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）的研究已成为人类后基因组时代的主要内容之一。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。文章系统地介绍了最新发展的几种SNP检测技术的原理和检测平台,详细阐述了等位基因特异性杂交、内切酶酶切技术、引物延伸法、寡核苷酸连接反应等SNP检测原理,以及平板读数仪、基因芯片、微球阵列技术和质谱仪等检测平台,并对SNP高通量检测技术的发展进行了展望。     

海洋生物单核苷酸多态性基因分型技术的研究进展

单核苷酸多态性(single nucleotide polymorphism,SNP)是一类广泛分布于基因组中由单个碱基差异引起的DNA序列变异,SNP标记是第三代分子标记的代表。随着大规模测序技术的快速发展,大量的候选SNP位点被发现,候选SNP位点的发掘需要合适的分型技术。从等位基因分型机制、反应方式和检测等位基因方法等方面介绍当前海洋生物SNP分型技术的研究进展,以期为不同试验目的的研究选择合适的SNP分型技术提供参考。     

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.     

等位基因特异性引物PCR技术及其应用研究

目的：研究建立等位基因特异性引物PCR技术体系,并将其应用于基因单核苷酸多态性研究工作。方法：通过美国国家生物信息中心(NCBI)的genBank获取基因序列及其相应位点的SNP信息。利用Primer5．0软件设计引物,并经NCBI的Blast2．0软件检验其特异性。结果：建立了单一等位基因特异性引物PCR(SASP—PCR)与嵌套式等位基因特异性引物PCR(NASP-PCR)两种技术,并应用于β2肾上腺素受体及内皮源性一氧化氮合酶基因单核苷酸多态性的研究,证实该技术的稳定性和优越性。结论：等位基因特异性引物PCR技术是一种更为简便、特异性较高、费用少的、便于推广的SNP检测方法,特别是在群体基因单核苷酸多态性研究中更有优势。     

人类基因组DNA单核苷酸多态性的检测方法

单核苷酸多态性（SNP）作为新的遗传标记对基因定位及相关疾病的研究意义重大。本文对近年来9种SNP检测方法的原理、应用及优缺点,包括基于FRET原理的Taqman法和分子灯塔法;基于分子杂交技术的寡核苷酸连接分析、等位基因特异性寡核苷酸探针杂交法、动态等位基因特异性杂交法及DNA芯片法;及质谱法、变性－高压液相色谱法和单个碱基延伸标记法进行综述。     

高分辨率熔解曲线HRM在SNP检测中的应用

自然界生物具有丰富多样性,其本质在于DNA序列的多样性,单核苷酸多态性(SNP)是DNA序列多样性的基本单位,亦为生物进化和选择的动力源。越来越多的研究表明,SNP与人类的疾病、动植物的生长性状显著相关,对SNP的检测亦逐渐成为疾病检测和生物分子育种的重要手段。与传统的SNP检测技术相比较,高分辨率熔解曲线(HRM)具有高通量、高灵敏度、高特异性、快速、操作简单和重复性好的特点,能够快捷、有效筛查生物群体中的SNP位点,将逐渐成为SNP检测的主流技术。本文对HRM原理、方法、特点和应用进行综述,以飨读者。     

用基因芯片检测单核苷酸多态性反应原理

基因芯片技术因其高通量、高效率的特点被用于第三代遗传标记单核苷酸多态性位点的筛选。近几年SNP芯片研究在反应原理方面取得了重大进展,其中包括基于核酸杂交反应的芯片、基于单碱基延伸反应的芯片、基于等位基因特异性引物延伸反应的芯片、基于“一步法”反应的芯片、基于引物连接反应的芯片、基于限制性内切酶反应的芯片、基于蛋白DNA结合反应的芯片,及基于荧光分子DNA结合反应的芯片。比较了上述各种芯片技术方法的优劣,为进一步科研工作的开展提供了参照,并综述了SNP芯片在疾病基因组学、药物遗传学和个体识别等科研领域的应用,且对其今后的发展方向进行了阐述。     

单核苷酸多态性检测技术研究进展

单核苷酸多态性(SNP)是个体中最普遍的遗传变异,主要是指在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性。SNP作为第三代遗传标记,具有遗传稳定性强、数量多、分布广等特点,被广泛应用于群体遗传学、疾病相关基因定位研究中,并在疾病的早期诊断、预防、治疗,研究遗传因素对药物代谢的影响,指导药物临床使用等方面发挥重要的作用。鉴于此,建立适合各级平台应用的准确、高效、稳定的SNP分型技术十分必要。目前SNP检测与分析技术众多,在原理上差别很大,适用范围也不尽相同。本文综述了目前临床应用较广的SNP分型方法,简要介绍了各种技术的检测原理和优缺点,并对SNP分型技术前景进行了展望。     

四引物扩增受阻突变体系PCR技术及其在动植物遗传育种研究中的应用

四引物扩增受阻突变体系PCR(Tetra-primer amplification refractory mutation system PCR,Tetra-primer ARMS PCR)技术是一种在普通PCR基础上发展起来的单核苷酸多态性(SNP)分型技术。该项技术综合了扩增受阻突变体系(Amplification refractory mutation system,ARMS)和四引物PCR(tetra-primer PCR)技术的优点,是对等位基因特异性PCR法的改良。它具有操作简便、分型快速、费用低廉等特点,在国内外生命科学领域尤其是遗传育种领域的应用越来越广泛。本文介绍了四引物扩增受阻突变体系PCR的技术原理及优势、结果检测手段和反应体系改进方法,并在此基础上对该技术在遗传育种研究中的应用进行综述。     

SNPs及其在水产动物遗传学与育种学研究中的应用

1 SNP简介1.1 SNP的概念单核苷酸多态性(Single nucleotide polymorphism,SNP)指基因组DNA序列中某个特定位点的单个核苷酸发生变异而引起的序列多态性,包括单碱基的转换、颠换、插入及缺失等形式,其中一种等位基因在群体中的频     

双色荧光杂交芯片在检测 P 1A 1M P I 基C 因多态性中的应用

目的：应用一种新的高通量SNP检测方法-双色荧光杂交芯片技术检测CYPIA1 MspI基因多态性。方法：收集江苏汉族人群原发性肺癌患者75例和相应对照77例,应用双色荧光杂交芯片技术检测了152例样本的CYPIAI基因MspI基因多态性,并应用PCR-RFLP技术验证双色荧光杂交芯片的特异性。结果：152例样本的CYPIAI基因双色荧光杂交芯片技术分型结果与PCR-RFLP结果完全相符,两种方法的基因型分型结果具有很好的一致性。结论：双色荧光杂交芯片技术是一个高通量SNP检测的良好工具,特异性高,在大规模人群SNP筛检中具有良好的发展前案。     

SNP allele frequency estimation in DNA pools and variance components analysis

The estimation of single nucleotide polymorphism (SNP) allele frequency in pooled DNA samples has been proposed as a cost-effective approach to whole genome association studies. However, the key issue is the allele frequency window in which a genotyping method operates and provides a statistically reliable answer. We assessed the homogeneous mass extend assay and estimated the variance associated with each experimental stage. We report that a relationship between estimated allele frequency and variance might exist, suggesting that high statistical power can be retained at low, as well as high, allele frequencies. Assuming this relationship, the formation of subpools consisting of 100 samples retains an effective sample size greater than 70% of the true sample size, with a savings of 11-fold the cost of an individual genotyping study, regardless of allele frequency.     

基于荧光定量PCR扩增反应的SNP测定法

建立一种利用荧光定量PCR扩增反应进行单核苷酸多态性(SNP)快速测定的方法.以人β肾上腺素受体2基因中的Arg16Gly为研究对象,利用荧光染料SYBRGreenⅠ标记定量PCR产物,通过PCR生长曲线和融解曲线分析结果进行SNP分型.为提高SNP测定的特异性,分别在野生型和突变型等位基因的特异性引物3′端倒数第3个碱基位置,引入了一个人为错配碱基,使引物的错误延伸率显著降低,大大提高了SNP分析的准确性.通过DNA测序验证荧光定量PCR对β肾上腺素受体2基因中Arg16Gly分型结果的准确率.实验结果表明,所建立的方法操作简便,结果准确,适合进行大规模样品的SNP检测工作.     

应用SNPstream技术检测MASP2基因的多态性

目的：应用一种高通量单核苷酸多态性（SNP）检测方法——SNPstream技术检测甘露聚糖结合凝集素相关丝氨酸蛋白酶-2（MASP2）基因的多态性。方法：收集北京汉族人群SARS病例96例和正常对照96例,用SNPstream技术检测样本的MASP2基因多态性,并用PCR产物直接测序技术对其中一个位点rs2273346进行分型,以验证SNPstream技术的准确性。结果：192例样本的MASP2基因rs2273346位点SNPstream技术分型结果与测序结果完全相符,2种方法的基因型分型结果具有很好的一致性。结论：SNPstream技术是高通量SNP检测的良好工具,准确性高,所需样本量低,在大规模人群SNP筛检中具有良好的发展前景。     

Further development of multiplex single nucleotide polymorphism typing method, the DigiTag2 assay

A number of single nucleotide polymorphisms (SNPs) are considered to be candidate susceptibility or resistance genetic factors for multifactorial disease. Genome-wide searches for disease susceptibility regions followed by high-resolution mapping of primary genes require cost-effective and highly reliable technology. To accomplish successful and low-cost typing for candidate SNPs, new technologies must be developed. We previously reported a multiplex SNP typing method, designated the DigiTag assay, that has the potential to analyze nearly any SNP with high accuracy and reproducibility. However, the DigiTag assay requires multiple washing steps in manipulation and uses genotyping probes modified with biotin for each target SNP. Here we describe the next version of the assay, DigiTag2, which works with simple protocols and uses unmodified genotyping probes. We investigated the feasibility of the DigiTag2 assay by genotyping 96 target SNPs spanning a 610-kb region of human chromosome 5. The DigiTag2 assay is suitable for genotyping an intermediate number of SNPs (tens to hundreds of sites) with a high conversion rate (>90%), high accuracy, and low cost.     

A rapid, microplate SNP genotype assay for the leptinob allele

A nonsense mutation in the mouse leptin gene causes genetic obesity. As a result of extensive research in the field of obesity, the use of leptinob mice is widespread. This mutation renders mice sterile, creating the need to breed heterozygous mice. For this reason, leptinob genotyping is necessary. To date, gel-based assays have been used for genotyping. Using the Invader Plus assay for single nucleotide polymorphism (SNP) detection, we have developed a gel-free microplate SNP assay for genotyping leptinwt and leptinob alleles.     

SNP haplotype tagging from DNA pools of two individuals

Background  

DNA pooling is a technique to reduce genotyping effort while incurring only minor losses in accuracy of allele frequency estimates for single nucleotide polymorphism (SNP) markers.     

Multiplex genotyping of PCR products with MassTag-labeled primers.

A simple mass spectrometric based assay, the PinPoint assay, has previously been described for typing single nucleotide polymorphisms. The identity of a polymorphism is determined by mass differences of single base extended genotyping primers as determined by MALDI-TOF mass spectrometry. A simple method for multiplexing the assay is described, employing multiple primers with 5'oligo(dT) sequences (MassTags) which serve to mass discriminate the peaks of multiple extended and non-extended primers. The assay is extremely rapid and requires no labeling reagents.     

Gene mapping in the wild with SNPs: guidelines and future directions

One of the biggest challenges facing evolutionary biologists is to identify and understand loci that explain fitness variation in natural populations. This review describes how genetic (linkage) mapping with single nucleotide polymorphism (SNP) markers can lead to great progress in this area. Strategies for SNP discovery and SNP genotyping are described and an overview of how to model SNP genotype information in mapping studies is presented. Finally, the opportunity afforded by new generation sequencing and typing technologies to map fitness genes by genome-wide association studies is discussed.