单核苷酸多态性分析在近交系大鼠遗传检测中的应用

目的研究SNP在近交系大鼠遗传检测中的应用。方法 选取大鼠20号染色体MHC所在P12区上的9个SNP位点,应用新建立的高保真酶特异性检测SNP基因分型技术对五种常用近交系大鼠(BN、F344、WKY、LEW、SHR)和两种新培育近交系大鼠(MIJ和HFJ)进行SNP多态性分析。结果五种常用近交系的SNP检测结果与Rat Genome Database网站提供的基因型数据一致,并检测确立了新品系的SNP基因型。同时绘制出七种近交系大鼠在该9个SNP位点的遗传扩增图谱。结论运用所筛选的9个SNP位点进行大鼠多态性分析,能够快速、可靠地对BN、F344、WKY、LEW、SHR及MIJ、HFJ进行遗传监测。     

等位基因特异PCR技术的研究与应用

生物的单核苷酸多态性(Single-nucleotide polymorphism,SNP)具有数量多、分布广、易于分型、稳定性强等优点,很适合于用做分子标记.等位基因特异PCR(Allele-specific PCR,AS-PCR)是根据SNP位点设计3'末端与SNP位点碱基互补或错配的特异PCR引物,通过凝胶电泳等方法检测PCR扩增产物的有或无,从而检测基因型中SNP的一种技术.经过不断地改进与完善,基于SNP的等位基因特异PCR标记已逐渐成为一种快速、简便、低成本、可靠、高通量的检测基因型SNP的方法.本文应用等位基因特异PCR技术,根据小麦TaDREB1基因在旱选10和鲁麦14的120(C→A)SNP成功地开发了一个SNP分子标记,证明了该方法的有效性和可行性.     

新疆4个绵羊品种内皮型一氧化氮合酶基因第8外显子的PCR-SSCP鉴定

目的:对新疆4个绵羊品种内皮型一氧化氮合酶(eNOS)基因第8外显子的多态性进行鉴定。方法:利用PCR-SSCP和测序的方法对76只中国美利奴羊、51只无角陶赛特羊、57只萨福克羊、37只哈萨克羊共4个绵羊品种进行单核苷酸多态性(SNP)检测,并用生物信息学方法对检测出的SNP进行统计分析。结果:在中国美利奴羊、无角陶赛特羊、萨福克羊、哈萨克羊中共检测到AA、AB、BB等3种基因型,AA基因型的频率分别为0.0526、0.0980、0.1754和0.2973,BB基因型的频率分别为0.6316、0.1961、0.5614和0.1892,AB基因型的频率分别为0.3158、0.7059、0.2632和0.5135。通过测序,在eNOS基因第8外显子上发现了一个新的SNP位点(ss974768653),位于绵羊eNOS基因第8外显子142 bp处(A142G)。结论:中国美利奴羊和哈萨克羊的多态性位点(P0.05)处于Hardy-Weinberg平衡状态,萨福克和无角陶赛特羊的多态性位点(P0.05)不处于Hardy-Weinberg平衡状态。     

甘南牦牛(Bos grunniens)FBXO32基因突变及其与胴体和肉质性状关联分析

为了分析甘南牦牛(Bos grunniens)肌肉萎缩盒蛋白32(F-box protein 32,FBXO32)基因的单核苷酸多态性(single nucleotide polymorphism, SNP)位点,以及基因型与胴体和肉质性状间的相关性,本研究以593头甘南牦牛为研究对象,采用混池测序和竞争性等位基因特异性PCR(kompetitive allele specific PCR, KASP)技术,检测了甘南牦牛FBXO32基因突变位点及基因型,分析了基因型与甘南牦牛胴体及肉质性状的相关性。结果表明,从甘南牦牛FBXO32基因检测到7个SNP位点,分别是位于5′UTR区的SNP1(g.267A>C)、外显子1区的SNP2(g.326G>T)、外显子8区的SNP3(g.31231G>C),以及3′UTR区的SNP4(g.31352G>A)、 SNP5(g.31424C>T)、 SNP6(g.31503A>C)和SNP7(g.31504A>G)。其中,SNP1、 SNP4、 SNP5、 SNP6与肌肉嫩度显著相关(P<0.05), ...     

Ⅱ型糖尿病易感基因研究的进展

Ⅱ型糖尿病是由基因和环境共同作用所引起的多基因遗传病,随着人类基因组计划的完成和单核苷酸多态性(single-nu-clcotide polymorphisms,SNP)扫描技术的发展,许多SNP位点被证实与Ⅱ型糖尿病相关,因此易感基因的检测是未来糖尿病预防和治疗的关键.本文主要介绍了目前Ⅱ型糖尿病易感基因的检测策略、方法、研究进展及今后的研究方向.     

大规模单核苷酸多态性分析与肿瘤易感性

单核苷酸多态性(SNPs)是人类基因组中最常见的变异形式。作为第三代遗传标记,SNP在基因定位、克隆、遗传多态性方面具有广泛应用,特别是作为基因诊断标记在预防医学中具有十分重要的作用。近年来,随着人类基因组计划的发展,数以百万计的SNP被陆续发现,并可在公共数据库中免费获得。SNP数量的快速增加和SNP检测方法的发展,为其在肿瘤易感性领城的应用提供了可能。在本综述中,我们介绍了几种高通量检测SNP的分析方法,总结了大规模SNP分析技术在肿瘤易感性中的应用,介绍了目前人们对于不同人群中的SNP分析、肿瘤易感基因、个体肿瘤易感性的理解,以及研究SNP标记与肿瘤易感性关系时存在的难点。     

单核苷酸多态性检测方法的研究进展

单核苷酸多态性(single nucleotide polymorphism,SNP)作为第三代遗传标记已经广泛应用于基因作图、疾病相关性分析、群体遗传学及药物研究等领域.因此建立高度自动化和高通量的SNP检测分析技术十分重要.简要介绍了国内外几种主要SNP检测技术的原理和检测分析手段,并对SNP高通量检测技术的发展进行了展望.     

TaqMan-MGB 探针检测GSTP1外显子5SNP可行性研究

目的：评估TaqMan-MGB探针基因分型方法检测已知SNP的可行性,并与传统的PCR-RFLP方法比较。方法：高通量的TaqMan-MGB探针基因分型方法已被用来检测单核苷酸多态性（SNP）。在321倒样本中,同时用TaqMan-MGB探针基因分型方法和PCR—RFLP方法检测GSTP1外显子5SNP。结果：2种方法所得结果完全一致。野生型（AA）226例（70．4％）,杂合子（AG）92例（28．7％）,纯合突变型3例（O．9％）。结论：TaqMan-MGB探针基因分型方法是一种能快速、高度特异性、高度自动化检测SNP的方法。可用于大规模的基因分型。     

一种高效筛选Mx GTPase效应区SNP方法

[目的]建立高效检测家禽Mx功能区SNP方法 ,改进PCR SSCP。[方法]以引进品种和地方品种为研究对象,筛选引物,优化程序,通过二次PCR改进SSCP方法对Mx基因GTPase效应区SNP进行检测。[结果]SSCP检测引物分型检测及SNP分析,检测结果与测序一致。来航鸡检测到阳性结果,且PA检测的多态位点均由编码区A/G替换引起,济宁百日鸡和来航鸡两个群体PIC为0.2515和0.2628,呈低度多态。[结论]χ~2独立性检验位点基因频率和基因型频率呈Hardy-Weinberg平衡,不同品种间位点8(S631N)基因型分布差异极显著(P<0.01)。改进后的PCR SSCP高效经济简便,为高通量后期检测、新位点筛选奠定基础。     

猪Nramp1基因外显子2的SNP位点筛选及生物信息学分析

本研究采用DNA混合池扩增并进行直接测序的方法,对3个引进猪种和3个地方猪种Nramp1基因外显子2的SNP位点进行筛选,并通过生物信息学软件对该基因的m RNA二级结构、蛋白质二级结构和功能进行预测。结果在2个引进猪种(大白猪,长白猪)和3个地方猪种(八眉猪,蕨麻猪,烟台黑猪)中共检测到2个SNP位点,分别为T~(62)C和A~(92)G,而在杜洛克猪中未检测到SNP位点。生物信息学分析结果表明,T~(62)C和A~(92)G位点均降低了RNA二级结构的稳定性,Nramp1基因可能在转录和信号转导过程中发挥着重要作用。本研究结果丰富了猪Nramp1基因库,为进一步研究Nramp1基因功能和筛选抗病育种相关SNP位点提供理论参考。     

The SNPlex genotyping system: a flexible and scalable platform for SNP genotyping.

We developed the SNPlex Genotyping System to address the need for accurate genotyping data, high sample throughput, study design flexibility, and cost efficiency. The system uses oligonucleotide ligation/polymerase chain reaction and capillary electrophoresis to analyze bi-allelic single nucleotide polymorphism genotypes. It is well suited for single nucleotide polymorphism genotyping efforts in which throughput and cost efficiency are essential. The SNPlex Genotyping System offers a high degree of flexibility and scalability, allowing the selection of custom-defined sets of SNPs for medium- to high-throughput genotyping projects. It is therefore suitable for a broad range of study designs. In this article we describe the principle and applications of the SNPlex Genotyping System, as well as a set of single nucleotide polymorphism selection tools and validated assay resources that accelerate the assay design process. We developed the control pool, an oligonucleotide ligation probe set for training and quality-control purposes, which interrogates 48 SNPs simultaneously. We present performance data from this control pool obtained by testing genomic DNA samples from 44 individuals. in addition, we present data from a study that analyzed 521 SNPs in 92 individuals. Combined, both studies show the SNPlex Genotyping system to have a 99.32% overall call rate, 99.95% precision, and 99.84% concordance with genotypes analyzed by TaqMan probe-based assays. The SNPlex Genotyping System is an efficient and reliable tool for a broad range of genotyping applications, supported by applications for study design, data analysis, and data management.     

用于“野生小家鼠来源一号染色体替换系”构建的PCR-LDR分型系统

目的 建立用于“野生小家鼠来源一号染色体替换系”构建的PCR-LDR (polymerase chain reaction and ligase detection reaction,PCR-LDR)分型系统.方法 采用易于判断的二元性遗传标记单核苷酸多态性位点( single nuclear polymorphism...     

Rapid genotyping of three polymorphisms in the LBP gene using a triplex pyrosequencing approach

We described a triplex polymerase chain reaction (PCR) and triplex pyrosequencing assay which allowed a simultaneous determination of three tag single nuleotide polymorphisms (tag SNPs) in the lipopolysaccharide-binding protein (LBP) gene: rs1780623, rs11536972 and rs2232618. This method enables a fast and cost-effective genotyping and a simultaneous determination of the three tag SNPs.     

Simultaneous genotyping of multiplex single nucleotide polymorphisms of the K-ras gene with a home-made kit

Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is important in the human genome project. Here an automated fluorescent method that can rapidly and accurately genotype multiplex known SNPs was developed by using a homemade kit, which has lower cost but higher resolution than commercial kit. With this method, oncogene K-ras was investigated, four known SNPs of K-ras gene exon 1 in 31 coloerctal cancer patients were detected. Results indicate that mutations were present in 8(26%) of 31 patients, and most mutations were localized in codon 12. The presence of these mutations is thought to be a critical step and plays an important role in human colorectal carcinogenesisas.     

Development of Genotyping Methods for Single Nucleotide Polymorphism in the Human Pancreatic Ribonuclease Gene (RNASE1) and Their Application to Population Studies

Two potential single nucleotide polymorphisms [SNPs; rs1804215 (G979T) and rs11545379 (G1169T)] have been identified in the human pancreatic ribonuclease, RNase 1, gene (RNASE1) that could give rise to an amino acid substitution in the protein, but relevant population data are not available. We have developed genotyping methods for each SNP using the mismatched PCR-restriction fragment length polymorphism technique. These methods are advantageous in comparison with other SNP genotyping methods because they are technically simpler and do not require specialized instruments. We applied these genotyping methods to examine the genotype distribution of each SNP in four populations, including Japanese populations living in two prefectures, an Ovambo population, and a Turkish population. In all the populations studied, however, only a single genotype for each SNP was found. Therefore, irrespective of differences in ethnic groups, RNASE1 might show markedly low heterogeneity in its genetic structure with regard to these SNPs.     

Touchdown thermocycling program enables a robust single nucleotide polymorphism typing method based on allele-specific real-time polymerase chain reaction

Different methods have been developed for single nucleotide polymorphism (SNP) typing during recent years. Allele-specific polymerase chain reaction (ASPCR) is a cost-saving method that scores SNPs by difference of the PCR efficiency of allele-specific primers. However, ASPCR for SNP typing is notoriously confounded for its locus-specific unpredictability and the laborious gel electrophoresis. In the current study, we investigated the real-time kinetics of ASPCR and found that a simple touchdown thermocycling protocol improved its specificity significantly. Combined with real-time PCR, we developed a homogeneous genotyping method and scored more than 1000 genotypes, including all transition and transversion SNPs. A clear genotyping result was identified and validated the robustness of the method. Optimization of reactions and intrinsic modification of allele-specific primers, a laborious process but one that is repeatedly reported to be inevitable for successful ASPCR, was proved to be unnecessary with our method. Accuracy was confirmed with mass spectrometry. These characters enabled real-time ASPCR with the touchdown thermocycling protocol being very competitive among various SNP typing methods for large-scale genetic studies.     

An alternative method for genotyping of the <Emphasis Type="Italic">ACE</Emphasis> I/D polymorphism

The mistyping of the angiotensin I-converting enzyme insertion/deletion (ACE I/D) has been well documented, and new methods have been suggested here to improve the genotyping efficiency. Buccal cell samples were collected from 157 young Caucasians, and genotyped using previously known and newly developed PCR amplification genotyping techniques, as well as PCR-RFLP tests for three single nucleotide polymorphisms (rs4327, rs4341 and rs4343). Inconsistent genotyping results were found when using only the PCR amplification genotyping techniques across repeated attempts (8% to 45%), however, individual SNP genotyping was highly consistent (100%). Two SNPs (rs4341 and rs4343) were in complete LD and SNP rs4327 was in high LD with the ACE I/D. The ACE I/D was in HW equilibrium in the portion of the population with consistent genotyping results, whereas the three SNPs were not in HW equilibrium. The mistyping of ACE I/D by only PCR amplification can be improved using alternative methods.     

SNPstream UHT: ultra-high throughput SNP genotyping for pharmacogenomics and drug discovery

Single nucleotide polymorphism (SNP) genotyping is playing an increasing role in genome mapping, pharmacogenetic studies, and drug discovery. To date, genome-wide scans and studies involving thousands of SNPs and samples have been hampered by the lack of a system that can perform genotyping with cost-effective throughput, accuracy, and reliability. To address this need, Orrhid has developed an automated, ultra-high throughput system, SNPstream UHT, which uses multiplexed PCR in conjunction with our next generation SNP-IT tag array single base extension genotyping technology The system employs oligonucleotide microarrays manufactured in a 384-well format on a novel glass-bottomed plate. Multiplexed PCR and genotyping are performed in homogeneous reactions, and assay results are read by direct two-color fluorescence on the SNPstream UHTArray Imager. The systems flexibility enables large projects involving thousands of SNPs and thousands of samples as well as small projects that have hundreds of SNPs and hundreds of samples to be done cost effectively. We have successfully demonstrated this system in greater than 1,000,000 genotyping assays with >96% of samples giving genotypes with >99% accuracy     

焦磷酸测序测定SNP的常见问题与解决方法

目的:探讨焦磷酸测序技术对单核苷酸多态性分型因测序图谱中存在的一些典型问题而导致分型结果不准确的解决方法。方法:以VKORC1基因1639 GA位点、CYP2C19基因636 GA位点及UGT1A1基因TA重复序列(TA)6(TA)7的多态性检测为例,分别采用优化PCR条件、改变测序时dNTP的加入顺序以及设立外标校正的方法来解决上述问题,从而提高焦测序对SNP分型的准确性。结果:通过升高PCR退火温度,可以显著提高VKORC1基因的扩增特异性,降低了测序图谱中非特异性信号峰强度;通过优化测序时dNTP的加入顺序,CYP2C19基因636 GA位点的准确分型结果可通过观察测序图谱中相关信号峰的有无而简单获得,避免了比较信号峰的相对强度;通过比较待测样本与已知基因型的外标样本的测序图谱来确定待测样本的基因型,提高了对UGT1A1基因TA重复序列(TA)6(TA)7多态性的分型准确性。结论:本文针对焦测序在测定SNP时的常见问题所提出的相应解决方法不仅简单、经济有效,而且在临床应用方面具有可靠性。     

PCR amplification on magnetic nanoparticles: application for high-throughput single nucleotide polymorphism genotyping

A novel approach for the genotyping of single nucleotide polymorphisms (SNPs) based on solidphase PCR on magnetic nanoparticles (MNPs) is described. PCR products were amplified directly on MNPs. The genotypes of a given SNP were differentiated by hybridization with a pair of allele-specific probes labeled with dual-color fluorescence (Cy3, Cy5). The results were analyzed by scanning the microarray printed with the denatured fluorescent probes on an unmodified glass slide. Electrophoresis analysis indicated that PCR could proceed successfully when MNPs-bound primers were used. Furthermore, nine different samples were genotyped and their fluorescent signals were quantified. Genotyping results showed that three genotypes for the locus were very easily discriminated. The fluorescent ratios (match probe:mismatch probe signal) of homozygous samples were over 9.3, whereas heterozygous samples had ratios near 1.0. Without any purification and concentration of PCR products, this new MNP-PCR based genotyping assay potentially provides a rapid, labor-saving method for genotyping of a large number of individuals.