SNPs基因分型技术

介绍了SNP的概念、特点,集中讨论了各种技术的原理及优缺点,并对目前SNP在遗传图的绘制、疾病防治、药物设计及法医学等方面的应用及研究进展进行了综述。     

三色荧光技术在SNP检测中的应用

利用三色荧光标记的A、C、T双脱氧核苷酸单碱基延伸的方法结合编码寡核苷酸芯片技术检测单核苷酸多态性 (SNP)的基因型。以beta地中海贫血样本基因 (HBB基因 )突变作为模型的研究结果显示该方法能同时对多位点的SNP进行检测。     

寡核苷酸DNA Microarray用于HLA DRB1基因分型的研究

对寡核苷酸DNA Microarray用于HLA DRB1基因分型的技术进行研究。常规的酚/氯仿法提取标准血样基因组DNA,在DRB1的exon2区域设计一对引物,经PCR扩增基因组相应区段并用Cy5-dCTP进行标记。设计寡核苷酸分型探针,将探针固定在APS-PDC法制作的DNA Microarray上,用标记的PCR产物与之杂交,扫描仪对杂交效果进行扫描,Imagene软件对杂交图像进行分析。共检测了33例标准血样的HLA DRB1基因型。检测结果证明研制的DNA Microarray准确、灵敏。DNA Microarray技术可以有效地检测DRB1等位基因,对比常规的PCR-SSP和PCR-SSO方法、分型基因芯片方法更为直观,并有集成化优势。     

应用红外荧光和加尾引物法进行向日葵SSR遗传分析中的多聚PCR和多聚凝胶电泳的优化

在向日葵(Helianthus annuus L.)自交系SSR遗传分析中,为了提高SSR荧光分析效率、简化分析程序和降低研究费用,我们进行了多聚PCR和多聚凝胶电泳两项技术的优化研究.结果表明,优化多聚PCR和多聚凝胶电泳的影响因子(如逐步降低的退火温度的循环数、各个多聚位点引物浓度的平衡、PCR缓冲液浓度以及Taq DNA多聚酶的浓度等)可以收到更好的实验效果.基于以上的优化研究,系统地提出了一套优化的加尾引物法的策略.同时,提出了在多聚PCR和多聚凝胶电泳中常常遇到的技术难题的有效防止和解决的方法.     

应用红外荧光和加尾引物法进行向日葵SSR遗传分析中的多聚PCR和多聚凝胶电泳的优化

在向日葵(Helianthus annuus L.)自交系SSR遗传分析中,为了提高SSR荧光分析效率、简化分析程序和降低研究费用,我们进行了多聚PCR和多聚凝胶电泳两项技术的优化研究。结果表明,优化多聚PCR和多聚凝胶电泳的影响因子(如逐步降低的退火温度的循环数、各个多聚位点引物浓度的平衡、PCR缓冲液浓度以及Taq DNA多聚酶的浓度等)可以收到更好的实验效果。基于以上的优化研究,系统地提出了一套优化的加尾引物法的策略。同时,提出了在多聚PCR和多聚凝胶电泳中常常遇到的技术难题的有效防止和解决的方法。     

Full Genotyping of a Highly Polymorphic Human Gene Trait by Time-Resolved Fluorescence Resonance Energy Transfer

The ability of detecting the subtle variations occurring, among different individuals, within specific DNA sequences encompassed in highly polymorphic genes discloses new applications in genomics and diagnostics. DQB1 is a gene of the HLA-II DQ locus of the Human Leukocyte Antigens (HLA) system. The polymorphisms of the trait of the DQB1 gene including codons 52–57 modulate the susceptibility to a number of severe pathologies. Moreover, the donor-receiver tissue compatibility in bone marrow transplantations is routinely assessed through crossed genotyping of DQB and DQA. For the above reasons, the development of rapid, reliable and cost-effective typing technologies of DQB1 in general, and more specifically of the codons 52–57, is a relevant although challenging task. Quantitative assessment of the fluorescence resonance energy transfer (FRET) efficiency between chromophores labelling the opposite ends of gene-specific oligonucleotide probes has proven to be a powerful tool to type DNA polymorphisms with single-nucleotide resolution. The FRET efficiency can be most conveniently quantified by applying a time-resolved fluorescence analysis methodology, i.e. time-correlated single-photon counting, which allows working on very diluted template specimens and in the presence of fluorescent contaminants. Here we present a full in-vitro characterization of the fluorescence responses of two probes when hybridized to oligonucleotide mixtures mimicking all the possible genotypes of the codons 52–57 trait of DQB1 (8 homozygous and 28 heterozygous). We show that each genotype can be effectively tagged by the combination of the fluorescence decay constants extrapolated from the data obtained with such probes.     

Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays

We present a genotyping method for simultaneously scoring 116,204 SNPs using oligonucleotide arrays. At call rates >99%, reproducibility is >99.97% and accuracy, as measured by inheritance in trios and concordance with the HapMap Project, is >99.7%. Average intermarker distance is 23.6 kb, and 92% of the genome is within 100 kb of a SNP marker. Average heterozygosity is 0.30, with 105,511 SNPs having minor allele frequencies >5%.     

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

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

Multiplex Genotyping of Allelic Variants of Genes Involved in Metabolizing Antileukemic Drugs

A biochip, primer set, and genotyping protocol were developed to simultaneously address 16 single nucleotide polymorphisms in antileukemic drug metabolism genes, including TPMT, ITPA, MTHFR, SLCO1B1, SLC19A1, NR3C1, GRIA1, ASNS, MTRR, and ABCB1. The genotyping procedure included a one-round multiplex polymerase chain reaction (PCR) with simultaneous incorporation of a fluorescent label into the PCR product and subsequent hybridization on a biochip with immobilized probes. The method was used to test 65 DNA samples of leukemia patients. Fluorescence signal intensity ratios in pairs of wildtype and respective mutant sequence probes were analyzed for all polymorphic markers and demonstrated high accuracy of genotyping. The reliability of genotype determination using the biochip was confirmed by direct Sanger sequencing.     

Ultrahigh-Density Linkage Map for Cultivated Cucumber (Cucumis sativus L.) Using a Single-Nucleotide Polymorphism Genotyping Array

Genotyping arrays are tools for high-throughput genotyping, which is beneficial in constructing saturated genetic maps and therefore high-resolution mapping of complex traits. Since the report of the first cucumber genome draft, genetic maps have been constructed mainly based on simple-sequence repeats (SSRs) or on combinations of SSRs and sequence-related amplified polymorphism (SRAP). In this study, we developed the first cucumber genotyping array consisting of 32,864 single-nucleotide polymorphisms (SNPs). These markers cover the cucumber genome with a median interval of ~2 Kb and have expected genotype calls in parents/F₁ hybridizations as a training set. The training set was validated with Fluidigm technology and showed 96% concordance with the genotype calls in the parents/F₁ hybridizations. Application of the genotyping array was illustrated by constructing a 598.7 cM genetic map based on a ‘9930’ × ‘Gy14’ recombinant inbred line (RIL) population comprised of 11,156 SNPs. Marker collinearity between the genetic map and reference genomes of the two parents was estimated at R² = 0.97. We also used the array-derived genetic map to investigate chromosomal rearrangements, regional recombination rate, and specific regions with segregation distortions. Finally, 82% of the linkage-map bins were polymorphic in other cucumber variants, suggesting that the array can be applied for genotyping in other lines. The genotyping array presented here, together with the genotype calls of the parents/F₁ hybridizations as a training set, should be a powerful tool in future studies with high-throughput cucumber genotyping. An ultrahigh-density linkage map constructed by this genotyping array on RIL population may be invaluable for assembly improvement, and for mapping important cucumber QTLs.     

Fidelity of SNP Array Genotyping Using Epstein Barr Virus-Transformed B-Lymphocyte Cell Lines: Implications for Genome-Wide Association Studies

Background

As availability of primary cells can be limited for genetic studies of human disease, lymphoblastoid cell lines (LCL) are common sources of genomic DNA. LCL are created in a transformation process that entails in vitro infection of human B-lymphocytes with the Epstein-Barr Virus (EBV).

Methodology/Principal Findings

To test for genotypic errors potentially induced by the Epstein-Barr Virus transformation process, we compared single nucleotide polymorphism (SNP) genotype calls in peripheral blood mononuclear cells (PBMC) and LCL from the same individuals. The average mismatch rate across 19 comparisons was 0.12% for SNPs with a population call rate of at least 95%, and 0.03% at SNPs with a call rate of at least 99%. Mismatch rates were not correlated across genotype subarrays run on all sample pairs.

Conclusions/Significance

Genotypic discrepancies found in PBMC and LCL pairs were not significantly different than control pairs, and were not correlated across subarrays. These results suggest that mismatch rates are minimal with stringent quality control, and that most genotypic discrepancies are due to technical artifacts rather than the EBV transformation process. Thus, LCL likely constitute a reliable DNA source for host genotype analysis.     

应用多重PCR技术检测性器官异常患者的性别决定基因

采用针对人SRY基因及X与Y染色体同源序列的两对引物进行多重聚合酶链反应技术检测204例新生儿脐血DNA,男性均显示590bp、 355bp及280bp 3条扩增带,女性只有590bp1条扩增带,性别鉴定准确率为100%。又检测47例性器官异常患,13例社会性别为女性者只出现590bp扩增带,与核型性别结果一致。 34例社会性别为男性者中,有2例SRY基因检测结果阴性,只出现590bp带,核型为46,XX,证明这两例患者社会性别不相符,经病理证实后应诊断为女性假两性畸形。 Abstract:This investigation adapted multiplex PCR technique with two pairs of primer to determine sex according to the human SRY gene and X,Y chromosome analogical sequence.We detected bellybutton blood DNA from 204 newborns.Male revealed three bands:590bp,355bp and 280bp,and females only had one band which was 590bp,the accuracy of sex determination was 100%.47 sexual abnormal patients were tested.The results showed that 13 cases,whose social sex were female,had one 590bp band which consistent with their chromosome type sex.In the rest 34 cases,whose social sex were male,2cases showed,on the contrary,only one 590bp band and their chromosome type were 46,XX.This proved that the two patients did not consistent with their social sex,and pathological analysis showed that they were female pseud-hermaphroditism.     

应用多重PCR技术检测性器官异常患者的性别决定基因

采用针对人SRY基因及X与Y染色体同源序列的两对引物进行多重聚合酶链反应技术检测204例新生儿脐血DNA,男性均显示590bp、 355bp及280bp 3条扩增带,女性只有590bp1条扩增带,性别鉴定准确率为100%。又检测47例性器官异常患,13例社会性别为女性者只出现590bp扩增带,与核型性别结果一致。 34例社会性别为男性者中,有2例SRY基因检测结果阴性,只出现590bp带,核型为46,XX,证明这两例患者社会性别不相符,经病理证实后应诊断为女性假两性畸形。 Abstract:This investigation adapted multiplex PCR technique with two pairs of primer to determine sex according to the human SRY gene and X,Y chromosome analogical sequence.We detected bellybutton blood DNA from 204 newborns.Male revealed three bands:590bp,355bp and 280bp,and females only had one band which was 590bp,the accuracy of sex determination was 100%.47 sexual abnormal patients were tested.The results showed that 13 cases,whose social sex were female,had one 590bp band which consistent with their chromosome type sex.In the rest 34 cases,whose social sex were male,2cases showed,on the contrary,only one 590bp band and their chromosome type were 46,XX.This proved that the two patients did not consistent with their social sex,and pathological analysis showed that they were female pseud-hermaphroditism.     

Design of a High Density SNP Genotyping Assay in the Pig Using SNPs Identified and Characterized by Next Generation Sequencing Technology

Background

The dissection of complex traits of economic importance to the pig industry requires the availability of a significant number of genetic markers, such as single nucleotide polymorphisms (SNPs). This study was conducted to discover several hundreds of thousands of porcine SNPs using next generation sequencing technologies and use these SNPs, as well as others from different public sources, to design a high-density SNP genotyping assay.

Methodology/Principal Findings

A total of 19 reduced representation libraries derived from four swine breeds (Duroc, Landrace, Large White, Pietrain) and a Wild Boar population and three restriction enzymes (AluI, HaeIII and MspI) were sequenced using Illumina''s Genome Analyzer (GA). The SNP discovery effort resulted in the de novo identification of over 372K SNPs. More than 549K SNPs were used to design the Illumina Porcine 60K+SNP iSelect Beadchip, now commercially available as the PorcineSNP60. A total of 64,232 SNPs were included on the Beadchip. Results from genotyping the 158 individuals used for sequencing showed a high overall SNP call rate (97.5%). Of the 62,621 loci that could be reliably scored, 58,994 were polymorphic yielding a SNP conversion success rate of 94%. The average minor allele frequency (MAF) for all scorable SNPs was 0.274.

Conclusions/Significance

Overall, the results of this study indicate the utility of using next generation sequencing technologies to identify large numbers of reliable SNPs. In addition, the validation of the PorcineSNP60 Beadchip demonstrated that the assay is an excellent tool that will likely be used in a variety of future studies in pigs.     

Development and Implementation of a Multiplex Single-Nucleotide Polymorphism Genotyping Assay for Detection of Virulence-Attenuating Mutations in the Listeria monocytogenes Virulence-Associated Gene inlA

The virulence factor internalin A (InlA) facilitates the uptake of Listeria monocytogenes by epithelial cells that express the human isoform of E-cadherin. Previous studies identified naturally occurring premature stop codon (PMSC) mutations in inlA and demonstrated that these mutations are responsible for virulence attenuation. We assembled >1,700 L. monocytogenes isolates from diverse sources representing 90 EcoRI ribotypes. A subset of this isolate collection was selected based on ribotype frequency and characterized by a Caco-2 cell invasion assay. The sequencing of inlA genes from isolates with attenuated invasion capacities revealed three novel inlA PMSCs which had not been identified previously among U.S. isolates. Since ribotypes include isolates with and without inlA PMSCs, we developed a multiplex single-nucleotide polymorphism (SNP) genotyping assay to detect isolates with virulence-attenuating PMSC mutations in inlA. The SNP genotyping assay detects all inlA PMSC mutations that have been reported worldwide and verified in this study to date by the extension of unlabeled primers with fluorescently labeled dideoxynucleoside triphosphates. We implemented the SNP genotyping assay to characterize human clinical and food isolates representing common ribotypes associated with novel inlA PMSC mutations. PMSCs in inlA were significantly (ribotypes DUP-1039C and DUP-1045B; P < 0.001) or marginally (ribotype DUP-1062D; P = 0.11) more common among food isolates than human clinical isolates. SNP genotyping revealed a fourth novel PMSC mutation among U.S. L. monocytogenes isolates, which was observed previously among isolates from France and Portugal. This SNP genotyping assay may be implemented by regulatory agencies and the food industry to differentiate L. monocytogenes isolates carrying virulence-attenuating PMSC mutations in inlA from strains representing the most significant health risk.     

Automated Genotyping of Biobank Samples by Multiplex Amplification of Insertion/Deletion Polymorphisms

The genomic revolution in oncology will entail mutational analyses of vast numbers of patient-matched tumor and normal tissue samples. This has meant an increased risk of patient sample mix up due to manual handling. Therefore, scalable genotyping and sample identification procedures are essential to pathology biobanks. We have developed an efficient alternative to traditional genotyping methods suited for automated analysis. By targeting 53 prevalent deletions and insertions found in human populations with fluorescent multiplex ligation dependent genome amplification, followed by separation in a capillary sequencer, a peak spectrum is obtained that can be automatically analyzed. 24 tumor-normal patient samples were successfully matched using this method. The potential use of the developed assay for forensic applications is discussed.     

Development of a Genotyping Method for Potato Scab Pathogens Based on Multiplex PCR

Scab disease significantly damages potato and other root crops. Streptomyces scabiei, S. acidiscabiei, and S. turgidiscabiei are the best-known causal agents of this disease. We have developed a novel genotyping method for these potato scab pathogens using multiplex PCR, whose benefits include rapid and easy detection of multiple species. We designed a species-specific primer set (6 primers, 3 pairs) for the 16S rRNA genes and 16S–23S ITS regions of these potato scab pathogens. The specificity of the primer set was confirmed by testing 18 strains containing potato scab pathogens, other Streptomyces species, and strains of other genera. The application of the developed method to potato field soil and potato tissue samples resulted in the clear detection and identification of pathogens. Since this method is applicable to a large number of environmental samples, it is expected to be useful for a high-throughput analysis of soil and plant tissues of scab disease.     

Development of Multiplex Genotyping Method of Polymorphic Markers of Genes Associated with Cognitive Abilities

A developed method of multiplex genotyping of polymorphic markers of genes associated with cognitive abilities and neuropsychiatric diseases is based on multilocus PCR and MALDI-TOF mass spectrometry of DNA molecules. The frequencies of 32 single-nucleotide markers localized in 24 genes are analyzed in a sample of elderly people from the Russian population of Tomsk. The data obtained are compared with data for populations from the 1000 Genomes Project.