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PubMed2001

The single strand conformational analysis of cattle and human single nucleotide polymorphisms may be biased towards specific sequence motifs that minimize local secondary structure of single strand DNA.

Single strand conformational polymorphisms (SSCP) resulting from point mutations were found to be associated preferentially with two DNA sequence motifs. These motifs are (1) three or more of the same base but in which the polymorphism is not due to length variation and (2) a region of polypurine or polypyrimidine bases. These motifs were identified after SSCP alleles from cattle were sequenced. The sequence difference and flanking sequence for each single nucleotide polymorphism are shown. The motifs were also found in SSCP from humans chosen at random from the literature, in which the alleles had been sequenced. There is a low level of complementarity of adjacent bases in these motifs and they should represent regions of low secondary structure in the single stranded DNA. Regions of high secondary structure, such as palindromes, were found in the same sample to have allelic variation that was not detected by SSC analysis. These results give a rule of thumb for selecting the particular part of a DNA fragment to be selected for testing for polymorphisms, but this rule clashes with rules used to design primers to amplify sequences using the PCR, namely, minimise hydrogen bonding within and between primers and reduce self-complementarity.

Alleles↗

PubMed2005

Factor V G1691A (Leiden) and prothrombin G20210A single-nucleotide polymorphisms in type 2 diabetes mellitus.

The association of the single nucleotide polymorphisms (SNPs) G1691A in coagulation factor V (FV)-Leiden and G20210A in prothrombin (PRT) genes with type 2 diabetes mellitus (T2DM) were analyzed in 112 T2DM patients (58 males, 54 females; mean age 55.24 +/- 13.5 years) and 249 healthy control subjects (118 males, 131 females; mean age 53.03 +/- 13.8 years). No association was found for FV-Leiden with T2DM, as the frequency of the G/G (82.1% vs. 85.5%), G/A (17.0% vs. 14.1%), and A/A (0.9% vs. 0.4%) genotypes was not different between patients and controls, respectively (P = 0.644). Similarly, lack of association of PRT G20210A with T2DM was seen among the population studied, and the frequency of the G/G (92.9% vs. 97.2%), G/A (6.3% vs. 2.8%), and A/A (0.9% vs. 0.0%) genotypes was similar among patients and controls, respectively (P = 0.094). Neither FV-Leiden nor PRT G20210A was associated with, and no evidence for interactions between these mutations was seen in, T2DM.

DNA↗

PubMed2005

[Study on single nucleotide polymorphism of TLR4 in Chinese population].

OBJECTIVE: To identify the single nucleotide polymorphisms(SNPs) in the regulatory and coding regions of human Toll-like receptor 4(TLR4) gene and to search for its new genetic makers. METHODS: The 5' flank region, exons, parts of the introns, as well as 3' flank region of TLR4 gene were sequenced to identify and characterize the SNPs in Chinese population. SNP genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism for 2 highly distributed SNPs. RESULTS: Five novel SNPs were identified through a 4.98 kb sequencing of TLR4 gene. Among them, three were in 5'flank region, two in 3'UTR. In the sample of Han population from Chongqing, the minor allele frequencies of two highly distributed SNPs were 0.266 and 0.404 respectively. CONCLUSION: Sampling analysis in Han population of Chongqing showed that the two highly distributed SNPs of TLR4 were common in Chinese population and could be used for genetic marker of TLR4 gene.

Adult↗

PubMed2005

Accuracy of haplotype reconstruction from haplotype-tagging single-nucleotide polymorphisms.

Many investigators are now using haplotype-tagging single-nucleotide polymorphism (htSNPs) as a way of screening regions of the genome for association with disease. A common approach is to genotype htSNPs in a study population and to use this information to draw inferences about each individual's haplotypic makeup, including SNPs that were not directly genotyped. To test the validity of this approach, we simulated the exercise of typing htSNPs in a large sample of individuals and compared the true and inferred haplotypes. The accuracy of haplotype inference varied, depending on the method of selecting htSNPs, the linkage-disequilibrium structure of the region, and the amount of missing data. At the stage of selection of htSNPs, haplotype-block-based methods required a larger number of htSNPs than did unstructured methods but gave lower levels of error in haplotype inference, particularly when there was a significant amount of missing data. We present a Web-based utility that allows investigators to compare the likely error rates of different sets of htSNPs and to arrive at an economical set of htSNPs that provides acceptable levels of accuracy in haplotype inference.

Biometry↗

PubMed2003

Nonparametric disequilibrium mapping of functional sites using haplotypes of multiple tightly linked single-nucleotide polymorphism markers.

As the speed and efficiency of genotyping single-nucleotide polymorphisms (SNPs) increase, using the SNP map, it becomes possible to evaluate the extent to which a common haplotype contributes to the risk of disease. In this study we propose a new procedure for mapping functional sites or regions of a candidate gene of interest using multiple linked SNPs. Based on a case-parent trio family design, we use expectation-maximization (EM) algorithm-derived haplotype frequency estimates of multiple tightly linked SNPs from both unambiguous and ambiguous families to construct a contingency statistic S for linkage disequilibrium (LD) analysis. In the procedure, a moving-window scan for functional SNP sites or regions can cover an unlimited number of loci except for the limitation of computer storage. Within a window, all possible widths of haplotypes are utilized to find the maximum statistic S* for each site (or locus). Furthermore, this method can be applied to regional or genome-wide scanning for determining linkage disequilibrium using SNPs. The sensitivity of the proposed procedure was examined on the simulated data set from the Genetic Analysis Workshop (GAW) 12. Compared with the conventional and generalized TDT methods, our procedure is more flexible and powerful.

Algorithms↗

PubMed2002

Utility and accuracy of template-directed dye-terminator incorporation with fluorescence-polarization detection for genotyping single nucleotide polymorphisms.

There are little independent data available about how well single nucleotide polymorphism (SNP) genotyping technologies perform in the typical molecular genetics laboratory. We evaluated the utility and accuracy of a widely used technology, template-directed dye-terminator incorporation with fluorescence-polarization detection (FP-TDI), in a sample of 177 SNPs selected solely on the basis of map location. Genotypes were generated without optimization using standard protocols. Overall, 81% of the SNPs we studied generated readable genotypes by FP-TDI. Thirty-two SNPs were genotyped in duplicate by PCR-RFLP orfluorescent dye-terminator sequencing. Out of a total of 631 duplicate genotypes, no true discrepancies were detected. The true error rate has a 95% chance of lying between 0 and 6 out of 1000 genotypes. We also tested for deviations from Hardy-Weinberg Equilibrium in 33 SNPs genotyped in 50 unrelated individuals, and no significant deviations were detected. Our FP-TDI data were readily adaptable to automated genotype calling using our own method of cluster analysis, which assigns a probability score to each genotype call. We conclude that FP-TDI is both efficient and accurate. The method can easily fill the needs of SNP genotyping projects at the scale typically used for regional or candidate-gene association studies.

Fluorescence Polarization↗

PubMed2005

A comparison of five methods for selecting tagging single-nucleotide polymorphisms.

Our goal was to compare methods for tagging single-nucleotide polymorphisms (tagSNPs) with respect to the power to detect disease association under differing haplotype-disease association models. We were also interested in the effect that SNP selection samples, consisting of either cases, controls, or a mixture, would have on power. We investigated five previously described algorithms for choosing tagSNPS: two that picked SNPs based on haplotype structure (Chapman-haplotypic and Stram), two that picked SNPs based on pair-wise allelic association (Chapman-allelic and Cousin), and one control method that chose equally spaced SNPs (Zhai). In two disease-associated regions from the Genetic Analysis Workshop 14 simulated data, we tested the association between tagSNP genotype and disease over the tagSNP sets chosen by each method for each sampling scheme. This was repeated for 100 replicates to estimate power. The two allelic methods chose essentially all SNPs in the region and had nearly optimal power. The two haplotypic methods chose about half as many SNPs. The haplotypic methods had poor performance compared to the allelic methods in both regions. We expected an improvement in power when the selection sample contained cases; however, there was only moderate variation in power between the sampling approaches for each method. Finally, when compared to the haplotypic methods, the reference method performed as well or worse in the region with ancestral disease haplotype structure.

Genetic Techniques↗

PubMed2006

Candidate single nucleotide polymorphism selection using publicly available tools: a guide for epidemiologists.

Single nucleotide polymorphisms (SNPs) are the most common form of human genetic variation, with millions present in the human genome. Because only 1% might be expected to confer more than modest individual effects in association studies, the selection of predictive candidate variants for complex disease analyses is formidable. Technologic advances in SNP discovery and the ever-changing annotation of the genome have led to massive informational resources that can be difficult to master across disciplines. A simplified guide is needed. Although methods for evaluating nonsynonymous coding SNPs are known, several other publicly available computational tools can be utilized to assess polymorphic variants in noncoding regions. As an example, the authors applied multiple methods to select SNPs in DNA double-strand break repair genes. They chose to evaluate SNPs that occurred among a preexisting set of 57 validated assays and to justify new assay development for 83 potential SNPs in the DNA-dependent protein kinase catalytic subunit. Of the 140 SNPs, the authors eliminated 119 variants with low or neutral predictions. The existing computational methods they used and the semiquantitative relative ranking strategy they developed can be adapted to a priori SNP selection or post hoc evaluation of variants identified in whole genome scans or within haplotype blocks associated with disease. The authors show a "real world" application of some existing bioinformatics tools for use in large epidemiologic studies and genetic analyses. They also reviewed alternative approaches that provide related information.

Algorithms↗

PubMed2003

Large-scale identification and analysis of genome-wide single-nucleotide polymorphisms for mapping in Arabidopsis thaliana.

Genetic markers such as single nucleotide polymorphisms (SNPs) are essential tools for positional cloning, association, or quantitative trait locus mapping and the determination of genetic relationships between individuals. We identified and characterized a genome-wide set of SNP markers by generating 10,706 expressed sequence tags (ESTs) from cDNA libraries derived from 6 different accessions, and by analysis of 606 sequence tagged sites (STS) from up to 12 accessions of the model flowering plant Arabidopsis thaliana. The cDNA libraries for EST sequencing were made from individuals that were stressed by various means to enrich for transcripts from genes expressed under such conditions. SNPs discovered in these sequences may be useful markers for mapping genes involved in interactions with the biotic and abiotic environment. The STS loci are distributed randomly over the genome. By comparison with the Col-0 genome sequence, we identified a total of 8051 SNPs and 637 insertion/deletion polymorphisms (InDel). Analysis of STS-derived SNPs shows that most SNPs are rare, but that it is possible to identify intermediate frequency framework markers that can be used for genetic mapping in many different combinations of accessions. A substantial proportion of SNPs located in ORFs caused a change of the encoded amino acid. A comparison of the density of our SNP markers among accessions in both the EST and STS datasets, revealed that Cvi-0 is the most divergent accession from Col-0 among the 12 accessions studied. All of these markers are freely available via the internet.

Arabidopsis↗

PubMed2004

Single nucleotide polymorphism (SNP) discovery in mammals: a targeted-gene approach.

Single nucleotide polymorphisms (SNPs) have rarely been exploited in nonhuman and nonmodel organism genetic studies. This is due partly to difficulties in finding SNPs in species where little DNA sequence data exist, as well as to a lack of robust and inexpensive genotyping methods. We have explored one SNP discovery method for molecular ecology, evolution, and conservation studies to evaluate the method and its limitations for population genetics in mammals. We made use of 'CATS' (or 'EPIC') primers to screen for novel SNPs in mammals. Most of these primer sets were designed from primates and/or rodents, for amplifying intron regions from conserved genes. We have screened 202 loci in 16 representatives of the major mammalian clades. Polymerase chain reaction (PCR) success correlated with phylogenetic distance from the human and mouse sequences used to design most primers; for example, specific PCR products from primates and the mouse amplified the most consistently and the marsupial and armadillo amplifications were least successful. Approximately 24% (opossum) to 65% (chimpanzee) of primers produced usable PCR product(s) in the mammals tested. Products produced generally high but variable levels of readable sequence and similarity to the expected genes. In a preliminary screen of chimpanzee DNA, 12 SNPs were identified from six (of 11) sequenced regions, yielding a SNP on average every 400 base pairs (bp). Given the progress in genome sequencing, and the large numbers of CATS-like primers published to date, this approach may yield sufficient SNPs per species for population and conservation genetic studies in nonmodel mammals and other organisms.

Animals↗

PubMed2003

Single-nucleotide polymorphism genotyping on optical thin-film biosensor chips.

Single-nucleotide polymorphisms (SNPs) constitute the bulk of human genetic variation and provide excellent markers to identify genetic factors contributing to complex disease susceptibility. A rapid, sensitive, and inexpensive assay is important for large-scale SNP scoring. Here we report the development of a multiplex SNP detection system using silicon chips coated to create a thin-film optical biosensor. Allele-discriminating, aldehyde-labeled oligonucleotides are arrayed and covalently attached to a hydrazinederivatized chip surface. Target sequences (e.g., PCR amplicons) then are hybridized in the presence of a mixture of biotinylated detector probes, one for each SNP, and a thermostable DNA ligase. After a stringent wash (0.01 M NaOH), ligation of biotinylated detector probes to perfectly matched capture oligomers is visualized as a color change on the chip surface (gold to blue/purple) after brief incubations with an anti-biotin IgG-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate. Testing of PCR fragments is completed in 30-40 min. Up to several hundred SNPs can be assayed on a 36-mm2 chip, and SNP scoring can be done by eye or with a simple digital-camera system. This assay is extremely robust, exhibits high sensitivity and specificity, and is format-flexible and economical. In studies of mutations associated with risk for venous thrombosis and genotyping/haplotyping of African-American samples, we document high-fidelity analysis with 0 misassignments in 500 assays performed in duplicate.

Biosensing Techniques↗

PubMed2007

Identification and analysis of single nucleotide polymorphisms (SNPs) in the mosquito Anopheles funestus, malaria vector.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the most common source of genetic variation in eukaryotic species and have become an important marker for genetic studies. The mosquito Anopheles funestus is one of the major malaria vectors in Africa and yet, prior to this study, no SNPs have been described for this species. Here we report a genome-wide set of SNP markers for use in genetic studies on this important human disease vector. RESULTS: DNA fragments from 50 genes were amplified and sequenced from 21 specimens of An. funestus. A third of specimens were field collected in Malawi, a third from a colony of Mozambican origin and a third form a colony of Angolan origin. A total of 494 SNPs including 303 within the coding regions of genes and 5 indels were identified. The physical positions of these SNPs in the genome are known. There were on average 7 SNPs per kilobase similar to that observed in An. gambiae and Drosophila melanogaster. Transitions outnumbered transversions, at a ratio of 2:1. The increased frequency of transition substitutions in coding regions is likely due to the structure of the genetic code and selective constraints. Synonymous sites within coding regions showed a higher polymorphism rate than non-coding introns or 3' and 5'flanking DNA with most of the substitutions in coding regions being observed at the 3rd codon position. A positive correlation in the level of polymorphism was observed between coding and non-coding regions within a gene. By genotyping a subset of 30 SNPs, we confirmed the validity of the SNPs identified during this study. CONCLUSION: This set of SNP markers represents a useful tool for genetic studies in An. funestus, and will be useful in identifying candidate genes that affect diverse ranges of phenotypes that impact on vector control, such as resistance insecticide, mosquito behavior and vector competence.

Animals↗

PubMed2004

MALDI-TOF mass spectrometric detection of multiplex single base extended primers. A study of 17 y-chromosome single-nucleotide polymorphisms.

One of the most promising techniques for typing of multiple single-nucleotide polymorphism (SNP) is detection of single base extension primers (SBE) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We present a new MALDI-TOF MS protocol for typing of multiple SNPs in a single reaction. Biotin-labeled ddNTPs were used in the SBE reaction and solid phase-bound monomeric avidin was used as capturing/purification scheme allowing the exclusive release of the SBE products under gentle conditions using 5% triethylamine. We dubbed this method monomeric avidin triethylamine purification. The biotin-labeled ddNTPs contained linkers with different masses ensuring a clear separation of the alleles even for SBE primers with a mass of 10 300 Da. Furthermore, only 25-350 fmol of SBE primers were necessary in order to obtain reproducible MALDI-TOF spectra. Similar signal intensities were obtained in the 5500-10 300 m/z mass range by increasing the concentration of the longer SBE primers in the reaction. To validate the technique, 17 Y-chromosome SNPs were analyzed in 200 males. The precision and accuracy of the mass determination were analyzed by parametric statistic, and the potential use of MALDI-TOF MS for SNP typing is discussed.

Alleles↗

PubMed2004

Localization of cancer susceptibility genes by genome-wide single-nucleotide polymorphism linkage-disequilibrium mapping.

With the large numbers of single nucleotide polymorphisms (SNPs) available and new technologies that permit high throughput genotyping, we have investigated the possibility of the localization of disease genes with genome-wide panels of SNP markers and taking advantage of the linkage-disequilibrium (LD) between the disease gene and closely linked markers. For this purpose, we selected cases from the Ashkenazi Jewish population, in which the mutant alleles are expected to be identical by descent from a common founder and the regions of LD encompassing these mutant alleles are large. As a validation of this approach for localization, we performed two trials: one in autosomal recessive Bloom syndrome, in which a unique mutation of the BLM gene is present at elevated frequencies in cases, and the other in autosomal dominant hereditary nonpolyposis colorectal cancer (HNPCC), in which a unique mutation of MSH2 is present at elevated frequencies. In the Bloom syndrome trial, we genotyped 3,258 SNPs in 10 Jewish Bloom syndrome cases and 31 non-Bloom syndrome Jewish persons as a comparison group. In the HNPCC trial, we genotyped 8,549 SNPS in 13 Jewish HNPCC cases whose colon cancers exhibited microsatellite instability and in 63 healthy Jews as a comparison group. To identify significant associations, we performed (a) Fisher's exact test comparing genotypes at each locus in cases versus controls and (b) a haplotype analysis by estimating the frequency of haplotypes with the expectation-maximization algorithm and comparing haplotype frequencies in cases versus controls by logistic regression and a maximum likelihood ratio method. In the Bloom syndrome trial, by Fisher's exact test, statistically significant association was detected at a single locus, TSC0754862, which is a locus 1.7 million bp from BLM. Two-locus, three-locus, and four-locus haplotypes that included TSC0754862 and flanked BLM were also statistically more frequent in cases versus controls. In the HNPCC trial, although a significant P value was not obtained by the single SNP genotype analysis, significant associations were detected for several multilocus haplotypes in an 11-million-bp region that contained the MSH2 gene. This work demonstrates the power of the LD mapping approach in an isolated population and its general applicability to the identification of novel cancer-causing genes.

Adenosine Triphosphatases↗

PubMed1998

dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics.

PCR-based detection of single nucleotide polymorphisms is a powerful tool for the plant geneticist. Cleaved amplified polymorphic sequence analysis is the most widely used approach for the detection of single nucleotide polymorphisms. However, this technique is limited to mutations which create or disrupt a restriction enzyme recognition site. This paper presents a modification of this technique where mismatches in a PCR primer are used to create a polymorphism based on the target mutation. This technique is useful for following known mutations in segregating populations and genetic mapping of isolated DNAs used for positional based cloning of new genes. In addition, a computer program has been developed that facilitates the design of these PCR primers.

Arabidopsis↗

PubMed1999

High-throughput single nucleotide polymorphism genotyping by fluorescent 5' exonuclease assay.

Single nucleotide polymorphisms (SNPs) are the class of DNA variants that are most common in the genome. Technologies are needed that allow relatively high-throughput, high-quality genotyping of SNPs, with rapid assay development for individual SNPs of interest. We have implemented an accurate and high-throughput SNP genotyping system using a commercially available fluorescent 5' exonuclease assay. Optimization of the assay system for low-volume reactions and low-probe concentrations has reduced assay costs by 75%. Using a simple assay optimization process, we successfully developed genotyping assays for 92% of assays attempted (309 out of 335) and have generated over 200,000 genotypes.

Alleles↗

PubMed2003

Different applications of polymerases with and without proofreading activity in single-nucleotide polymorphism analysis.

With the completion of the human genome project, single-nucleotide polymorphisms (SNPs) have become the focus of intense study in biomedical research. Polymerase-mediated primer extension has been employed in a variety of SNP assays. However, these SNP assays using polymerase without proofreading function are compromised by their low reliability. Using a newly developed short amplicon harboring restriction enzyme site, EcoR-I, we were able to compare the single-base discrimination abilities of polymerases with and without proofreading function in primer extension in a broad range of annealing temperatures. Thermodynamic analysis demonstrated a striking single-nucleotide discrimination ability of polymerases with proofreading function. Using unmodified 3'-end allele-specific primers, only template-dependent products were generated by polymerase with proofreading activity. This powerful single-base discrimination ability of exo(+) polymerases was further evaluated in primer extension using three types of 3' terminally modified allele-specific primers. As compared with the poor fidelity in primer extension of polymerases lacking 3' exonuclease activity, this study provides convincing evidence that the use of proofreading polymerases in combination with 3'-end modified allele-specific primers can be a powerful new strategy for the development of SNP assays.

Base Sequence↗

PubMed2005

Microsatellite linkage analysis, single-nucleotide polymorphisms, and haplotype associations with ECB21 in the COGA data.

This study, part of the Genetic Analysis Workshop 14 (GAW14), explored real Collaborative Study on the Genetics of Alcoholism data for linkage and association mapping between genetic polymorphisms (microsatellite and single-nucleotide polymorphisms (SNPs)) and beta (16.5-20 Hz) oscillations of the brain rhythms (ecb21). The ecb21 phenotype underwent the statistical adjustments for the age of participants, and for attaining a normal distribution. A total of 1,000 subjects' available phenotypes were included in linkage analysis with microsatellite markers. Linkage analysis was performed only for chromosome 4 where a quantitative trait locus with 5.01 LOD score had been previously reported. Previous findings related this location with the gamma-aminobutyric acid type A (GABAA) receptor. At the same location, our analysis showed a LOD score of 2.2. This decrease in the LOD score is the result of a drastic reduction (one-third) of the available GAW14 phenotypic data. We performed SNP and haplotype association analyses with the same phenotypic data under the linkage peak region on chromosome 4. Seven Affymetrix and two Illumina SNPs showed significant associations with ecb21 phenotype. A haplotype, a combination of SNPs TSC0044171 and TSC0551006 (the latter almost under the region of GABAA genes), showed a significant association with ecb21 (p = 0.015) and a relatively high frequency in the sample studied. Our results affirmed that the GABA region has potential of harboring genes that contribute quantitatively to the beta oscillation of the brain rhythms. The inclusion of the remaining 614 subjects, which in the GAW14 had missing data for the ecb21, can improve the strength of the associations as they have already shown that they contribute quite important information in the linkage analysis.

Alcoholism↗