMID:19103647

  10. Single nucleotide polymorphisms in the CXCR1 gene and its association with clinical mastitis incidence in Polish Holstein-Friesian cows.

    PubMed

    Pokorska, J; Dusza, M; Kułaj, D; Żukowski, K; Makulska, J

    2016-01-01

    The aim of this study was to identify the association between single nucleotide polymorphisms (SNPs) in the bovine chemokine receptor (CXCR1) gene and the resistance or susceptibility of cows to mastitis. The analysis of the CXCR1 polymorphism was carried out using polymerase chain reaction restriction fragment length polymorphism analysis for six SNP mutations (c.+291C>T, c.+365T>C, c.+816C>A, c.+819G>A, +1093C>T, and +1373C>A), of which four were located within the coding region and two in the 3'UTR region of the CXCR1 gene. Genetic material from 146 Polish Holstein-Friesian cows was analyzed after dividing into two groups depending on the incidence of clinical mastitis. Identified polymorphisms were in linkage disequilibrium and formed two linkage groups. Three haplotypes (CCCATA, TTAGCC, CTCGCC), forming six haplotype combinations, were detected. The logistic regression showed a significant association between the CC genotype at c.+365T>C and susceptibility of cows to clinical mastitis (P = 0.047). The frequency of haplotype combination 1/1 (CCCATA/CCCATA) was not significantly higher in cows susceptible to mastitis (P = 0.062). Of the identified SNP mutations, only c.+365T>C is a nonsynonymous mutation that induces a change in the coded protein [GCC (Ala) to GTC (Val) at the 122nd amino acid]. This amino acid change can result in changes in receptor function, which may be a reason for the increased mastitis incidence observed in cows with polymorphism at this site. PMID:27173275

  11. Naked-eye fingerprinting of single nucleotide polymorphisms on psoriasis patients

    NASA Astrophysics Data System (ADS)

    Valentini, Paola; Marsella, Alessandra; Tarantino, Paolo; Mauro, Salvatore; Baglietto, Silvia; Congedo, Maurizio; Paolo Pompa, Pier

    2016-05-01

    We report a low-cost test, based on gold nanoparticles, for the colorimetric (naked-eye) fingerprinting of a panel of single nucleotide polymorphisms (SNPs), relevant for the personalized therapy of psoriasis. Such pharmacogenomic tests are not routinely performed on psoriasis patients, due to the high cost of standard technologies. We demonstrated high sensitivity and specificity of our colorimetric test by validating it on a cohort of 30 patients, through a double-blind comparison with two state-of-the-art instrumental techniques, namely reverse dot blotting and sequencing, finding 100% agreement. This test offers high parallelization capabilities and can be easily generalized to other SNPs of clinical relevance, finding broad utility in diagnostics and pharmacogenomics.We report a low-cost test, based on gold nanoparticles, for the colorimetric (naked-eye) fingerprinting of a panel of single nucleotide polymorphisms (SNPs), relevant for the personalized therapy of psoriasis. Such pharmacogenomic tests are not routinely performed on psoriasis patients, due to the high cost of standard technologies. We demonstrated high sensitivity and specificity of our colorimetric test by validating it on a cohort of 30 patients, through a double-blind comparison with two state-of-the-art instrumental techniques, namely reverse dot blotting and sequencing, finding 100% agreement. This test offers high parallelization capabilities and can be easily generalized to other SNPs of clinical relevance, finding broad utility in diagnostics and pharmacogenomics. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr02200f

  12. The evolution of lineage-specific clusters of single nucleotide substitutions in the human genome.

    PubMed

    Xu, Ke; Wang, Jianrong; Elango, Navin; Yi, Soojin V

    2013-10-01

    Genomic regions harboring large numbers of human-specific single nucleotide substitutions are of significant interest since they are potential genomic foci underlying the evolution of human-specific traits as well as human adaptive evolution. Previous studies aimed to identify such regions either used pre-defined genomic locations such as coding sequences and conserved genomic elements or employed sliding window methods. Such approaches may miss clusters of substitutions occurring in regions other than those pre-defined locations, or not be able to distinguish human-specific clusters of substitutions from regions of generally high substitution rates. Here, we conduct a 'maximal segment' analysis to scan the whole human genome to identify clusters of human-specific substitutions that occurred since the divergence of the human and the chimpanzee genomes. This method can identify species-specific clusters of substitutions while not relying on pre-defined regions. We thus identify thousands of clusters of human-specific single nucleotide substitutions. The evolution of such clusters is driven by a combination of several different evolutionary processes including increased regional mutation rate, recombination-associated processes, and positive selection. These newly identified regions of human-specific substitution clusters include large numbers of previously identified human accelerated regions, and exhibit significant enrichments of genes involved in several developmental processes. Our study provides a useful tool to study the evolution of the human genome. PMID:23770436

  13. Single Nucleotide Variants in the Protein C Pathway and Mortality in Dialysis Patients

    PubMed Central

    Ocak, Gürbey; Drechsler, Christiane; Vossen, Carla Y.; Vos, Hans L.; Rosendaal, Frits R.; Reitsma, Pieter H.; Hoffmann, Michael M.; März, Winfried; Ouwehand, Willem H.; Krediet, Raymond T.; Boeschoten, Elisabeth W.; Dekker, Friedo W.; Wanner, Christoph; Verduijn, Marion

    2014-01-01

    Background The protein C pathway plays an important role in the maintenance of endothelial barrier function and in the inflammatory and coagulant processes that are characteristic of patients on dialysis. We investigated whether common single nucleotide variants (SNV) in genes encoding protein C pathway components were associated with all-cause 5 years mortality risk in dialysis patients. Methods Single nucleotides variants in the factor V gene (F5 rs6025; factor V Leiden), the thrombomodulin gene (THBD rs1042580), the protein C gene (PROC rs1799808 and 1799809) and the endothelial protein C receptor gene (PROCR rs867186, rs2069951, and rs2069952) were genotyped in 1070 dialysis patients from the NEtherlands COoperative Study on the Adequacy of Dialysis (NECOSAD) cohort) and in 1243 dialysis patients from the German 4D cohort. Results Factor V Leiden was associated with a 1.5-fold (95% CI 1.1–1.9) increased 5-year all-cause mortality risk and carriers of the AG/GG genotypes of the PROC rs1799809 had a 1.2-fold (95% CI 1.0–1.4) increased 5-year all-cause mortality risk. The other SNVs in THBD, PROC, and PROCR were not associated with 5-years mortality. Conclusion Our study suggests that factor V Leiden and PROC rs1799809 contributes to an increased mortality risk in dialysis patients. PMID:24816905

  14. Gallium plasmonic nanoparticles for label-free DNA and single nucleotide polymorphism sensing.

    PubMed

    Marín, Antonio García; García-Mendiola, Tania; Bernabeu, Cristina Navio; Hernández, María Jesús; Piqueras, Juan; Pau, Jose Luis; Pariente, Félix; Lorenzo, Encarnación

    2016-05-01

    A label-free DNA and single nucleotide polymorphism (SNP) sensing method is described. It is based on the use of the pseudodielectric function of gallium plasmonic nanoparticles (GaNPs) deposited on Si (100) substrates under reversal of the polarization handedness condition. Under this condition, the pseudodielectric function is extremely sensitive to changes in the surrounding medium of the nanoparticle surface providing an excellent sensing platform competitive to conventional surface plasmon resonance. DNA sensing has been carried out by immobilizing a thiolated capture probe sequence from Helicobacter pylori onto GaNP/Si substrates; complementary target sequences of Helicobacter pylori can be quantified over the range of 10 pM to 3.0 nM with a detection limit of 6.0 pM and a linear correlation coefficient of R(2) = 0.990. The selectivity of the device allows the detection of a single nucleotide polymorphism (SNP) in a specific sequence of Helicobacter pylori, without the need for a hybridization suppressor in solution such as formamide. Furthermore, it also allows the detection of this sequence in the presence of other pathogens, such as Escherichia coli in the sample. The broad applicability of the system was demonstrated by the detection of a specific gene mutation directly associated with cystic fibrosis in large genomic DNA isolated from blood cells. PMID:27120517

  15. A Locked Nucleic Acid Probe Based on Selective Salt-Induced Effect Detects Single Nucleotide Polymorphisms

    PubMed Central

    Zhang, Jing; Wu, Huizhe; Chen, Qiuchen; Zhao, Pengfei; Zhao, Haishan; Yao, Weifan; Wei, Minjie

    2015-01-01

    Detection of single based genetic mutation by using oligonucleotide probes is one of the common methods of detecting single nucleotide polymorphisms at known loci. In this paper, we demonstrated a hybridization system which included a buffer solution that produced selective salt-induced effect and a locked nucleic acid modified 12 nt oligonucleotide probe. The hybridization system is suitable for hybridization under room temperature. By using magnetic nanoparticles as carriers for PCR products, the SNPs (MDR1 C3435T/A) from 45 volunteers were analyzed, and the results were consistent with the results from pyrophosphoric acid sequencing. The method presented in this paper differs from the traditional method of using molecular beacons to detect SNPs in that it is suitable for research institutions lacking real-time quantitative PCR detecting systems, to detect PCR products at room temperature. PMID:26347880

  16. Multiplex single-nucleotide polymorphism typing of the human Y chromosome using TaqMan probes

    PubMed Central

    2011-01-01

    Background The analysis of human Y-chromosome variation in the context of population genetics and forensics requires the genotyping of dozens to hundreds of selected single-nucleotide polymorphisms (SNPs). In the present study, we developed a 121-plex (121 SNPs in a single array) TaqMan array capable of distinguishing most haplogroups and subhaplogroups on the Y-chromosome human phylogeny in Europe. Results We present data from 264 samples from several European areas and ethnic groups. The array developed in this study shows >99% accuracy of assignation to the Y human phylogeny (with an average call rate of genotypes >96%). Conclusions We have created and evaluated a robust and accurate Y-chromosome multiplex which minimises the possible errors due to mixup when typing the same sample in several independent reactions. PMID:21627798

  17. Genetic Aberrations in Childhood Acute Lymphoblastic Leukaemia: Application of High-Density Single Nucleotide Polymorphism Array

    PubMed Central

    Sulong, Sarina

    2010-01-01

    Screening of the entire human genome using high-density single nucleotide polymorphism array (SNPA) has become a powerful technique used in cancer genetics and population genetics studies. The GeneChip® Mapping Array, introduced by Affymetrix, is one SNPA platform utilised for genotyping studies. This GeneChip system allows researchers to gain a comprehensive view of cancer biology on a single platform for the quantification of chromosomal amplifications, deletions, and loss of heterozygosity or for allelic imbalance studies. Importantly, this array analysis has the potential to reveal novel genetic findings involved in the multistep development of cancer. Given the importance of genetic factors in leukaemogenesis and the usefulness of screening the whole genome, SNPA analysis has been utilised in many studies to characterise genetic aberrations in childhood acute lymphoblastic leukaemia. PMID:22135543

  18. Estimating population size using single-nucleotide polymorphism-based pedigree data.

    PubMed

    Spitzer, Robert; Norman, Anita J; Schneider, Michael; Spong, Göran

    2016-05-01

    Reliable population estimates are an important aspect of sustainable wildlife management and conservation but can be difficult to obtain for rare and elusive species. Here, we test a new census method based on pedigree reconstruction recently developed by Creel and Rosenblatt (2013). Using a panel of 96 single-nucleotide polymorphisms (SNPs), we genotyped fecal samples from two Swedish brown bear populations for pedigree reconstruction. Based on 433 genotypes from central Sweden (CS) and 265 from northern Sweden (NS), the population estimates (N = 630 for CS, N = 408 for NS) fell within the 95% CI of the official estimates. The precision and accuracy improved with increasing sampling intensity. Like genetic capture-mark-recapture methods, this method can be applied to data from a single sampling session. Pedigree reconstruction combined with noninvasive genetic sampling may thus augment population estimates, particularly for rare and elusive species for which sampling may be challenging. PMID:27096081

  19. Pyrosequencing with di-base addition for single nucleotide polymorphism genotyping.

    PubMed

    Pu, Dan; Mao, Chengguang; Cui, Lunbiao; Shi, Zhiyang; Xiao, Pengfeng

    2016-05-01

    We develop color code-based pyrosequencing with di-base addition for analysis of single nucleotide polymorphisms (SNPs). When a di-base is added into the polymerization, one or several two-color code(s) containing the type and the number of incorporated nucleotides will be produced. The code information obtained in a single run is useful to genotype SNPs as each allelic variant will give a specific pattern compared to the two other variants. Special care has to be taken while designing the di-base dispensation order. Here, we present a detailed protocol for establishing sequence-specific di-base addition to avoid nonsynchronous extension at the SNP sites. By using this technology, as few as 50 copies of DNA templates were accurately sequenced. Higher signals were produced and thus a relatively lower sample amount was required. Furthermore, the read length of per flow was increased, making simultaneous identification of multiple SNPs in a single sequencing run possible. Validation of the method was performed by using templates with two SNPs covering 37 bp and with three SNPs covering 58 bp as well as 82 bp. These SNPs were successfully genotyped by using only a sequencing primer in a single PCR/sequencing run. Our results demonstrated that this technology could be potentially developed into a powerful methodology to accurately determine SNPs so as to diagnose clinical settings. Graphical Abstract Conventional pyrosequencing adds one base (A, G, C, or T) at a time to determine the SNP site (left). Pyrosequencing with di-base addition adds di-base AG, AC, AT, CT, GC or GT at a time to determine the SNP site (right). Higher signals at SNP site will be produced due to the addition of di-bases. PMID:26935928

  20. Unraveling the complexity of the interactions of DNA nucleotides with gold by single molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Bano, Fouzia; Sluysmans, Damien; Wislez, Arnaud; Duwez, Anne-Sophie

    2015-11-01

    Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct adsorption behavior of the deoxyribonucleotides (i.e., a nitrogenous base, a deoxyribose sugar, and a phosphate group) and on the factors that govern the DNA-gold bond strength. Here, using single molecule force spectroscopy, we investigated the interaction of the four individual nucleotides, adenine, guanine, cytosine, and thymine, with gold. Experiments were performed in three salinity conditions and two surface dwell times to reveal the factors that influence nucleotide-Au bond strength. Force data show that, at physiological ionic strength, adenine-Au interactions are stronger, asymmetrical and independent of surface dwell time as compared to cytosine-Au and guanine-Au interactions. We suggest that in these conditions only adenine is able to chemisorb on gold. A decrease of the ionic strength significantly increases the bond strength for all nucleotides. We show that moderate ionic strength along with longer surface dwell period suggest weak chemisorption also for cytosine and guanine.Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct

  1. Study of single-nucleotide polymorphisms by means of electrical conductance measurements

    PubMed Central

    Hihath, Joshua; Xu, Bingqian; Zhang, Peiming; Tao, Nongjian

    2005-01-01

    Understanding the complexities of DNA has been a hallmark of science for over a half century, and one of the important topics in DNA research is recognizing the occurrence of mutations in the base-stack. In this article, we present a study of SNPs by direct-contact electrical measurements to a single DNA duplex. We have used short, 11- and 12-bp dsDNA to investigate the change in conductance that occurs if a single base pair, a single base, or two separate bases in the stack are modified. All measurements are carried out in aqueous solution with the DNA chemically bound to the electrodes. These measurements demonstrate that the presence of a single base pair mismatch can be identified by the conductance of the molecule and can cause a change in the conductance of dsDNA by as much as an order of magnitude, depending on the specific details of the double helix and the single nucleotide polymorphism. PMID:16284253

  2. Probing genomic diversity and evolution of Escherichia coli O157 by single nucleotide polymorphisms

    PubMed Central

    Zhang, Wei; Qi, Weihong; Albert, Thomas J.; Motiwala, Alifiya S.; Alland, David; Hyytia-Trees, Eija K.; Ribot, Efrain M.; Fields, Patricia I.; Whittam, Thomas S.; Swaminathan, Bala

    2006-01-01

    Infections by Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) are the predominant cause of bloody diarrhea and hemolytic uremic syndrome in the United States. In silico comparison of the two complete STEC O157 genomes (Sakai and EDL933) revealed a strikingly high level of sequence identity in orthologous protein-coding genes, limiting the use of nucleotide sequences to study the evolution and epidemiology of this bacterial pathogen. To systematically examine single nucleotide polymorphisms (SNPs) at a genome scale, we designed comparative genome sequencing microarrays and analyzed 1199 chromosomal genes (a total of 1,167,948 bp) and 92,721 bp of the large virulence plasmid (pO157) of eleven outbreak-associated STEC O157 strains. We discovered 906 SNPs in 523 chromosomal genes and observed a high level of DNA polymorphisms among the pO157 plasmids. Based on a uniform rate of synonymous substitution for Escherichia coli and Salmonella enterica (4.7 × 10−9 per site per year), we estimate that the most recent common ancestor of the contemporary β-glucuronidase-negative, non-sorbitolfermenting STEC O157 strains existed ca. 40 thousand years ago. The phylogeny of the STEC O157 strains based on the informative synonymous SNPs was compared to the maximum parsimony trees inferred from pulsed-field gel electrophoresis and multilocus variable numbers of tandem repeats analysis. The topological discrepancies indicate that, in contrast to the synonymous mutations, parts of STEC O157 genomes have evolved through different mechanisms with highly variable divergence rates. The SNP loci reported here will provide useful genetic markers for developing high-throughput methods for fine-resolution genotyping of STEC O157. Functional characterization of nucleotide polymorphisms should shed new insights on the evolution, epidemiology, and pathogenesis of STEC O157 and related pathogens. PMID:16606700

  3. Fluorescence detection of single-nucleotide polymorphism with single-strand triplex-forming DNA probes.

    PubMed

    Li, Xinpeng; Wang, Yuan; Guo, Jiajie; Tang, Xinjing

    2011-12-16

    Triple-helix-forming oligonucleotides (TFOs) are widespread in the genome and have been found in regulatory regions, especially in promoter zones and recombination hotspots of DNA. To specifically detect these polypurine sequences, we designed and synthesized two dual pyrene-labeled single-strand oligonucleotide probes (TFO-FPs) consisting of recognition, linker, and detection sequences. The hybridization processes of TFO-FPs with target polypurine oligonucleotides involve both Watson-Crick and Hoogsteen base-pairings. Through double sensing of oligonucleotide sequences, single mutations of target oligonucleotides are detected by monitoring changes in pyrene fluorescence. The high specificities of the probes are maintained over a wide temperature range without sacrifice of hybridization kinetics. PMID:22095630

  4. Subtyping of Salmonella enterica Subspecies I Using Single-Nucleotide Polymorphisms in Adenylate Cyclase.

    PubMed

    Guard, Jean; Abdo, Zaid; Byers, Sara Overstreet; Kriebel, Patrick; Rothrock, Michael J

    2016-07-01

    Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single-nucleotide polymorphisms were characterized within adenylate cyclase (cyaA). The National Center for Biotechnology Information (NCBI) database had 378 cyaA sequences from S. enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4, and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele-specific primer extension. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a ∼89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases, and genes used for multilocus sequence typing indicated that cyaA is an appropriate gene for assessing epidemiological trends of Salmonella because of its relative stability in nucleotide composition. PMID:27035032

  5. Assessment of the Geographic Origins of Pinewood Nematode Isolates via Single Nucleotide Polymorphism in Effector Genes

    PubMed Central

    Figueiredo, Joana; Simões, Maria José; Gomes, Paula; Barroso, Cristina; Pinho, Diogo; Conceição, Luci; Fonseca, Luís; Abrantes, Isabel; Pinheiro, Miguel; Egas, Conceição

    2013-01-01

    The pinewood nematode, Bursaphelenchus xylophilus, is native to North America but it only causes damaging pine wilt disease in those regions of the world where it has been introduced. The accurate detection of the species and its dispersal routes are thus essential to define effective control measures. The main goals of this study were to analyse the genetic diversity among B. xylophilus isolates from different geographic locations and identify single nucleotide polymorphism (SNPs) markers for geographic origin, through a comparative transcriptomic approach. The transcriptomes of seven B. xylophilus isolates, from Continental Portugal (4), China (1), Japan (1) and USA (1), were sequenced in the next generation platform Roche 454. Analysis of effector gene transcripts revealed inter-isolate nucleotide diversity that was validated by Sanger sequencing in the genomic DNA of the seven isolates and eight additional isolates from different geographic locations: Madeira Island (2), China (1), USA (1), Japan (2) and South Korea (2). The analysis identified 136 polymorphic positions in 10 effector transcripts. Pairwise comparison of the 136 SNPs through Neighbor-Joining and the Maximum Likelihood methods and 5-mer frequency analysis with the alignment-independent bilinear multivariate modelling approach correlated the SNPs with the isolates geographic origin. Furthermore, the SNP analysis indicated a closer proximity of the Portuguese isolates to the Korean and Chinese isolates than to the Japanese or American isolates. Each geographic cluster carried exclusive alleles that can be used as SNP markers for B. xylophilus isolate identification. PMID:24391785

  6. Single nucleotide markers of D-loop for identification of Indian wild pig (Sus scrofa cristatus)

    PubMed Central

    Srivastava, Gaurav Kumar; Rajput, Nidhi; Jadav, Kajal Kumar; Shrivastav, Avadh Bihari; Joshi, Himanshu R.

    2015-01-01

    Aim: Partial fragment of D-loop region extending from 35 to 770 were compared with corresponding sequences of 16 wild pigs and 9 domestic pig breeds from different parts of the world for detection of single nucleotide polymorphism (SNP) markers in the region. The paper also reappraises SNP markers from two fragments of cytochrome b gene and a fragment 12S rRNA gene distinguishing the Indian wild pig from other pig species of the world. Materials and Methods: Deoxyribonucleic acid (DNA) was isolated from 14 and 12 tissue samples of Indian wild and domestic pigs, respectively, collected from Central India for characterization of the D-loop DNA sequences using universal primers. The sequences obtained were aligned along with the retrieved sequences to analyze species-specific SNP marker. Results: A total of 58 mitochondrial D-loop gene sequences of pig races were aligned to identify 1349 polymorphic sites in the fragment from nucleotide positions 35-770 bp and four SNP markers were identified to differentiate Indian wild pig from all the sequences investigated in this study. With the inclusion of cytochrome b gene and 12S rRNA gene fragments, the present study contributes to the total 15 SNP markers, which have been identified in the mitochondrial fragment of 1936 bp for identification of Indian wild pig. Conclusion: SNP markers have advantages over other marker types and do not require subsequent standardization to compare data across studies or laboratories. PMID:27047129

  7. Assessment of the geographic origins of pinewood nematode isolates via single nucleotide polymorphism in effector genes.

    PubMed

    Figueiredo, Joana; Simões, Maria José; Gomes, Paula; Barroso, Cristina; Pinho, Diogo; Conceição, Luci; Fonseca, Luís; Abrantes, Isabel; Pinheiro, Miguel; Egas, Conceição

    2013-01-01

    The pinewood nematode, Bursaphelenchus xylophilus, is native to North America but it only causes damaging pine wilt disease in those regions of the world where it has been introduced. The accurate detection of the species and its dispersal routes are thus essential to define effective control measures. The main goals of this study were to analyse the genetic diversity among B. xylophilus isolates from different geographic locations and identify single nucleotide polymorphism (SNPs) markers for geographic origin, through a comparative transcriptomic approach. The transcriptomes of seven B. xylophilus isolates, from Continental Portugal (4), China (1), Japan (1) and USA (1), were sequenced in the next generation platform Roche 454. Analysis of effector gene transcripts revealed inter-isolate nucleotide diversity that was validated by Sanger sequencing in the genomic DNA of the seven isolates and eight additional isolates from different geographic locations: Madeira Island (2), China (1), USA (1), Japan (2) and South Korea (2). The analysis identified 136 polymorphic positions in 10 effector transcripts. Pairwise comparison of the 136 SNPs through Neighbor-Joining and the Maximum Likelihood methods and 5-mer frequency analysis with the alignment-independent bilinear multivariate modelling approach correlated the SNPs with the isolates geographic origin. Furthermore, the SNP analysis indicated a closer proximity of the Portuguese isolates to the Korean and Chinese isolates than to the Japanese or American isolates. Each geographic cluster carried exclusive alleles that can be used as SNP markers for B. xylophilus isolate identification. PMID:24391785

  8. Single-nucleotide resolution mapping of m6A and m6Am throughout the transcriptome

    PubMed Central

    Linder, Bastian; Grozhik, Anya V.; Olarerin-George, Anthony O.; Meydan, Cem; Mason, Christopher E.; Jaffrey, Samie R.

    2015-01-01

    N6-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate. Current m6A mapping approaches localize m6A residues to 100–200 nt-long regions of transcripts. The precise position of m6A in mRNAs cannot be identified on a transcriptome-wide level because there are no chemical methods to distinguish between m6A and adenosine. Here we show that anti-m6A antibodies can induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA crosslinking and reverse transcription. We find these antibodies similarly induce mutational signatures at N6,2′-O-dimethyladenosine (m6Am), a nucleotide found at the first encoded position of certain mRNAs. Using these mutational signatures, we map m6A and m6Am at single-nucleotide resolution in human and mouse mRNA and identify snoRNAs as a novel class of m6A-containing ncRNAs. PMID:26121403

  9. Investigation of single nucleotide polymorphism loci susceptible to degradation by ultraviolet light.

    PubMed

    Machida, Mitsuyo; Taki, Takashi; Shimada, Ryo; Kibayashi, Kazuhiko

    2016-10-01

    DNA in biological fluids is often degraded by environmental factors. Given that single nucleotide polymorphism (SNP) analyses require shorter amplicons than short tandem repeat (STR) analyses do, their use in human identification using degraded samples has recently attracted attention. Although various SNP loci are used to analyze degraded samples, it is unclear which ones are more appropriate. To characterize and identify SNP loci that are susceptible or resistant to degradation, we artificially degraded DNA, obtained from buccal swabs from 11 volunteers, by exposure to ultraviolet (UV) light for different durations (254 nm for 5, 15, 30, 60, or 120 min) and analyzed the resulting SNP loci. DNA degradation was assessed using gel electrophoresis, STR, and SNP profiling. DNA fragmentation occurred within 5 min of UV irradiation, and successful STR and SNP profiling decreased with increasing duration. However, 73% of SNP loci were still detected correctly in DNA samples irradiated for 120 min, a dose that rendered STR loci undetectable. The unsuccessful SNP typing and the base call failure of nucleotides neighboring the SNPs were traced to rs1031825, and we found that this SNP was susceptible to UV light. When comparing the detection efficiencies of STR and SNP loci, SNP typing was more successful than STR typing, making it effective when using degraded DNA. However, it is important to use rs1031825 with caution when interpreting SNP analyses of degraded DNA. PMID:27570235

  10. Subtyping of Salmonella enterica Subspecies I Using Single-Nucleotide Polymorphisms in Adenylate Cyclase

    PubMed Central

    Abdo, Zaid; Byers, Sara Overstreet; Kriebel, Patrick; Rothrock, Michael J.

    2016-01-01

    Abstract Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single-nucleotide polymorphisms were characterized within adenylate cyclase (cyaA). The National Center for Biotechnology Information (NCBI) database had 378 cyaA sequences from S. enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4, and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele-specific primer extension. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a ∼89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases, and genes used for multilocus sequence typing indicated that cyaA is an appropriate gene for assessing epidemiological trends of Salmonella because of its relative stability in nucleotide composition. PMID:27035032

  11. Distribution of Fitness Effects Caused by Single-Nucleotide Substitutions in Bacteriophage f1

    PubMed Central

    Peris, Joan B.; Davis, Paulina; Cuevas, José M.; Nebot, Miguel R.; Sanjuán, Rafael

    2010-01-01

    Empirical knowledge of the fitness effects of mutations is important for understanding many evolutionary processes, yet this knowledge is often hampered by several sources of measurement error and bias. Most of these problems can be solved using site-directed mutagenesis to engineer single mutations, an approach particularly suited for viruses due to their small genomes. Here, we used this technique to measure the fitness effect of 100 single-nucleotide substitutions in the bacteriophage f1, a filamentous single-strand DNA virus. We found that approximately one-fifth of all mutations are lethal. Viable ones reduced fitness by 11% on average and were accurately described by a log-normal distribution. More than 90% of synonymous substitutions were selectively neutral, while those affecting intergenic regions reduced fitness by 14% on average. Mutations leading to amino acid substitutions had an overall mean deleterious effect of 37%, which increased to 45% for those changing the amino acid polarity. Interestingly, mutations affecting early steps of the infection cycle tended to be more deleterious than those affecting late steps. Finally, we observed at least two beneficial mutations. Our results confirm that high mutational sensitivity is a general property of viruses with small genomes, including RNA and single-strand DNA viruses infecting animals, plants, and bacteria. PMID:20382832

  12. Gene comparison based on the repetition of single-nucleotide structure patterns.

    PubMed

    Qi, Zhao-Hui; Du, Ming-Hui; Qi, Xiao-Qin; Zheng, Li-Juan

    2012-10-01

    According to the repetition structure patterns of single-nucleotides, we propose a novel digital representation method to characterize primary DNA sequences. Based on this representation we give a new RP-SP (repeat and space) vector to compute the distance of different sequences. The examination of similarities/dissimilarities among different sequences illustrates the utility of the proposed RP-SP vector distance. Then, we use the proposed RP-SP vector method to analyze two groups of genomes, 15 E. coli genomes and 31 mitochondrial genomes. For comparison, we also apply other alignment-free methods to the two groups of genomes. The results show that the proposed method can distinguish characteristics of different genomes and used to reconstruct the phylogenetic tree of different genomes. PMID:22902300

  13. Chemical Gradient-mediated Melting Curve Analysis for Genotyping of Single Nucleotide Polymorphisms

    PubMed Central

    Russom, Aman; Irimia, Daniel; Toner, Mehmet

    2009-01-01

    This report describes a microfluidic solid-phase Chemical Gradient-mediated Melting Curve Analysis (CGMCA) method for single nucleotide polymorphism (SNP) analysis. The method is based on allele-specific denaturation to discriminate mismatched (MM) from perfectly matched (PM) DNA duplexes upon exposure to linear chemical gradient. PM and MM DNA duplexes conjugated on beads are captured in a microfluidic gradient generator device designed with dams, keeping the beads trapped perpendicular to a gradient generating channel. Two denaturants, formamide and urea, were tested for their ability to destabilize the DNA duplex by competing with Watson-Crick pairing. Upon exposure to the chemical gradient, rapid denaturing profile was monitored in real time using fluorescence microscopy. The results show that the two duplexes exhibit different kinetics of denaturation profiles, enabling discrimination of MM from PM DNA duplexes to score SNP. PMID:19593749

  14. Exploiting the CRISPR/Cas9 PAM Constraint for Single-Nucleotide Resolution Interventions

    PubMed Central

    Li, Yi; Mendiratta, Saurabh; Ehrhardt, Kristina; Kashyap, Neha; White, Michael A.; Bleris, Leonidas

    2016-01-01

    CRISPR/Cas9 is an enabling RNA-guided technology for genome targeting and engineering. An acute DNA binding constraint of the Cas9 protein is the Protospacer Adjacent Motif (PAM). Here we demonstrate that the PAM requirement can be exploited to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on the wild-type alleles. Specifically, we target the heterozygous G13A activating mutation of KRAS in colorectal cancer cells and we show reversal of drug resistance to a MEK small-molecule inhibitor. Our study introduces a new paradigm in genome editing and therapeutic targeting via the use of gRNA to guide Cas9 to a desired protospacer adjacent motif. PMID:26788852

  15. Genotyping of single nucleotide polymorphisms related to attention-deficit hyperactivity disorder.

    PubMed

    Tortajada-Genaro, Luis A; Mena, Salvador; Niñoles, Regina; Puigmule, Marta; Viladevall, Laia; Maquieira, Ángel

    2016-03-01

    Pharmacological treatment of several diseases, such as attention-deficit hyperactivity disorder (ADHD), presents marked variability in efficiency and its adverse effects. The genotyping of specific single nucleotide polymorphisms (SNPs) can support the prediction of responses to drugs and the genetic risk of presenting comorbidities associated with ADHD. This study presents two rapid and affordable microarray-based strategies to discriminate three clinically important SNPs in genes ADRA2A, SL6CA2, and OPRM1 (rs1800544, rs5569, and rs1799971, respectively). These approaches are allele-specific oligonucleotide hybridization (ASO) and a combination of allele-specific amplification (ASA) and solid-phase hybridization. Buccal swab and blood samples taken from ADHD patients and controls were analyzed by ASO, ASA, and a gold-reference method. The results indicated that ASA is superior in genotyping capability and analytical performance. PMID:26832728

  16. Epidemic population structure of extraintestinal pathogenic Escherichia coli determined by single nucleotide polymorphism pyrosequencing.

    PubMed

    Fernández-Romero, Natalia; Romero-Gómez, María Pilar; Gómez-Gil, María Rosa; Mingorance, Jesús

    2011-10-01

    We have developed an MLST-based scheme for typing Escherichia coli isolates using pyrosequencing of single nucleotide polymorphic positions (SNP). The SNP sequences are converted into allelic patterns and analyzed using the same approach used for MLST analyses. We have tested the method in two unselected collections of clinical isolates of E. coli obtained from blood and urine cultures. The two collections had a similar structure, 25% of the profiles (representing 68% of the isolates) were common to both, and 62% of the profiles (nearly 20% of the isolates) were unique. The four major profiles accounted for 44% of the isolates, and among these the most frequent one was related to the pandemic ST131 clone. The method is easy to implement and might be useful for typing large microbial collections. PMID:21723423

  17. Single nucleotide polymorphisms of Toll-like receptors and susceptibility to infectious diseases

    PubMed Central

    Skevaki, C; Pararas, M; Kostelidou, K; Tsakris, A; Routsias, J G

    2015-01-01

    Toll-like receptors (TLRs) are the best-studied family of pattern-recognition receptors (PRRs), whose task is to rapidly recognize evolutionarily conserved structures on the invading microorganisms. Through binding to these patterns, TLRs trigger a number of proinflammatory and anti-microbial responses, playing a key role in the first line of defence against the pathogens also promoting adaptive immunity responses. Growing amounts of data suggest that single nucleotide polymorphisms (SNPs) on the various human TLR proteins are associated with altered susceptibility to infection. This review summarizes the role of TLRs in innate immunity, their ligands and signalling and focuses on the TLR SNPs which have been linked to infectious disease susceptibility. PMID:25560985

  18. Candidate single-nucleotide polymorphisms and cerebral palsy: A case-control study

    PubMed Central

    HE, XIAO-GUANG; PENG, QI; CHEN, YAN-HUA; HE, TING; HUANG, HUI; MA, ZE-KE; FAN, XUE-JIN; LUO, LING; LIU, SHAO-JI; LU, XIAO-MEI

    2015-01-01

    Certain genetic polymorphisms have been suggested to be associated with cerebral palsy; the candidate genes are involved in thrombophilia, inflammation and preterm labor, but the mechanism remains to be elucidated. The aim of the present study was to investigate the associations between selected single-nucleotide polymorphisms (SNPs) and cerebral palsy among children. A case-control study was conducted, including 74 infants with cerebral palsy (case group) and 99 healthy infants (control group). The distributions of the allele and genotype frequencies were examined for the total cerebral palsy patient population in addition to subgroups divided according to gestational age (preterm versus full-term). The results showed that the rs1042714 variant in adrenergic receptor β-2 (ADRB2) and heterozygosity for ADRB2 were associated with the cerebral palsy risk among the preterm infants. No significant differences in the allele or genotype frequencies were observed between the total cerebral palsy patient population and controls for the eight SNPs investigated. PMID:26623029

  19. Pre-Steady-State Kinetic Analysis of Single-Nucleotide Incorporation by DNA Polymerases.

    PubMed

    Su, Yan; Peter Guengerich, F

    2016-01-01

    Pre-steady-state kinetic analysis is a powerful and widely used method to obtain multiple kinetic parameters. This protocol provides a step-by-step procedure for pre-steady-state kinetic analysis of single-nucleotide incorporation by a DNA polymerase. It describes the experimental details of DNA substrate annealing, reaction mixture preparation, handling of the RQF-3 rapid quench-flow instrument, denaturing polyacrylamide DNA gel preparation, electrophoresis, quantitation, and data analysis. The core and unique part of this protocol is the rationale for preparation of the reaction mixture (the ratio of the polymerase to the DNA substrate) and methods for conducting pre-steady-state assays on an RQF-3 rapid quench-flow instrument, as well as data interpretation after analysis. In addition, the methods for the DNA substrate annealing and DNA polyacrylamide gel preparation, electrophoresis, quantitation and analysis are suitable for use in other studies. © 2016 by John Wiley & Sons, Inc. PMID:27248785

  20. Novel single nucleotide polymorphism of UGT1A9 gene in Japanese.

    PubMed

    Fujita, Ken-ichi; Ando, Yuichi; Nagashima, Fumio; Yamamoto, Wataru; Endo, Hisashi; Kodama, Keiji; Araki, Kazuhiro; Miya, Toshimichi; Narabayashi, Masaru; Sasaki, Yasutsuna

    2006-02-01

    We sequenced from 5'-franking region to intron 1 (to 337 bp downstream from exon 1) of the UDP-glucuronosyltransferase (UGT) 1A9 gene prepared from 55 Japanese cancer patients. Seven single nucleotide polymorphisms (SNPs) were found. Two of them were UGT1A9 -118(T)n (n=10) and UGT1A9*5, and four were reported SNPs in intron 1 of UGT1A9 gene (89540C>T, 89549G>A, 89616T>A and 89710A>C). A novel SNP (89587T>C) was found. The sequence is as follows: SNP, 050824FujitaK001; Gene Name, UGT1A9; Accession Number, AF297093; Length, 25 bases; 5'-CCTTCTTGAAGAT/CATGTATTTATAA-3'. Two patients were heterozygous for the mutant allele, resulting in the allele frequency of 1.82%. PMID:16547398

  1. Large-scale detection and application of expressed sequence tag single nucleotide polymorphisms in Nicotiana.

    PubMed

    Wang, Y; Zhou, D; Wang, S; Yang, L

    2015-01-01

    Single nucleotide polymorphisms (SNPs) are widespread in the Nicotiana genome. Using an alignment and variation detection method, we developed 20,607,973 SNPs, based on the expressed sequence tag sequences of 10 Nicotiana species. The replacement rate was much higher than the transversion rate in the SNPs, and SNPs widely exist in the Nicotiana. In vitro verification indicated that all of the SNPs were high quality and accurate. Evolutionary relationships between 15 varieties were investigated by polymerase chain reaction with a special primer; the specific 302 locus of these sequence results clearly indicated the origin of Zhongyan 100. A database of Nicotiana SNPs (NSNP) was developed to store and search for SNPs in Nicotiana. NSNP is a tool for researchers to develop SNP markers of sequence data. PMID:26214460

  2. Gallium plasmonic nanoparticles for label-free DNA and single nucleotide polymorphism sensing

    NASA Astrophysics Data System (ADS)

    Marín, Antonio García; García-Mendiola, Tania; Bernabeu, Cristina Navio; Hernández, María Jesús; Piqueras, Juan; Pau, Jose Luis; Pariente, Félix; Lorenzo, Encarnación

    2016-05-01

    A label-free DNA and single nucleotide polymorphism (SNP) sensing method is described. It is based on the use of the pseudodielectric function of gallium plasmonic nanoparticles (GaNPs) deposited on Si (100) substrates under reversal of the polarization handedness condition. Under this condition, the pseudodielectric function is extremely sensitive to changes in the surrounding medium of the nanoparticle surface providing an excellent sensing platform competitive to conventional surface plasmon resonance. DNA sensing has been carried out by immobilizing a thiolated capture probe sequence from Helicobacter pylori onto GaNP/Si substrates; complementary target sequences of Helicobacter pylori can be quantified over the range of 10 pM to 3.0 nM with a detection limit of 6.0 pM and a linear correlation coefficient of R2 = 0.990. The selectivity of the device allows the detection of a single nucleotide polymorphism (SNP) in a specific sequence of Helicobacter pylori, without the need for a hybridization suppressor in solution such as formamide. Furthermore, it also allows the detection of this sequence in the presence of other pathogens, such as Escherichia coli in the sample. The broad applicability of the system was demonstrated by the detection of a specific gene mutation directly associated with cystic fibrosis in large genomic DNA isolated from blood cells.A label-free DNA and single nucleotide polymorphism (SNP) sensing method is described. It is based on the use of the pseudodielectric function of gallium plasmonic nanoparticles (GaNPs) deposited on Si (100) substrates under reversal of the polarization handedness condition. Under this condition, the pseudodielectric function is extremely sensitive to changes in the surrounding medium of the nanoparticle surface providing an excellent sensing platform competitive to conventional surface plasmon resonance. DNA sensing has been carried out by immobilizing a thiolated capture probe sequence from Helicobacter pylori

  3. Naked-eye fingerprinting of single nucleotide polymorphisms on psoriasis patients.

    PubMed

    Valentini, Paola; Marsella, Alessandra; Tarantino, Paolo; Mauro, Salvatore; Baglietto, Silvia; Congedo, Maurizio; Paolo Pompa, Pier

    2016-06-01

    We report a low-cost test, based on gold nanoparticles, for the colorimetric (naked-eye) fingerprinting of a panel of single nucleotide polymorphisms (SNPs), relevant for the personalized therapy of psoriasis. Such pharmacogenomic tests are not routinely performed on psoriasis patients, due to the high cost of standard technologies. We demonstrated high sensitivity and specificity of our colorimetric test by validating it on a cohort of 30 patients, through a double-blind comparison with two state-of-the-art instrumental techniques, namely reverse dot blotting and sequencing, finding 100% agreement. This test offers high parallelization capabilities and can be easily generalized to other SNPs of clinical relevance, finding broad utility in diagnostics and pharmacogenomics. PMID:27174795

  4. Developing single nucleotide polymorphism (SNP) markers from transcriptome sequences for identification of longan (Dimocarpus longan) germplasm

    PubMed Central

    Wang, Boyi; Tan, Hua-Wei; Fang, Wanping; Meinhardt, Lyndel W; Mischke, Sue; Matsumoto, Tracie; Zhang, Dapeng

    2015-01-01

    Longan (Dimocarpus longan Lour.) is an important tropical fruit tree crop. Accurate varietal identification is essential for germplasm management and breeding. Using longan transcriptome sequences from public databases, we developed single nucleotide polymorphism (SNP) markers; validated 60 SNPs in 50 longan germplasm accessions, including cultivated varieties and wild germplasm; and designated 25 SNP markers that unambiguously identified all tested longan varieties with high statistical rigor (P<0.0001). Multiple trees from the same clone were verified and off-type trees were identified. Diversity analysis revealed genetic relationships among analyzed accessions. Cultivated varieties differed significantly from wild populations (Fst=0.300; P<0.001), demonstrating untapped genetic diversity for germplasm conservation and utilization. Within cultivated varieties, apparent differences between varieties from China and those from Thailand and Hawaii indicated geographic patterns of genetic differentiation. These SNP markers provide a powerful tool to manage longan genetic resources and breeding, with accurate and efficient genotype identification. PMID:26504559

  5. Single Nucleotide Polymorphism Genotyping and Distribution of Coxiella burnetii Strains from Field Samples in Belgium

    PubMed Central

    Dal Pozzo, Fabiana; Renaville, Bénédicte; Martinelle, Ludovic; Renaville, Robert; Thys, Christine; Smeets, François; Kirschvink, Nathalie; Grégoire, Fabien; Delooz, Laurent; Czaplicki, Guy

    2015-01-01

    The genotypic characterization of Coxiella burnetii provides useful information about the strains circulating at the farm, region, or country level and may be used to identify the source of infection for animals and humans. The aim of the present study was to investigate the strains of C. burnetii circulating in caprine and bovine Belgian farms using a single nucleotide polymorphism (SNP) technique. Direct genotyping was applied to different samples (bulk tank milk, individual milk, vaginal swab, fetal product, and air sample). Besides the well-known SNP genotypes, unreported ones were found in bovine and caprine samples, increasing the variability of the strains found in the two species in Belgium. Moreover, multiple genotypes were detected contemporarily in caprine farms at different years of sampling and by using different samples. Interestingly, certain SNP genotypes were detected in both bovine and caprine samples, raising the question of interspecies transmission of the pathogen. PMID:26475104

  6. Identification of a Novel Single Nucleotide Polymorphism in Porcine Beta-Defensin-1 Gene.

    PubMed

    Pruthviraj, D R; Usha, A P; Venkatachalapathy, R T

    2016-03-01

    Porcine beta-defensin-1 (PBD-1) gene plays an important role in the innate immunity of pigs. The peptide encoded by this gene is an antimicrobial peptide that has direct activity against a wide range of microbes. This peptide is involved in the co-creation of an antimicrobial barrier in the oral cavity of pigs. The objective of the present study was to detect polymorphisms, if any, in exon-1 and exon-2 regions of PBD-1 gene in Large White Yorkshire (LWY) and native Ankamali pigs of Kerala, India. Blood samples were collected from 100 pigs and genomic DNA was isolated using phenol chloroform method. The quantity of DNA was assessed in a spectrophotometer and quality by gel electrophoresis. Exon-1 and exon-2 regions of PBD-1 gene were amplified by polymerase chain reaction (PCR) and the products were subjected to single strand conformation polymorphism (SSCP) analysis. Subsequent silver staining of the polyacrylamide gels revealed three unique SSCP banding patterns in each of the two exons. The presence of single nucleotide polymorphisms (SNPs) was confirmed by nucleotide sequencing of the PCR products. A novel SNP was found in the 5'-UTR region of exon-1 and a SNP was detected in the mature peptide coding region of exon-2. In exon-1, the pooled population frequencies of GG, GT, and TT genotypes were 0.67, 0.30, and 0.03, respectively. GG genotype was predominant in both the breeds whereas TT genotype was not detected in LWY breed. Similarly, in exon-2, the pooled population frequencies of AA, AG, and GG genotypes were 0.50, 0.27, and 0.23, respectively. AA genotype was predominant in LWY pigs whereas GG genotype was predominant in native pigs. These results suggest that there exists a considerable genetic variation at PBD-1 locus and further association studies may help in development of a PCR based genotyping test to select pigs with better immunity. PMID:26950860

  7. Characterization of frequencies and distribution of single nucleotide insertions/deletions in the human genome.

    PubMed

    Tan, Ene-Choo; Li, Haixia

    2006-07-19

    Most of the studies on single nucleotide variations are on substitutions rather than insertions/deletions. In this study, we examined the distribution and characteristics of single nucleotide insertions/deletions (SNindels), using data available from dbSNP for all the human chromosomes. There are almost 300,000 SNindels in the database, of which only 0.8% are validated. They occur at the frequency of 0.887 per 10 kb on average for the whole genome, or approximately 1 for every 11,274 bp. More than half occur in regions with mononucleotide repeats the longest of which is 47 bases. Overall the mononucleotide repeats involving C and G are much shorter than those for A and T. About 12% are surrounded by palindromes. There is general correlation between chromosome size and total number for each chromosome. Inter-chromosomal variation in density ranges from 0.6 to 21.7 per kilobase. The overall spectrum shows very high proportion of SNindel of types -/A and -/T at over 81%. The proportion of -/A and -/T SNindels for each chromosome is correlated to its AT content. Less than half of the SNindels are within or near known genes and even fewer (<0.183%) in coding regions, and more than 1.4% of -/C and -/G are in coding compared to 0.2% for -/A and -/T types. SNindels of -/A and -/T types make up 80% of those found within untranslated regions but less than 40% of those within coding regions. A separate analysis using the subset of 2324 validated SNindels showed slightly less AT bias of 74%, SNindels not within mononucleotide repeats showed even less AT bias at 58%. Density of validated SNindels is 0.007/10 kb overall and 90% are found within or near genes. Among all chromosomes, Y has the lowest numbers and densities for all SNindels, validated SNindels, and SNindels not within repeats. PMID:16781088

  8. Identification of a Novel Single Nucleotide Polymorphism in Porcine Beta-Defensin-1 Gene

    PubMed Central

    Pruthviraj, D. R.; Usha, A. P.; Venkatachalapathy, R. T.

    2016-01-01

    Porcine beta-defensin-1 (PBD-1) gene plays an important role in the innate immunity of pigs. The peptide encoded by this gene is an antimicrobial peptide that has direct activity against a wide range of microbes. This peptide is involved in the co-creation of an antimicrobial barrier in the oral cavity of pigs. The objective of the present study was to detect polymorphisms, if any, in exon-1 and exon-2 regions of PBD-1 gene in Large White Yorkshire (LWY) and native Ankamali pigs of Kerala, India. Blood samples were collected from 100 pigs and genomic DNA was isolated using phenol chloroform method. The quantity of DNA was assessed in a spectrophotometer and quality by gel electrophoresis. Exon-1 and exon-2 regions of PBD-1 gene were amplified by polymerase chain reaction (PCR) and the products were subjected to single strand conformation polymorphism (SSCP) analysis. Subsequent silver staining of the polyacrylamide gels revealed three unique SSCP banding patterns in each of the two exons. The presence of single nucleotide polymorphisms (SNPs) was confirmed by nucleotide sequencing of the PCR products. A novel SNP was found in the 5′-UTR region of exon-1 and a SNP was detected in the mature peptide coding region of exon-2. In exon-1, the pooled population frequencies of GG, GT, and TT genotypes were 0.67, 0.30, and 0.03, respectively. GG genotype was predominant in both the breeds whereas TT genotype was not detected in LWY breed. Similarly, in exon-2, the pooled population frequencies of AA, AG, and GG genotypes were 0.50, 0.27, and 0.23, respectively. AA genotype was predominant in LWY pigs whereas GG genotype was predominant in native pigs. These results suggest that there exists a considerable genetic variation at PBD-1 locus and further association studies may help in development of a PCR based genotyping test to select pigs with better immunity. PMID:26950860

  9. 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.

  10. A single nucleotide polymorphism in an exon dictates allele dependent differential splicing of episialin mRNA.

    PubMed Central

    Ligtenberg, M J; Gennissen, A M; Vos, H L; Hilkens, J

    1991-01-01

    The episialin gene (MUC1) encodes an epithelial mucin containing a variable number of repeats with a length of twenty amino acids, resulting in many different alleles that can be subdivided into two size classes. The episialin pre-mRNA uses either one of two neighbouring splice acceptor sites for exon 2, which mainly encodes the repeats. Using the genetic polymorphism of the episialin gene to identify different alleles, we show here that the splice site recognition is allele dependent and is based on a single A/G nucleotide difference in exon 2 eight nucleotides downstream of the second splice acceptor site. Transfection experiments confirm that this polymorphic nucleotide regulates the splice site selection. The identity of this nucleotide is in most cases correlated with one of the size classes of the alleles, indicating that mutations altering the number of repeats seldom arise by unequal cross-over between the repeat regions. Images PMID:2014168

  11. Method: a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus

    PubMed Central

    2012-01-01

    Background The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus. Method This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors. Result Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA. Conclusion The molecular typing method presented is one tool that could be incorporated into the forensic

  12. Kelvin probe force microscopy of DNA-capped nanoparticles for single-nucleotide polymorphism detection

    NASA Astrophysics Data System (ADS)

    Lee, Hyungbeen; Lee, Sang Won; Lee, Gyudo; Lee, Wonseok; Lee, Jeong Hoon; Hwang, Kyo Seon; Yang, Jaemoon; Lee, Sang Woo; Yoon, Dae Sung

    2016-07-01

    Kelvin probe force microscopy (KPFM) is a robust toolkit for profiling the surface potential (SP) of biomolecular interactions between DNAs and/or proteins at the single molecule level. However, it has often suffered from background noise and low throughput due to instrumental or environmental constraints, which is regarded as limiting KPFM applications for detection of minute changes in the molecular structures such as single-nucleotide polymorphism (SNP). Here, we show KPFM imaging of DNA-capped nanoparticles (DCNP) that enables SNP detection of the BRCA1 gene owing to sterically well-adjusted DNA-DNA interactions that take place within the confined spaces of DCNP. The average SP values of DCNP interacting with BRCA1 SNP were found to be lower than the DCNP reacting with normal (non-mutant) BRCA1 gene. We also demonstrate that SP characteristics of DCNP with different substrates (e.g., Au, Si, SiO2, and Fe) provide us with a chance to attenuate or augment the SP signal of DCNP without additional enhancement of instrumentation capabilities.Kelvin probe force microscopy (KPFM) is a robust toolkit for profiling the surface potential (SP) of biomolecular interactions between DNAs and/or proteins at the single molecule level. However, it has often suffered from background noise and low throughput due to instrumental or environmental constraints, which is regarded as limiting KPFM applications for detection of minute changes in the molecular structures such as single-nucleotide polymorphism (SNP). Here, we show KPFM imaging of DNA-capped nanoparticles (DCNP) that enables SNP detection of the BRCA1 gene owing to sterically well-adjusted DNA-DNA interactions that take place within the confined spaces of DCNP. The average SP values of DCNP interacting with BRCA1 SNP were found to be lower than the DCNP reacting with normal (non-mutant) BRCA1 gene. We also demonstrate that SP characteristics of DCNP with different substrates (e.g., Au, Si, SiO2, and Fe) provide us with a

  13. IDO1 and IDO2 Non-Synonymous Gene Variants: Correlation with Crohn's Disease Risk and Clinical Phenotype

    PubMed Central

    Zhang, Yuanhao; Sayuk, Gregory S.; Li, Ellen; Ciorba, Matthew A.

    2014-01-01

    Background Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. Genetic polymorphisms can confer CD risk and influence disease phenotype. Indoleamine 2,3 dioxygenase-1 (IDO1) is one of the most over-expressed genes in CD and mediates potent anti-inflammatory effects via tryptophan metabolism along the kynurenine pathway. We aimed to determine whether non-synonymous polymorphisms in IDO1 or IDO2 (a gene paralog) are important either as CD risk alleles or as modifiers of CD phenotype. Methods Utilizing a prospectively collected database, clinically phenotyped CD patients (n = 734) and non-IBD controls (n = 354) were genotyped for established IDO1 and IDO2 non-synonymous single nucleotide polymorphisms (SNPs) and novel genetic variants elucidated in the literature. Allelic frequencies between CD and non-IBD controls were compared. Genotype-phenotype analysis was conducted. IDO1 enzyme activity was assessed by calculating the serum kynurenine to tryptophan ratio (K/T). Results IDO1 SNPs were rare (1.7% non-IBD vs 1.1% CD; p = NS) and not linked to Crohn's disease diagnosis in this population. IDO1 SNPs did however associate with a severe clinical course, presence of perianal disease, extraintestinal manifestations and a reduced serum K/T ratio during active disease suggesting lower IDO1 function. IDO2 minor allele variants were common and one of them, rs45003083, associated with reduced risk of Crohn's disease (p = 0.025). No IDO2 SNPs associated with a particular Crohn's disease clinical phenotype. Conclusions This work highlights the functional importance of IDO enzymes in human Crohn's disease and establishes relative rates of IDO genetic variants in a US population. PMID:25541686

  14. A common single-nucleotide variant in T is strongly associated with chordoma.

    PubMed

    Pillay, Nischalan; Plagnol, Vincent; Tarpey, Patrick S; Lobo, Samira B; Presneau, Nadège; Szuhai, Karoly; Halai, Dina; Berisha, Fitim; Cannon, Stephen R; Mead, Simon; Kasperaviciute, Dalia; Palmen, Jutta; Talmud, Philippa J; Kindblom, Lars-Gunnar; Amary, M Fernanda; Tirabosco, Roberto; Flanagan, Adrienne M

    2012-11-01

    Chordoma is a rare malignant bone tumor that expresses the transcription factor T. We conducted an association study of 40 individuals with chordoma and 358 ancestry-matched controls, with replication in an independent cohort. Whole-exome and Sanger sequencing of T exons showed strong association of the common nonsynonymous SNP rs2305089 with chordoma risk (allelic odds ratio (OR) = 6.1, 95% confidence interval (CI) = 3.1-12.1; P = 4.4 × 10(-9)), a finding that is exceptional in cancers with a non-Mendelian mode of inheritance. PMID:23064415

  15. On-chip detection of a single nucleotide polymorphism without polymerase amplification

    PubMed Central

    Han, Jinhee; Tan, Matthew; Sudheendra, Lakshmana; Weiss, Robert H.; Kennedy, Ian M.

    2014-01-01

    A nanoparticle-assembled photonic crystal (PC) array was used to detect single nucleotide polymorphism (SNP). The assay platform with PC nanostructure enhanced the fluorescent signal from nanoparticle-hybridized DNA complexes due to phase matching of excitation and emission. Nanoparticles coupled with probe DNA were trapped into nanowells in an array by using an electrophoretic particle entrapment system. The PC/DNA assay platform was able to identify a 1 base pair (bp) difference in synthesized nucleotide sequences that mimicked the mutation seen in a feline model of human autosomal dominant polycystic kidney disease (PKD) with a sensitivity of 0.9 fg/mL (50 aM)-sensitivity, which corresponds to 30 oligos/array. The reliability of the PC/DNA assay platform to detect SNP in a real sample was demonstrated by using genomic DNA (gDNA) extracted from the urine and blood of two PKD− wild type and three PKD positive cats. The standard curves for PKD positive (PKD+) and negative (PKD−) DNA were created using two feline-urine samples. An additional three urine samples were analyzed in a similar fashion and showed satisfactory agreement with the standard curve, confirming the presence of the mutation in affected urine. The limit of detection (LOD) was 0.005 ng/mL which corresponds to 6 fg per array for gDNA in urine and blood. The PC system demonstrated the ability to detect a number of genome equivalents for the PKD SNP that was very similar to the results reported with real time polymerase chain reaction (PCR). The favorable comparison with quantitative PCR suggests that the PC technology may find application well beyond the detection of the PKD SNP, into areas where a simple, cheap and portable nucleic acid analysis is desirable. PMID:25580203

  16. Identification of single nucleotide polymorphisms from the transcriptome of an organism with a whole genome duplication

    PubMed Central

    2013-01-01

    Background The common ancestor of salmonid fishes, including rainbow trout (Oncorhynchus mykiss), experienced a whole genome duplication between 20 and 100 million years ago, and many of the duplicated genes have been retained in the trout genome. This retention complicates efforts to detect allelic variation in salmonid fishes. Specifically, single nucleotide polymorphism (SNP) detection is problematic because nucleotide variation can be found between the duplicate copies (paralogs) of a gene as well as between alleles. Results We present a method of differentiating between allelic and paralogous (gene copy) sequence variants, allowing identification of SNPs in organisms with multiple copies of a gene or set of genes. The basic strategy is to: 1) identify windows of unique cDNA sequences with homology to each other, 2) compare these unique cDNAs if they are not shared between individuals (i.e. the cDNA is homozygous in one individual and homozygous for another cDNA in the other individual), and 3) give a “SNP score” value between zero and one to each candidate sequence variant based on six criteria. Using this strategy we were able to detect about seven thousand potential SNPs from the transcriptomes of several clonal lines of rainbow trout. When directly compared to a pre-validated set of SNPs in polyploid wheat, we were also able to estimate the false-positive rate of this strategy as 0 to 28% depending on parameters used. Conclusions This strategy has an advantage over traditional techniques of SNP identification because another dimension of sequencing information is utilized. This method is especially well suited for identifying SNPs in polyploids, both outbred and inbred, but would tend to be conservative for diploid organisms. PMID:24237905

  17. Developing single nucleotide polymorphism markers for the identification of pineapple (Ananas comosus) germplasm

    PubMed Central

    Zhou, Lin; Matsumoto, Tracie; Tan, Hua-Wei; Meinhardt, Lyndel W; Mischke, Sue; Wang, Boyi; Zhang, Dapeng

    2015-01-01

    Pineapple (Ananas comosus [L.] Merr.) is the third most important tropical fruit in the world after banana and mango. As a crop with vegetative propagation, genetic redundancy is a major challenge for efficient genebank management and in breeding. Using expressed sequence tag and nucleotide sequences from public databases, we developed 213 single nucleotide polymorphism (SNP) markers and validated 96 SNPs by genotyping the United States Department of Agriculture - Agricultural Research Service pineapple germplasm collection, maintained in Hilo, Hawaii. The validation resulted in designation of a set of 57 polymorphic SNP markers that revealed a high rate of duplicates in this pineapple collection. Twenty-four groups of duplicates were detected, encompassing 130 of the total 170 A cosmos accessions. The results show that somatic mutation has been the main source of intra-cultivar variations in pineapple. Multivariate clustering and a model-based population stratification suggest that the modern pineapple cultivars are comprised of progenies that are derived from different wild Ananas botanical varieties. Parentage analysis further revealed that both A. comosus var. bracteatus and A. comosus var. ananassoides are likely progenitors of pineapple cultivars. However, the traditional classification of cultivated pineapple into horticultural groups (e.g. ‘Cayenne’, ‘Spanish’, ‘Queen’) was not well supported by the present study. These SNP markers provide robust and universally comparable DNA fingerprints; thus, they can serve as an efficient genotyping tool to assist pineapple germplasm management, propagation of planting material, and pineapple cultivar protection. The high rate of genetic redundancy detected in this pineapple collection suggests the potential impact of applying this technology on other clonally propagated perennial crops. PMID:26640697

  18. Developing single nucleotide polymorphism markers for the identification of pineapple (Ananas comosus) germplasm.

    PubMed

    Zhou, Lin; Matsumoto, Tracie; Tan, Hua-Wei; Meinhardt, Lyndel W; Mischke, Sue; Wang, Boyi; Zhang, Dapeng

    2015-01-01

    Pineapple (Ananas comosus [L.] Merr.) is the third most important tropical fruit in the world after banana and mango. As a crop with vegetative propagation, genetic redundancy is a major challenge for efficient genebank management and in breeding. Using expressed sequence tag and nucleotide sequences from public databases, we developed 213 single nucleotide polymorphism (SNP) markers and validated 96 SNPs by genotyping the United States Department of Agriculture - Agricultural Research Service pineapple germplasm collection, maintained in Hilo, Hawaii. The validation resulted in designation of a set of 57 polymorphic SNP markers that revealed a high rate of duplicates in this pineapple collection. Twenty-four groups of duplicates were detected, encompassing 130 of the total 170 A cosmos accessions. The results show that somatic mutation has been the main source of intra-cultivar variations in pineapple. Multivariate clustering and a model-based population stratification suggest that the modern pineapple cultivars are comprised of progenies that are derived from different wild Ananas botanical varieties. Parentage analysis further revealed that both A. comosus var. bracteatus and A. comosus var. ananassoides are likely progenitors of pineapple cultivars. However, the traditional classification of cultivated pineapple into horticultural groups (e.g. 'Cayenne', 'Spanish', 'Queen') was not well supported by the present study. These SNP markers provide robust and universally comparable DNA fingerprints; thus, they can serve as an efficient genotyping tool to assist pineapple germplasm management, propagation of planting material, and pineapple cultivar protection. The high rate of genetic redundancy detected in this pineapple collection suggests the potential impact of applying this technology on other clonally propagated perennial crops. PMID:26640697

  19. Single nucleotide polymorphism of FSHβ gene associated with reproductive traits in Japanese flounder ( Paralichthys olivaceus)

    NASA Astrophysics Data System (ADS)

    He, Feng; Wen, Haishen; Yu, Dahui; Li, Jifang; Shi, Bao; Chen, Caifang; Zhang, Jiaren; Jin, Guoxiong; Chen, Xiaoyan; Shi, Dan; Yang, Yanping

    2010-12-01

    Follicle stimulating hormone β (FSHβ) of Japanese flounder ( Paralichthys olivaceus) plays a key role in the regulation of gonadal development. This study aimed to investigate molecular genetic characteristics of the FSHβ gene and elucidate the effects of single nucleotide polymorphisms (SNPs) of FSHβ on reproductive traits in Japanese flounder. We used polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequencing of the FSHβ gene in 60 individuals. We identified only an SNP (T/C) in the coding region of exon3 of FSHβ. The SNP (T/C) did not lead to amino acid changes at the position 340 bp of FSHβ gene. Statistical analysis showed that the SNP was significantly associated with testosterone (T) level and gonadosomatic index (GSI) ( P < 0.05). Individuals with genotype TC of the SNP had significantly higher serum T levels and GSI ( P < 0.05) than that of genotype CC. Therefore, FSHβ gene could be a useful molecular marker in selection for prominent reproductive trait in Japanese Flounder.

  20. A single natural nucleotide mutation alters bacterial pathogen host-tropism

    PubMed Central

    Ward, Melissa J.; Selva, Laura; Guinane, Caitriona M.; González-Muñoz, Beatriz M.; Tristan, Anne; Foster, Simon J; Fitzgerald, J. Ross; Penadés, José R.

    2015-01-01

    The capacity of microbial pathogens to alter their host-tropism leading to epidemics in distinct host-species populations is a global public and veterinary health concern. In order to investigate the molecular basis of a bacterial host-switching event in a tractable host-species, we traced the evolutionary trajectory of the common rabbit clone of Staphylococcus aureus. We report that it evolved through a likely human-to-rabbit host jump over 40 years ago, and that only a single natural nucleotide mutation was required and sufficient to convert a human-specific S. aureus strain into one which could infect rabbits. Related mutations were identified at the same locus in other rabbit strains of distinct clonal origin, consistent with convergent evolution. This first report of a single mutation that was sufficient to alter the host-tropism of a micro-organism during its evolution highlights the capacity of some pathogens to readily expand into novel host-species populations. PMID:25685890

  1. Detection of mandarin in orange juice by single-nucleotide polymorphism qPCR assay.

    PubMed

    Aldeguer, Miriam; López-Andreo, María; Gabaldón, José A; Puyet, Antonio

    2014-02-15

    A dual-probe real time PCR (qPCR) DNA-based analysis was devised for the identification of mandarin in orange juice. A single nucleotide polymorphism at the trnL-trnF intergenic region of the chloroplast chromosome was confirmed in nine orange (Citrus sinensis) and thirteen commercial varieties of mandarin, including Citrus reticulata and Citrus unshiu species and a mandarin × tangelo hybrid. Two short minor-groove binding fluorescent probes targeting the polymorphic sequence were used in the dual-probe qPCR, which allowed the detection of both species in single-tube reactions. The similarity of PCR efficiencies allowed a simple estimation of the ratio mandarin/orange in the juice samples, which correlated to the measured difference of threshold cycle values for both probes. The limit of detection of the assay was 5% of mandarin in orange juice, both when the juice was freshly prepared (not from concentrate) or reconstituted from concentrate, which would allow the detection of fraudulently added mandarin juice. The possible use of the dual-probe system for quantitative measurements was also tested on fruit juice mixtures. qPCR data obtained from samples containing equal amounts of mandarin and orange juice revealed that the mandarin target copy number was approximately 2.6-fold higher than in orange juice. The use of a matrix-adapted control as calibrator to compensate the resulting C(T) bias allowed accurate quantitative measurements to be obtained. PMID:24128588

  2. 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

  3. The human BARX2 gene: genomic structure, chromosomal localization, and single nucleotide polymorphisms.

    PubMed

    Hjalt, T A; Murray, J C

    1999-12-15

    The BARX genes 1 and 2 are Bar class homeobox genes expressed in craniofacial structures during development. In this report, we present the genomic structure, chromosomal localization, and polymorphic markers in BARX2. The gene has four exons, ranging in size from 85 to 1099 bp. BARX2 is localized on human chromosome 11q25, as determined by radiation hybrid mapping. In the mouse, Barx2 is coexpressed with Pitx2 in several tissues. Based on the coexpression, BARX2 was assumed to be a candidate gene for those cases of Rieger syndrome that cannot be associated with mutations of PITX2. Mutations in PITX2 cause some cases of Rieger syndrome, an autosomal dominant disorder affecting eyes, teeth, and umbilicus. DNA from Rieger patients was subjected to single-strand conformation polymorphism screening of the BARX2 coding region. Three single nucleotide polymorphisms were found in a normal population, although no etiologic mutations were detectable in over 100 cases of Rieger syndrome or in individuals with related ocular disorders. PMID:10644443

  4. Kelvin probe force microscopy of DNA-capped nanoparticles for single-nucleotide polymorphism detection.

    PubMed

    Lee, Hyungbeen; Lee, Sang Won; Lee, Gyudo; Lee, Wonseok; Lee, Jeong Hoon; Hwang, Kyo Seon; Yang, Jaemoon; Lee, Sang Woo; Yoon, Dae Sung

    2016-07-14

    Kelvin probe force microscopy (KPFM) is a robust toolkit for profiling the surface potential (SP) of biomolecular interactions between DNAs and/or proteins at the single molecule level. However, it has often suffered from background noise and low throughput due to instrumental or environmental constraints, which is regarded as limiting KPFM applications for detection of minute changes in the molecular structures such as single-nucleotide polymorphism (SNP). Here, we show KPFM imaging of DNA-capped nanoparticles (DCNP) that enables SNP detection of the BRCA1 gene owing to sterically well-adjusted DNA-DNA interactions that take place within the confined spaces of DCNP. The average SP values of DCNP interacting with BRCA1 SNP were found to be lower than the DCNP reacting with normal (non-mutant) BRCA1 gene. We also demonstrate that SP characteristics of DCNP with different substrates (e.g., Au, Si, SiO2, and Fe) provide us with a chance to attenuate or augment the SP signal of DCNP without additional enhancement of instrumentation capabilities. PMID:27127876

  5. Facile method for automated genotyping of single nucleotide polymorphisms by mass spectrometry.

    PubMed

    Sauer, Sascha; Gelfand, David H; Boussicault, Francis; Bauer, Keith; Reichert, Fred; Gut, Ivo G

    2002-03-01

    In the future, analysis of single nucleotide polymorphisms (SNPs) should become a powerful tool for many genetic applications in areas such as association studies, pharmacogenetics and traceability in the agro-alimentary sector. A number of technologies have been developed for high-throughput genotyping of SNPs. Here we present the simplified GOOD assay for SNP genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI). The simplified GOOD assay is a single-tube, purification-free, three-step procedure consisting of PCR, primer extension and phosphodiesterase II digestion followed by mass spectrometric analysis. Due to the application of charge-tag technology, no sample purification is required prior to the otherwise very impurity-sensitive MALDI analysis. The use of methylphosphonate containing primers and ddNTPs or alpha-S-ddNTPs together with a novel DNA polymerase derived from Thermotoga maritima for primer extension allow the fluent preparation of negatively charge-tagged, allele-specific products. A key feature of this polymerase is its preference for ddNTPs and alpha-S-ddNTPs over dNTPs. The simplified GOOD assay was run with automatic liquid handling at the lowest manageable volumes, automatic data acquisition and interpretation. We applied this novel procedure to genotyping SNPs of candidate genes for hypertension and cardiovascular disease. PMID:11861927

  6. Wireless electrochemiluminescence bipolar electrode array for visualized genotyping of single nucleotide polymorphism.

    PubMed

    Khoshfetrat, Seyyed Mehdi; Ranjbari, Mitra; Shayan, Mohsen; Mehrgardi, Masoud A; Kiani, Abolfazl

    2015-08-18

    The development of simple, inexpensive, hand-held, user-friendly biosensor for high throughput and multiplexed genotyping of various single nucleotide polymorphisms (SNPs) in a single run experiment by a nonspecialist user is the main challenge in the analysis of DNA. Visualizing the signal and possibility to monitor SNPs by a digital camera opens a new horizon for the routine applications. In the present manuscript, a novel wireless electrochemiluminescence (ECL) DNA array is introduced for the visualized genotyping of different SNPs on the basis of ECL of luminol/hydrogen peroxide system on a bipolar electrode (BPE) array platform. After modification of anodic poles of the array with the DNA probe and its hybridization with the targets, genotyping of various SNPs is carried out by exposing the array to different monobase modified luminol-platinum nanoparticles (M-L-PtNPs). Upon the hybridization of M-L-PtNPs to mismatch sites, the ECL of luminol is followed using a photomultiplier tube (PMT) or digital camera and the images are analyzed by ImageJ software. This biosensor can detect even thermodynamically stable SNP (G-T mismatches) in the range of 2-600 pM. Also, by combining the advantages of BPE and the high visual sensitivity of ECL, it could be easily expected to achieve sensitive screening of different SNPs. The present biosensor demonstrates the capability for the discrimination between PCR products of normal, heterozygous, and homozygous beta thalassemia genetic disorders. PMID:26176414

  7. High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification

    PubMed Central

    Faruqi, A Fawad; Hosono, Seiyu; Driscoll, Mark D; Dean, Frank B; Alsmadi, Osama; Bandaru, Rajanikanta; Kumar, Gyanendra; Grimwade, Brian; Zong, Qiuling; Sun, Zhenyu; Du, Yuefen; Kingsmore, Stephen; Knott, Tim; Lasken, Roger S

    2001-01-01

    Background Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation. Results SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe. Conclusions Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring. PMID:11511324

  8. Phylogenetic discovery bias in Bacillus anthracis using single-nucleotide polymorphisms from whole-genome sequencing

    PubMed Central

    Pearson, Talima; Busch, Joseph D.; Ravel, Jacques; Read, Timothy D.; Rhoton, Shane D.; U'Ren, Jana M.; Simonson, Tatum S.; Kachur, Sergey M.; Leadem, Rebecca R.; Cardon, Michelle L.; Van Ert, Matthew N.; Huynh, Lynn Y.; Fraser, Claire M.; Keim, Paul

    2004-01-01

    Phylogenetic reconstruction using molecular data is often subject to homoplasy, leading to inaccurate conclusions about phylogenetic relationships among operational taxonomic units. Compared with other molecular markers, single-nucleotide polymorphisms (SNPs) exhibit extremely low mutation rates, making them rare in recently emerged pathogens, but they are less prone to homoplasy and thus extremely valuable for phylogenetic analyses. Despite their phylogenetic potential, ascertainment bias occurs when SNP characters are discovered through biased taxonomic sampling; by using whole-genome comparisons of five diverse strains of Bacillus anthracis to facilitate SNP discovery, we show that only polymorphisms lying along the evolutionary pathway between reference strains will be observed. We illustrate this in theoretical and simulated data sets in which complex phylogenetic topologies are reduced to linear evolutionary models. Using a set of 990 SNP markers, we also show how divergent branches in our topologies collapse to single points but provide accurate information on internodal distances and points of origin for ancestral clades. These data allowed us to determine the ancestral root of B. anthracis, showing that it lies closer to a newly described “C” branch than to either of two previously described “A” or “B” branches. In addition, subclade rooting of the C branch revealed unequal evolutionary rates that seem to be correlated with ecological parameters and strain attributes. Our use of nonhomoplastic whole-genome SNP characters allows branch points and clade membership to be estimated with great precision, providing greater insight into epidemiological, ecological, and forensic questions. PMID:15347815

  9. Single nucleotide polymorphisms and haplotypes associated with feed efficiency in beef cattle

    PubMed Central

    2013-01-01

    Background General, breed- and diet-dependent associations between feed efficiency in beef cattle and single nucleotide polymorphisms (SNPs) or haplotypes were identified on a population of 1321 steers using a 50 K SNP panel. Genomic associations with traditional two-step indicators of feed efficiency – residual feed intake (RFI), residual average daily gain (RADG), and residual intake gain (RIG) – were compared to associations with two complementary one-step indicators of feed efficiency: efficiency of intake (EI) and efficiency of gain (EG). Associations uncovered in a training data set were evaluated on independent validation data set. A multi-SNP model was developed to predict feed efficiency. Functional analysis of genes harboring SNPs significantly associated with feed efficiency and network visualization aided in the interpretation of the results. Results For the five feed efficiency indicators, the numbers of general, breed-dependent, and diet-dependent associations with SNPs (P-value < 0.0001) were 31, 40, and 25, and with haplotypes were six, ten, and nine, respectively. Of these, 20 SNP and six haplotype associations overlapped between RFI and EI, and five SNP and one haplotype associations overlapped between RADG and EG. This result confirms the complementary value of the one and two-step indicators. The multi-SNP models included 89 SNPs and offered a precise prediction of the five feed efficiency indicators. The associations of 17 SNPs and 7 haplotypes with feed efficiency were confirmed on the validation data set. Nine clusters of Gene Ontology and KEGG pathway categories (mean P-value < 0.001) including, 9nucleotide binding; ion transport, phosphorous metabolic process, and the MAPK signaling pathway were overrepresented among the genes harboring the SNPs associated with feed efficiency. Conclusions The general SNP associations suggest that a single panel of genomic variants can be used regardless of breed and diet. The breed- and diet

  10. Empirically derived subgroups in rheumatoid arthritis: association with single-nucleotide polymorphisms on chromosome 6

    PubMed Central

    Wilcox, Marsha A; McAfee, Andrew T

    2007-01-01

    Rheumatoid arthritis (RA) is a disorder with important public health implications. It is possible that there are clinically distinctive subtypes of the disorder with different genetic etiologies. We used the data provided to the participants in the Genetic Analysis Workshop 15 to evaluate and describe clinically based subgroups and their genetic associations with single-nucleotide polymorphisms (SNPs) on chromosome 6, which harbors the HLA region. Detailed two- and three-SNP haplotype analyses were conducted in the HLA region. We used demographic, clinical self-report, and biomarker data from the entire sample (n = 8477) to identify and characterize the subgroups. We did not use the RA diagnosis itself in the identification of the subgroups. Nuclear families (715 families, 1998 individuals) were used to examine the genetic association with the HLA region. We found five distinct subgroups in the data. The first comprised unaffected family members. Cluster 2 was a mix of affected and unaffected in which patients endorsed symptoms not corroborated by physicians. Clusters 3 through 5 represented a severity continuum in RA. Cluster 5 was characterized by early onset severe disease. Cluster 2 showed no association on chromosome 6. Clusters 3 through 5 showed association with 17 SNPs on chromosome 6. In the HLA region, Cluster 3 showed single-, two-, and three-SNP association with the centromeric side of the region in an area of linkage disequilibrium. Cluster 5 showed both single- and two-SNP association with the telomeric side of the region in a second area of linkage disequilibrium. It will be important to replicate the subgroup structure and the association findings in an independent sample. PMID:18466517

  11. Characterization of single-nucleotide-polymorphism markers for Plasmopara viticola, the causal agent of grapevine downy mildew.

    PubMed

    Delmotte, F; Machefer, V; Giresse, X; Richard-Cervera, S; Latorse, M P; Beffa, R

    2011-11-01

    We report 34 new nuclear single-nucleotide-polymorphism (SNP) markers that have been developed from an expressed sequence tag library of Plasmopara viticola, the causal agent of grapevine downy mildew. This newly developed battery of markers will provide useful additional genetic tools for population genetic studies of this important agronomic species. PMID:21926208

  12. Single nucleotide polymorphisms in candidate genes associated with fertilizing ability of sperm and subsequent embryonic development in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fertilization and development of the preimplantation embryo is under genetic control. The goal of the current study was to test 434 single nucleotide polymorphisms (SNPs) for association with genetic variation in fertilization and early embryonic development. The approach was to produce embryos from...

  13. High-throughput single nucleotide polymorphism genotyping for breeding applications in rice using the BeadXpress platform

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiplexed single nucleotide polymorphism (SNP) markers have the potential to increase the speed and cost-effectiveness of genotyping, provided that an optimal SNP density is used for each application. To test the efficiency of multiplexed SNP genotyping for diversity, mapping and breeding applicat...

  14. Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Identification of single nucleotide polymorphisms (SNPs) for specific genes involved in reproduction might improve reliability of genomic estimates for these low- heritability traits. Semen from 550 Holstein bulls of high (>= 1.7; n=288) or low (<= -2; n = 262) daughter pregnancy rate (DPR) was geno...

  15. Single Nucleotide Polymorphisms in ABCG5 and ABCG8 are associated with changes in cholestrol metabolism during weight loss

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: To examine whether changes in cholesterol lowering and metabolism after weight loss were affected by single nucleotide polymorphisms (SNPs) in ABCG5 and ABCG8 genes. Methods and Results: Thirty-five hypercholesterolemic women lost 11.7 +/- 2.5 kg (P<0.001). Cholesterol kinetics were ass...

  16. Use of the Illumina GoldenGate assay for single nucleotide polymorphism (SNP) genotyping in cereal crops

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The highly parallel genotyping assay, such as the GoldenGate assay developed by Illumina, capable of interrogating up to 3,072 single nucleotide polymorphisms (SNPs) simultaneously, has greatly facilitated the genome-wide studies particularly for crops with large and complex genome structures. In th...

  17. Characterization of polyploid wheat genomic diversity using a high-density 90 000 single nucleotide polymorphism array

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High-density single nucleotide polymorphism (SNP) genotyping chips are a powerful tool for studying genomic patterns of diversity, inferring ancestral relationships among individuals in populations and studying marker-trait associations in mapping experiments. We developed a genotyping array includ...

  18. Empirical Comparison of Simple Sequence Repeats and Single Nucleotide Polymorphisms in Assessment of Maize Diversity and Relatedness

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While Simple Sequence Repeats (SSRs) are extremely useful genetic markers, recent advances in technology have produced a shift toward use of single nucleotide polymorphisms (SNPs). The different mutational properties of these two classes of markers result in differences in heterozygosities and allel...

  19. Ultrahigh-density linkage map for cultivated cucumber (Cucumis sativus L.) using a single-nucleotide polymorphism genotyping array

    Technology Transfer Automated Retrieval System (TEKTRAN)

    With the low cost of single nucleotide polymorphism (SNP) discovery, use of SNP markers for SNP array development is becoming more affordable. The SNP array is a very useful tool for high throughput genotyping and has a number of applications such as genome-wide association studies (GWAS). Since the...

  20. Single nucleotide polymorphisms in uracil-processing genes, intake of one-carbon nutrients and breast cancer risk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background/Objectives: The misincorporation of uracil into DNA leads to genomic instability. In a previous study, some of us identified four common single nucleotide polymorphisms (SNPs) in uracil-processing genes (rs2029166 and rs7296239 in SMUG1, rs34259 in UNG and rs4775748 in DUT) that were asso...

  1. Effects of bovine cytochrome P450 single nucleotide polymorphism, forage type, and body condition on production traits in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Relating single nucleotide polymorphisms (SNP) to cows with acceptable productivity could benefit cattle breeders especially in areas where tall fescue is the predominant forage. This study aimed to 1) identify SNPs in bovine cytochrome P450 3A28 (CYP3A28) and 2) determine associations between SNP g...

  2. A ferrofluid-based homogeneous assay for highly sensitive and selective detection of single-nucleotide polymorphisms.

    PubMed

    Shen, Wei; Lim, Cai Le; Gao, Zhiqiang

    2013-09-21

    A simple and low-cost colorimetric assay utilizing ferrofluidic nanoparticulate probes (FNPs) and a ligase for single-nucleotide polymorphism genotyping is described. Excellent sensitivity and selectivity were accomplished through the engagement of the FNPs and a ligase chain reaction. PMID:23923128

  3. Assessing the association of single nucleotide polymorphisms at the thyroglobulin gene with carcass traits in beef cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to assess the association of single nucleotide polymorphisms in the thyroglobulin gene, including a previously reported marker in current industry use, with marbling score in beef cattle. Three populations, designated GPE6, GPE7, and GPE8, were studied. The GPE6 pop...

  4. Association of a single nucleotide polymorphism (SNP) of calpain 1 (CAPN1) gene with meat tenderness of yak.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The association of a single nucleotide polymorphism (SNP) of calpain 1 (CAPN1) gene with shear force of 2.54 cm steaks from M. longissimus dorsi from Gannan yaks (Bos grunniens, n=181) was studied. Yaks were harvested at 2, 3, and 4 yr of age (n=51, 59, and 71, respectively), and samples of each yak...

  5. Comparison of single nucleotide polymorphisms and simple sequence repeats in genotype identification and diversity assessment of cacao germplasm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Accurate identification of individual genotypes in an efficient manner is especially important for cacao (Theobroma cacao L.) germplasm conservation and breeding. The development of single nucleotide polymorphism (SNP) markers in cacao offers the opportunity to use a high throughput genotyping syste...

  6. A high-density simple sequence repeat and single nucleotide polymorphism genetic map of the tetraploid cotton genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cotton genome complexity was investigated with a saturated molecular genetic map that combined several sets of microsatellites or simple sequence repeats (SSR) and the first major public set of single nucleotide polymorphism (SNP) markers in cotton genomes (Gossypium spp.), and that was constructed ...

  7. Detection of single-nucleotide variations by monitoring the blinking of fluorescence induced by charge transfer in DNA.

    PubMed

    Kawai, Kiyohiko; Majima, Tetsuro; Maruyama, Atsushi

    2013-08-19

    Charge transfer dynamics in DNA: Photo-induced charge separation and charge-recombination dynamics in DNA was assessed by monitoring the blinking of fluorescence. Single nucleotide variations, mismatch and one base deletion, were differentiated based on the length of the off-time of the blinking, which corresponds to the lifetime of the charge-separated state. PMID:23846860

  8. Species diagnostic single-nucleotide polymorphism and sequence-tagged site markers for the parasitic WASP Genus Nasonia (Hymenoptera: Ptermalidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed, identified and evaluated eight single nucleotide polymorphism (SNP) and three sequence-tagged site (STS) markers in nuclear gene sequences of the wasp genus Nasonia (Hymenoptera). We studied variation of these markers in natural populations of the closely related and regionally sympatr...

  9. Selection of single nucleotide polymorphisms and genotype quality for genomic prediction of genetic merit in dairy cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A process to prepare high-density genotypic data for use in genomic prediction of genetic merit was developed. Marker genotypes from over 51,000 single nucleotide polymorphisms (SNP) were generated for 3,139 Holstein bulls on the Illumina Bovine SNP50™ chip. The SNP were categorized by minor allele ...

  10. Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array.

    PubMed

    Fuller, Carl W; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Bibillo, Arek; Stranges, P Benjamin; Dorwart, Michael; Tao, Chuanjuan; Li, Zengmin; Guo, Wenjing; Shi, Shundi; Korenblum, Daniel; Trans, Andrew; Aguirre, Anne; Liu, Edward; Harada, Eric T; Pollard, James; Bhat, Ashwini; Cech, Cynthia; Yang, Alexander; Arnold, Cleoma; Palla, Mirkó; Hovis, Jennifer; Chen, Roger; Morozova, Irina; Kalachikov, Sergey; Russo, James J; Kasianowicz, John J; Davis, Randy; Roever, Stefan; Church, George M; Ju, Jingyue

    2016-05-10

    DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5'-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods. PMID:27091962

  11. Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

    PubMed Central

    Fuller, Carl W.; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Bibillo, Arek; Stranges, P. Benjamin; Dorwart, Michael; Tao, Chuanjuan; Li, Zengmin; Guo, Wenjing; Shi, Shundi; Korenblum, Daniel; Trans, Andrew; Aguirre, Anne; Liu, Edward; Harada, Eric T.; Pollard, James; Bhat, Ashwini; Cech, Cynthia; Yang, Alexander; Arnold, Cleoma; Palla, Mirkó; Hovis, Jennifer; Chen, Roger; Morozova, Irina; Kalachikov, Sergey; Russo, James J.; Kasianowicz, John J.; Davis, Randy; Roever, Stefan; Church, George M.; Ju, Jingyue

    2016-01-01

    DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods. PMID:27091962

  12. Varietal identification of tea (Camellia sinensis) using nanofluidic array of single nucleotide polymorphism (SNP) markers

    PubMed Central

    Fang, Wan-Ping; Meinhardt, Lyndel W; Tan, Hua-Wei; Zhou, Lin; Mischke, Sue; Zhang, Dapeng

    2014-01-01

    Apart from water, tea is the world’s most widely consumed beverage. Tea is produced in more than 50 countries with an annual production of approximately 4.7 million tons. The market segment for specialty tea has been expanding rapidly owing to increased demand, resulting in higher revenues and profits for tea growers and the industry. Accurate varietal identification is critically important to ensure traceability and authentication of premium tea products, which in turn contribute to on-farm conservation of tea genetic diversity. Using a set of single nucleotide polymorphism (SNP) markers developed from the expressed sequence tag (EST) database of Camilla senensis, we genotyped deoxyribonucleic acid (DNA) samples extracted from a diverse group of tea varieties, including both fresh and processed commercial loose-leaf teas. The validation led to the designation of 60 SNPs that unambiguously identified all 40 tested tea varieties with high statistical rigor (p<0.0001). Varietal authenticity and genetic relationships among the analyzed cultivars were further characterized by ordination and Bayesian clustering analysis. These SNP markers, in combination with a high-throughput genotyping protocol, effectively established and verified specific DNA fingerprints for all tested tea varieties. This method provides a powerful tool for variety authentication and quality control for the tea industry. It is also highly useful for the management of tea genetic resources and breeding, where accurate and efficient genotype identification is essential. PMID:26504544

  13. A STAT6 Intronic Single-Nucleotide Polymorphism is Associated with Clinical Malaria in Ghanaian Children

    PubMed Central

    Amoako-Sakyi, Daniel; Adukpo, Selorme; Kusi, Kwadwo A.; Dodoo, Daniel; Ofori, Michael F.; Adjei, George O.; Edoh, Dominic E.; Asmah, Richard H.; Brown, Charles; Adu, Bright; Obiri-Yeboah, Dorcas; Futagbi, Godfred; Abubakari, Sharif Buari; Troye-Blomberg, Marita; Akanmori, Bartholomew D.; Goka, Bamenla Q.; Arko-Mensah, John; Gyan, Ben A.

    2016-01-01

    Malaria pathogenesis may be influenced by IgE responses and cytokine cross-regulation. Several mutations in the IL-4/STAT6 signaling pathway can alter cytokine cross-regulation and IgE responses during a Plasmodium falciparum malarial infection. This study investigated the relationship between a STAT6 intronic single-nucleotide polymorphism (rs3024974), total IgE, cytokines, and malaria severity in 238 Ghanaian children aged between 0.5 and 13 years. Total IgE and cytokine levels were measured by ELISA, while genotyping was done by polymerase chain reaction-restriction fragment length polymorphism (RFLP). Compared with healthy controls, heterozygosity protected against clinical malaria: uncomplicated malaria (odds ratios [OR] = 0.13, P < 0.001), severe malarial anemia (OR = 0.18, P < 0.001), and cerebral malaria (OR = 0.39, P = 0.022). Levels of total IgE significantly differed among malaria phenotypes (P = 0.044) and rs3024974 genotypes (P = 0.037). Neither cytokine levels nor IL-6/IL-10 ratios were associated with malaria phenotypes or rs3024974 genotypes. This study suggests a role for rs3024974 in malaria pathogenesis and offers further insights into an IL-4/STAT6 pathway mutation in malaria pathogenesis. PMID:27279750

  14. Human Aldo-Keto Reductases: Function, Gene Regulation, and Single Nucleotide Polymorphisms

    PubMed Central

    Penning, Trevor M.; Drury, Jason E.

    2007-01-01

    Aldo-Keto Reductases (AKRs) are a superfamily of NAD(P)H linked oxidoreductases that are generally monomeric 34- 37 kDa proteins present in all phyla. The superfamily consists of 15 families, which contains 151 members (www.med.upenn.edu/akr). Thirteen human AKRs exist that use endogenous substrates (sugar and lipid aldehydes, prostaglandins, retinals and steroid hormones), and in many instances they regulate nuclear receptor signaling. Exogenous substrates include metabolites implicated in chemical carcinogenesis: NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), polycyclic aromatic hydrocarbon trans-dihydrodiols, and aflatoxin dialdehyde. Promoter analysis of the human genes identifies common elements involved in their regulation which include osmotic response elements, antioxidant response elements, xenobiotic response elements, AP-1 sites and steroid response elements. The human AKRs are highly polymorphic, and in some instances single nucleotide polymorphisms (SNPs) of high penetrance exist. This suggests that there will be inter-individual variation in endogenous and xenobiotic metabolism which in turn affect susceptibility to nuclear receptor signaling and chemical carcinogenesis. PMID:17537398

  15. CASP3 gene single-nucleotide polymorphism (rs72689236) and Kawasaki disease in Taiwanese children.

    PubMed

    Kuo, Ho-Chang; Yu, Hong-Ren; Juo, Suh-Hang Hank; Yang, Kuender D; Wang, Yu-Shiuan; Liang, Chi-Di; Chen, Wei-Chiao; Chang, Wei-Pin; Huang, Chien-Fu; Lee, Chiu-Ping; Lin, Li-Yan; Liu, Yu-Chen; Guo, Yuh-Cherng; Chiu, Chien-Chih; Chang, Wei-Chiao

    2011-02-01

    Kawasaki disease (KD) is characterized by systemic vasculitis of unknown etiology. A study from Japan reported that G to A substitution of a single-nucleotide polymorphism (SNP) located in the 5'-untranslated region of caspase 3 (CASP3) (rs72689236), which was associated with nuclear factor of activated T cell-mediated T-cell activation, is responsible for susceptibility to KD. This study was conducted to investigate whether the polymorphism of CASP3 is responsible for susceptibility and coronary artery lesion (CAL) formation in KD in the Taiwanese population. A total of 1092 subjects (341 KD patients and 751 controls) were investigated to identify an SNP of rs72689236 using Invader assays (Third Wave Technologies). Our data provided a borderline significant association between the genotypes and allele frequency of rs72689236 in control subjects and KD patients (P=0.0535 under the dominant model; P=0.0575 under the allelic model). The A allele of rs72689236 in KD patients and in patients with CAL and intravenous immunoglobulin resistance was seen in a higher frequency. Importantly, a significant association was obtained between rs72689236 and KD patients with aneurysm formation (P=0.009, under the recessive model). The A allele of rs72689236 is very likely to be a risk allele in the development of aneurysm in patients with KD. PMID:21160486

  16. Single nucleotide polymorphism in the neuroplastin locus associates with cortical thickness and intellectual ability in adolescents

    PubMed Central

    Desrivières, S; Lourdusamy, A; Tao, C; Toro, R; Jia, T; Loth, E; Medina, L M; Kepa, A; Fernandes, A; Ruggeri, B; Carvalho, F M; Cocks, G; Banaschewski, T; Barker, G J; Bokde, A L W; Büchel, C; Conrod, P J; Flor, H; Heinz, A; Gallinat, J; Garavan, H; Gowland, P; Brühl, R; Lawrence, C; Mann, K; Martinot, M L P; Nees, F; Lathrop, M; Poline, J-B; Rietschel, M; Thompson, P; Fauth-Bühler, M; Smolka, M N; Pausova, Z; Paus, T; Feng, J; Schumann, G

    2015-01-01

    Despite the recognition that cortical thickness is heritable and correlates with intellectual ability in children and adolescents, the genes contributing to individual differences in these traits remain unknown. We conducted a large-scale association study in 1583 adolescents to identify genes affecting cortical thickness. Single-nucleotide polymorphisms (SNPs; n=54 837) within genes whose expression changed between stages of growth and differentiation of a human neural stem cell line were selected for association analyses with average cortical thickness. We identified a variant, rs7171755, associating with thinner cortex in the left hemisphere (P=1.12 × 10−7), particularly in the frontal and temporal lobes. Localized effects of this SNP on cortical thickness differently affected verbal and nonverbal intellectual abilities. The rs7171755 polymorphism acted in cis to affect expression in the human brain of the synaptic cell adhesion glycoprotein-encoding gene NPTN. We also found that cortical thickness and NPTN expression were on average higher in the right hemisphere, suggesting that asymmetric NPTN expression may render the left hemisphere more sensitive to the effects of NPTN mutations, accounting for the lateralized effect of rs7171755 found in our study. Altogether, our findings support a potential role for regional synaptic dysfunctions in forms of intellectual deficits. PMID:24514566

  17. A high-density single nucleotide polymorphism map for Neurospora crassa.

    PubMed

    Lambreghts, Randy; Shi, Mi; Belden, William J; Decaprio, David; Park, Danny; Henn, Matthew R; Galagan, James E; Bastürkmen, Meray; Birren, Bruce W; Sachs, Matthew S; Dunlap, Jay C; Loros, Jennifer J

    2009-02-01

    We report the discovery and validation of a set of single nucleotide polymorphisms (SNPs) between the reference Neurospora crassa strain Oak Ridge and the Mauriceville strain (FGSC 2555), of sufficient density to allow fine mapping of most loci. Sequencing of Mauriceville cDNAs and alignment to the completed genomic sequence of the Oak Ridge strain identified 19,087 putative SNPs. Of these, a subset was validated by cleaved amplified polymorphic sequence (CAPS), a simple and robust PCR-based assay that reliably distinguishes between SNP alleles. Experimental confirmation resulted in the development of 250 CAPS markers distributed evenly over the genome. To demonstrate the applicability of this map, we used bulked segregant analysis followed by interval mapping to locate the csp-1 mutation to a narrow region on LGI. Subsequently, we refined mapping resolution to 74 kbp by developing additional markers, resequenced the candidate gene, NCU02713.3, in the mutant background, and phenocopied the mutation by gene replacement in the WT strain. Together, these techniques demonstrate a generally applicable and straightforward approach for the isolation of novel genes from existing mutants. Data on both putative and validated SNPs are deposited in a customized public database at the Broad Institute, which encourages augmentation by community users. PMID:19015548

  18. Single nucleotide polymorphisms across a species' range: implications for conservation studies of Pacific salmon.

    PubMed

    Seeb, L W; Templin, W D; Sato, S; Abe, S; Warheit, K; Park, J Y; Seeb, J E

    2011-03-01

    Studies of the oceanic and near-shore distributions of Pacific salmon, whose migrations typically span thousands of kilometres, have become increasingly valuable in the presence of climate change, increasing hatchery production and potentially high rates of bycatch in offshore fisheries. Genetics data offer considerable insights into both the migratory routes as well as the evolutionary histories of the species. However, these types of studies require extensive data sets from spawning populations originating from across the species' range. Single nucleotide polymorphisms (SNPs) have been particularly amenable for multinational applications because they are easily shared, require little interlaboratory standardization and can be assayed through increasingly efficient technologies. Here, we discuss the development of a data set for 114 populations of chum salmon through a collaboration among North American and Asian researchers, termed PacSNP. PacSNP is focused on developing the database and applying it to problems of international interest. A data set spanning the entire range of species provides a unique opportunity to examine patterns of variability, and we review issues associated with SNP development. We found evidence of ascertainment bias within the data set, variable linkage relationships between SNPs associated with ancestral groupings and outlier loci with alleles associated with latitude. PMID:21429175

  19. A molecular beacon microarray based on a quantum dot label for detecting single nucleotide polymorphisms.

    PubMed

    Guo, Qingsheng; Bai, Zhixiong; Liu, Yuqian; Sun, Qingjiang

    2016-03-15

    In this work, we report the application of streptavidin-coated quantum dot (strAV-QD) in molecular beacon (MB) microarray assays by using the strAV-QD to label the immobilized MB, avoiding target labeling and meanwhile obviating the use of amplification. The MBs are stem-loop structured oligodeoxynucleotides, modified with a thiol and a biotin at two terminals of the stem. With the strAV-QD labeling an "opened" MB rather than a "closed" MB via streptavidin-biotin reaction, a sensitive and specific detection of label-free target DNA sequence is demonstrated by the MB microarray, with a signal-to-background ratio of 8. The immobilized MBs can be perfectly regenerated, allowing the reuse of the microarray. The MB microarray also is able to detect single nucleotide polymorphisms, exhibiting genotype-dependent fluorescence signals. It is demonstrated that the MB microarray can perform as a 4-to-2 encoder, compressing the genotype information into two outputs. PMID:26397421

  20. From Single Nucleotide Polymorphisms to Constant Immunosuppression: Mesenchymal Stem Cell Therapy for Autoimmune Diseases

    PubMed Central

    Galipeau, Jacques; Nooka, Ajay K.

    2013-01-01

    The regenerative abilities and the immunosuppressive properties of mesenchymal stromal cells (MSCs) make them potentially the ideal cellular product of choice for treatment of autoimmune and other immune mediated disorders. Although the usefulness of MSCs for therapeutic applications is in early phases, their potential clinical use remains of great interest. Current clinical evidence of use of MSCs from both autologous and allogeneic sources to treat autoimmune disorders confers conflicting clinical benefit outcomes. These varied results may possibly be due to MSC use across wide range of autoimmune disorders with clinical heterogeneity or due to variability of the cellular product. In the light of recent genome wide association studies (GWAS), linking predisposition of autoimmune diseases to single nucleotide polymorphisms (SNPs) in the susceptible genetic loci, the clinical relevance of MSCs possessing SNPs in the critical effector molecules of immunosuppression is largely undiscussed. It is of further interest in the allogeneic setting, where SNPs in the target pathway of MSC's intervention may also modulate clinical outcome. In the present review, we have discussed the known critical SNPs predisposing to disease susceptibility in various autoimmune diseases and their significance in the immunomodulatory properties of MSCs. PMID:24350294

  1. Mining for single nucleotide polymorphisms and insertions / deletions in expressed sequence tag libraries of oil palm.

    PubMed

    Riju, Aykkal; Chandrasekar, Arumugam; Arunachalam, Vadivel

    2007-01-01

    The oil palm is a tropical oil bearing tree. Recently EST-derived SNPs and SSRs are a free by-product of the currently expanding EST (Expressed Sequence Tag) data bases. The development of high-throughput methods for the detection of SNPs (Single Nucleotide Polymorphism) and small indels (insertion / deletion) has led to a revolution in their use as molecular markers. Available (5452) Oil palm EST sequences were mined from dbEST of NCBI. CAP3 program was used to assemble EST sequences into contigs. Candidate SNPs and Indel polymorphisms were detected using the perl script auto_snip version 1.0 which has used 576 ESTs for detecting SNPs and Indel sites. We found 1180 SNP sites and 137 indel polymorphisms with frequency 1.36 SNPs / 100 bp. Among the six tissues from which the EST libraries had been generated, mesocarp had high frequency of 2.91 SNPs and indels per 100 bp whereas the zygotic embryos had lowest frequency of 0.15 per 100 bp. We also used the Shannon index to analyze the proportion of ten possible types of SNP/indels. ESTs from tissues of normal apex showed highest values of Shannon index (0.60) whereas abnormal apex had least value (0.02). The present report deals the use of Shannon index for comparing SNP/ indel frequencies mined from ESTlibraries and also confirm that the frequency of SNP occurrence in oil palm to use them as markers for genetic studies. PMID:21670789

  2. High-density single-nucleotide polymorphism maps of the human genome

    PubMed Central

    Miller, Raymond D.; Phillips, Michael S.; Jo, Inho; Donaldson, Miriam A.; Studebaker, Joel F.; Addleman, Nicholas; Alfisi, Steven V.; Ankener, Wendy M.; Bhatti, Hamid A.; Callahan, Chad E.; Carey, Benjamin J.; Conley, Cheryl L.; Cyr, Justin M.; Derohannessian, Vram; Donaldson, Rachel A.; Elosua, Carolina; Ford, Stacey E.; Forman, Angela M.; Gelfand, Craig A.; Grecco, Nicole M.; Gutendorf, Susan M.; Hock, Cricket R.; Hozza, Mark J.; Hur, Soyoung; In, Sun Mi; Jackson, Diana L.; Jo, Sangmee Ahn; Jung, Sung-Chul; Kim, Sook; Kimm, Kuchan; Kloss, Ellen F.; Koboldt, Daniel C.; Kuebler, Jennifer M.; Kuo, Feng-Shen; Lathrop, Jessica A.; Lee, Jong-Keuk; Leis, Kathy L.; Livingston, Stephanie A.; Lovins, Elizabeth G.; Lundy, Maria L.; Maggan, Sima; Minton, Matthew; Mockler, Michael A.; Morris, David W.; Nachtman, Eric P.; Oh, Bermseok; Park, Chan; Park, Chang-Wook; Pavelka, Nicholas; Perkins, Adrienne B.; Restine, Stephanie L.; Sachidanandam, Ravi; Reinhart, Andrew J.; Scott, Kathryn E.; Shah, Gira J.; Tate, Jatana M.; Varde, Shobha A.; Walters, Amy; White, J. Rebecca; Yoo, Yeon-Kyeong; Lee, Jong-Eun; Boyce-Jacino, Michael T.; Kwok, Pui-Yan

    2007-01-01

    Here we report a large, extensively characterized set of single-nucleotide polymorphisms (SNPs) covering the human genome. We determined the allele frequencies of 55,018 SNPs in African Americans, Asians (Japanese–Chinese), and European Americans as part of The SNP Consortium’s Allele Frequency Project. A subset of 8333 SNPs was also characterized in Koreans. Because these SNPs were ascertained in the same way, the data set is particularly useful for modeling. Our results document that much genetic variation is shared among populations. For autosomes, some 44% of these SNPs have a minor allele frequency ≥10% in each population, and the average allele frequency differences between populations with different continental origins are less than 19%. However, the several percentage point allele frequency differences among the closely related Korean, Japanese, and Chinese populations suggest caution in using mixtures of well-established populations for case–control genetic studies of complex traits. We estimate that ~7% of these SNPs are private SNPs with minor allele frequencies <1%. A useful set of characterized SNPs with large allele frequency differences between populations (>60%) can be used for admixture studies. High-density maps of high-quality, characterized SNPs produced by this project are freely available. PMID:15961272

  3. Identification, validation and survey of a single nucleotide polymorphism (SNP) associated with pungency in Capsicum spp.

    PubMed

    Garcés-Claver, Ana; Fellman, Shanna Moore; Gil-Ortega, Ramiro; Jahn, Molly; Arnedo-Andrés, María S

    2007-11-01

    A single nucleotide polymorphism (SNP) associated with pungency was detected within an expressed sequence tag (EST) of 307 bp. This fragment was identified after expression analysis of the EST clone SB2-66 in placenta tissue of Capsicum fruits. Sequence alignments corresponding to this new fragment allowed us to identify an SNP between pungent and non-pungent accessions. Two methods were chosen for the development of the SNP marker linked to pungency: tetra-primer amplification refractory mutation system-PCR (tetra-primer ARMS-PCR) and cleaved amplified polymorphic sequence. Results showed that both methods were successful in distinguishing genotypes. Nevertheless, tetra-primer ARMS-PCR was chosen for SNP genotyping because it was more rapid, reliable and less cost-effective. The utility of this SNP marker for pungency was demonstrated by the ability to distinguish between 29 pungent and non-pungent cultivars of Capsicum annuum. In addition, the SNP was also associated with phenotypic pungent character in the tested genotypes of C. chinense, C. baccatum, C. frutescens, C. galapagoense, C. eximium, C. tovarii and C. cardenasi. This SNP marker is a faster, cheaper and more reproducible method for identifying pungent peppers than other techniques such as panel tasting, and allows rapid screening of the trait in early growth stages. PMID:17882396

  4. Analysis of endogenous nucleotides by single cell capillary electrophoresis-mass spectrometry

    PubMed Central

    Liu, Jing-Xin; Aerts, Jordan T.; Rubakhin, Stanislav S.; Zhang, Xin-Xiang; Sweedler, Jonathan V.

    2015-01-01

    Analytical technologies that enable investigations at the single cell level facilitate a range of studies; here a lab-fabricated capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) platform was used to analyze anionic metabolites from individual Aplysia californica neurons. The system employs a customized coaxial sheath-flow nanospray interface connected to a separation capillary, with the sheath liquid and separation buffer optimized to ensure a stable spray. The method provided good repeatability of separation and reliable detection sensitivity for 16 mono-, di- and triphosphate nucleosides. For a range of anionic analytes, including cyclic adenosine monophosphate (cAMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP), the detection limits were in the low nanomolar range (<22 nM). A large Aplysia R2 neuron was used to demonstrate the ability of CE-ESI-MS to quantitatively characterize anionic metabolites within individual cells, with 15 nucleotides and derivatives detected. Following the method validation process, we probed smaller, 60-μm diameter Aplysia sensory neurons where sample stacking was used as a simple on-line analyte preconcentration approach. The calculated energy balance ([ATP] + 0.5 × [ADP])/([AMP] + [ADP] + [ATP]) of these cells was comparable with the value obtained from bulk samples. PMID:25212237

  5. Single nucleotide polymorphisms to discriminate different classes of hybrid between wild Atlantic salmon and aquaculture escapees.

    PubMed

    Pritchard, Victoria L; Erkinaro, Jaakko; Kent, Matthew P; Niemelä, Eero; Orell, Panu; Lien, Sigbjørn; Primmer, Craig R

    2016-09-01

    Many wild Atlantic salmon (Salmo salar) populations are threatened by introgressive hybridization from domesticated fish that have escaped from aquaculture facilities. A detailed understanding of the hybridization dynamics between wild salmon and aquaculture escapees requires discrimination of different hybrid classes; however, markers currently available to discriminate the two types of parental genome have limited power to do this. Using a high-density Atlantic salmon single nucleotide polymorphism (SNP) array, in combination with pooled-sample allelotyping and an Fst outlier approach, we identified 200 SNPs that differentiated an important Atlantic salmon stock from the escapees potentially hybridizing with it. By simulating multiple generations of wild-escapee hybridization, involving wild populations in two major phylogeographic lineages and a genetically diverse set of escapees, we showed that both the complete set of SNPs and smaller subsets could reliably assign individuals to different hybrid classes up to the third hybrid (F3) generation. This set of markers will be a useful tool for investigating the genetic interactions between native wild fish and aquaculture escapees in many Atlantic salmon populations. PMID:27606009

  6. Genomewide linkage scan of schizophrenia in a large multicenter pedigree sample using single nucleotide polymorphisms

    PubMed Central

    Holmans, PA; Riley, B; Pulver, AE; Owen, MJ; Wildenauer, DB; Gejman, PV; Mowry, BJ; Laurent, C; Kendler, KS; Nestadt, G; Williams, NM; Schwab, SG; Sanders, AR; Nertney, D; Mallet, J; Wormley, B; Lasseter, VK; O’Donovan, MC; Duan, J; Albus, M; Alexander, M; Godard, S; Ribble, R; Liang, KY; Norton, N; Maier, W; Papadimitriou, G; Walsh, D; Jay, M; O’Neill, A; Lerer, FB; Dikeos, D; Crowe, RR; Silverman, JM; Levinson, DF

    2008-01-01

    A genomewide linkage scan was carried out in eight clinical samples of informative schizophrenia families. After all quality control checks, the analysis of 707 European-ancestry families included 1,615 affected and 1,602 unaffected genotyped individuals, and the analysis of all 807 families included 1900 affected and 1839 unaffected individuals. Multipoint linkage analysis with correction for marker-marker linkage disequilibrium was carried out with 5,861 single nucleotide polymorphisms (SNPs; Illumina 4.0 linkage map). Suggestive evidence for linkage (European families) was observed on chromosomes 8p21, 8q24.1, 9q34 and 12q24.1 in non-parametric and/or parametric analyses. In a logistic regression allele-sharing analysis of linkage allowing for intersite heterogeneity, genomewide significant evidence for linkage was observed on chromosome 10p12. Significant heterogeneity was also observed on chromosome 22q11.1. Evidence for linkage across family sets and analyses was most consistent on chromosome 8p21, with a one-lod support interval that does not include the candidate gene NRG1, suggesting that one or more other susceptibility loci might exist in the region. In this era of genomewide association and deep resequencing studies, consensus linkage regions deserve continued attention, given that linkage signals can be produced by many types of genomic variation, including any combination of multiple common or rare SNPs or copy number variants in a region. PMID:19223858

  7. Single nucleotide polymorphisms of TNFAIP3 are associated with systemic lupus erythematosus in Han Chinese population.

    PubMed

    Han, J-W; Wang, Y; Li, H-B; Alateng, C; Bai, Y-H; Sun, Z-Q; Lv, X-X; Wu, R-N

    2016-04-01

    The polymorphisms of tumour necrosis factor alpha-induced protein 3 (TNFAIP3) have been found to associate with several autoimmune diseases. This study aimed to explore the association of single nucleotide polymorphisms (SNPs) of TNFAIP3 gene with systemic lupus erythematosus (SLE) in Han Chinese. Thirty-two SNPs were genotyped in 284 patients with SLE and 630 controls using the ligation detection reaction (LDR) method. The quality control steps and statistical analyses were performed using the plink 1.07 package and haploview software. We found that 13 SNPs in TNFAIP3 showed significant association with SLE (P < 1.85 × 10(-3) ), and all of them were in high linkage disequilibrium (LD). After conditioning on the SNP rs2230926, other 12 SNPs did not show association (P > 0.27). All 13 SNPs showed most significant association in the dominant model. In haplotype analysis, a long risk SNP haplotype (GCCCGTGTCATGG) showed most significant association (P = 1.00 × 10(-4) ). In conclusion, our data suggest that TNFAIP3 is a susceptible gene for SLE in the Han Chinese population. PMID:26846592

  8. Development of a cassava core collection based on single nucleotide polymorphism markers.

    PubMed

    Oliveira, E J; Ferreira, C F; Santos, V S; Oliveira, G A F

    2014-01-01

    Single nucleotide polymorphism (SNP) markers were used in the largest cassava (Manihot esculenta Crantz) germplasm collection from Brazil to develop core collections based on the maximization strategy. Subsets with 61, 64, 84, 128, 256, and 384 cassava accessions were selected and named PoHEU, MST64, PoRAN, MST128, MST256, and MST384, respectively. All the 798 alleles identified by 402 SNP markers in the entire collection were captured in all core collections. Only small alterations in the diversity parameters were observed for the different core collections compared with the complete collection. Because of the optimal adjustment of the validation parameters representative of the complete collection, the absence of genotypes with high genetic similarity and the maximization of the genetic distances between accessions of the PoHEU core collection, which contained 4.7% of the accessions of the complete collection, maximized the genetic conservation of this important cassava collection. Furthermore, the development of this core collection will allow concentrated efforts toward future characterization and agronomic evaluation of accessions to maximize the diversity and genetic gains in cassava breeding programs. PMID:25158266

  9. Single nucleotide polymorphism-based microarray analysis for the diagnosis of hydatidiform moles.

    PubMed

    Xie, Yingjun; Pei, Xiaojuan; Dong, Yu; Wu, Huiqun; Wu, Jianzhu; Shi, Huijuan; Zhuang, Xuying; Sun, Xiaofang; He, Jialing

    2016-07-01

    In clinical diagnostics, single nucleotide polymorphism (SNP)-based microarray analysis enables the detection of copy number variations (CNVs), as well as copy number neutral regions, that are absent of heterozygosity throughout the genome. The aim of the present study was to evaluate the effectiveness and sensitivity of SNP‑based microarray analysis in the diagnosis of hydatidiform mole (HM). By using whole‑genome SNP microarray analysis, villous genotypes were detected, and the ploidy of villous tissue was determined to identify HMs. A total of 66 villous tissues and two twin tissues were assessed in the present study. Among these samples, 11 were triploid, one was tetraploid, 23 were abnormal aneuploidy, three were complete genome homozygosity, and the remaining ones were normal ploidy. The most noteworthy finding of the present study was the identification of six partial HMs and three complete HMs from those samples that were not identified as being HMs on the basis of the initial diagnosis of experienced obstetricians. This study has demonstrated that the application of an SNP‑based microarray analysis was able to increase the sensitivity of diagnosis for HMs with partial and complete HMs, which makes the identification of these diseases at an early gestational age possible. PMID:27151252

  10.  Monozygotic twins with NASH cirrhosis: cumulative effect of multiple single nucleotide polymorphisms?

    PubMed

    Grove, Jane I; Austin, Mark; Tibble, Jeremy; Aithal, Guruprasad P; Verma, Sumita

    2016-01-01

     Multiple genetic and environmental factors interact to determine an individual's predisposition to non-alcoholic fatty liver disease and its phenotypic characteristics. Association studies have found a number of alleles associated with the development of non-alcoholic steatohepatitis. Our aim was to investigate whether multiple risk-associated alleles may be present in affected monozygotic twins, indicating underlying genetic predisposition to non-alcoholic steatohepatitis. We determined the genotype of 14 candidate gene polymorphisms (at 11 unlinked loci) in a set of monozygotic twins who presented with cirrhosis within 18 months of each other. Genotyping revealed multiple single nucleotide polymorphisms at 9 independent loci in genes PNPLA3, APOC3, GCKR, TRIB1, LYPLAL1, PPP1R3B, COL13A1, and EFCAB4B, previously implicated in contributing to non-alcoholic steatohepatitis pathogenesis. In conclusion, this case series illustrates the potential cumulative effect of multiple polymorphisms in the development and potential progression of a complex trait such as NASH cirrhosis. PMID:26845607