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.     

单核苷酸多态性检测方法研究进展

：单核苷酸多态性（singlenucleotidepolymorphism,SNP）是指在基因组水平上由单个核苷酸的变异引起的一种DNA序列多态性。SNP作为第三代分子标记,具有数量多、分布广等特点,已成为人类后基因组时代的主要研究内容之一。单核苷酸多态性在医学研究、临床诊断、药物开发与合理用药、法医学、遗传学的发展方面具有重要意义。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。各种SNP分型检测方法都由等位基因特异性的识别反应和等住基因识别产物的分析检测两个部分组成。本文系统的介绍了引物延伸反应、序列杂交反应、酶连接反应、酶切割反应、核酸链构象差异反应等SNP检测的等位基因特异性的识别原理,以及质谱、荧光共振和偏振信号、化学发光、毛细管电泳测序、生物传感器等分析检测手段,并简要介绍了相关识别原理和分析检测手段的优缺点及应用范围,并对SNP检测技术的发展进行了展望。     

实时检测限制性内切酶活性的发夹型荧光探针

基于分子信标的原理 ,设计了一种发夹型荧光探针作为限制性内切酶作用的专一底物 ,来研究限制性内切酶的剪切作用。检测BglII和NcoI表明 ,这种探针不仅可以灵敏、实时地指示内切酶的活性 ,采用不同荧光标记的探针还可以同时特异地显示双酶切反应中两个酶各自的活性。并且以Rotor Gene 2 0 0 0实时荧光PCR仪作仪器检测 ,实现了高通量的操作。利用其特有的作变温曲线的功能 ,能很好的区分限制性内切酶与发夹型荧光探针的特异剪切与非特异的结合     

基于PCR-LDR平台的近交系小鼠遗传质量快速检测方法

目的建立基于PCR-LDR平台的近交系小鼠SNP快速分型方法,用于检测实验小鼠的遗传质量与品系纯度。方法利用可移植性极高的PCR-LDR技术,以常见近交系小鼠为研究对象,选取了21条染色体上的45个SNP位点,分别设计引物和探针,经过筛选和验证,建立了多重PCR-LDR(polymerase chain reaction and ligase detection reaction,PCR-LDR)分型方案。结果四组多重PCR-LDR可实现45个SNP位点的基因分型,其中43个、44个与45个SNP在样本中的检出率分别为100%、90.9%与36.4%。所有样本经分型确定为纯合体,并得到了常见近交系小鼠SNP位点信息。结论实现了常见近交系小鼠快速、高通量的基因分型,可用于遗传质量检测和品系鉴定。     

高通量SNP基因分型技术研究进展

在后基因组时代,单核苷酸多态性研究已迅速成为了生物医学许多领域的焦点。发展可靠、敏感、经济、稳定、高通量的SNP基因分型技术已迫在眉睫。本文主要着重于高通量SNP基因分型技术的原理、利弊以及这些技术在这个领域过去几年中的进展。     

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。     

Genome Sequencer FLX引领快速基因组测序时代的到来

焦磷酸法最早是应用在检测DNA甲基化和SNP位点分析的一项技术,在2005年底,此技术被用来进行基因组序列的测定,已经成为下一代高通量测序中最成熟的一种技术.本篇文章着重介绍了采用焦磷酸测序法的罗氏公司最新一代高通量基因组测序系统GS FLX的技术原理,操作过程和广泛的应用范围.     

TILLING技术在功能基因组学中的应用

TILLING(定向诱导基因组局部突变)技术是近年发展起来的一种高通量筛选化学诱变的点突变的技术,它利用专一识别点突变的核酸酶结合PCR来检测单核苷酸多态性(SNP)。TILLING技术起源于植物基因组研究,逐渐扩展到动物及人类功能基因组学的研究中。无论是筛选突变体还是研究特定基因的重要性,TILLING都具有高通量、自动化的优势。随着此项技术应用范围的扩展,从诱变剂和内切酶的选择到具体的操作方式,以及结果的识别和统计方法,都有了不少改进。在其他相关学科不断发展的大环境下,TILLING技术也在不断发展,其在功能基因组学研究中的作用也会更显著。     

变性高效液相色谱技术在单核苷酸多态性研究中的应用

人类基因组的单核苷酸多态性(SNPs)研究已成为后基因组时代最重要的内容和目的之一,随之而来的迫切任务是需要适合于自动化且高通量检测SNP的技术。变性高效液相色谱(DHPLC)是近几年发展起来的高效、快速筛检SNP的技术,因其检测SNP的高灵敏度、低成本以及全自动化操作等优点而备受关注。     

核酸检测技术在水产养殖病原诊断中的应用

目前核酸检测技术在水产养殖疾病诊断中已广泛应用。现就国内外研究发展动向,对分子杂交技术、PCR技术、PCR分子杂交联用技术、PCR免疫学联用技术、16SrRNA技术和限制性酶切技术等方法进行综述,详细阐明了各个技术的原理、特点及应用。病原体遗传背景的研究状况为决定是否应用这些方法的关键。     

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.     

高通量飞行时间质谱基因分型方法的研究

为了考察飞行时间质谱基因分型方法 (MALDI-TOF) 的位点分型成功率和分型结果质量的关系,分析了 96 个 SNPs 位点的近 10 000 个基因分型数据 (用 MALDI-TOF “4 重”实验方法检测 ). 结果显示,位点分型成功率和分型结果的质量显著正相关 . 分型成功率低于 82% 的 SNP 位点,其高质量结果占的比例开始逐渐降低 . 提示 82% 的分型成功率可以作为衡量分型结果质量的数据点 . 为了进一步提高通量并降低成本,在 MALDI-TOF “ 4 重”实验方法的基础上,发展了两种“准 8 重”实验方法 . 用新的实验方法检测了 95 个样本的 32 个 SNPs 位点 . 结果显示“混合准 8 重”实验方法与“ 4 重”实验方法相比无显著差异,而“复点准 8 重”的结果差于“ 4 重”分型方法 .     

Automated SNP genotype clustering algorithm to improve data completeness in high-throughput SNP genotyping datasets from custom arrays

High-throughput SNP genotyping platforms use automated genotype calling algorithms to assign genotypes. While these algorithms work efficiently for individual platforms, they are not compatible with other platforms, and have individual biases that result in missed genotype calls. Here we present data on the use of a second complementary SNP genotype clustering algorithm. The algorithm was originally designed for individual fluorescent SNP genotyping assays, and has been optimized to permit the clustering of large datasets generated from custom-designed Affymetrix SNP panels. In an analysis of data from a 3K array genotyped on 1,560 samples, the additional analysis increased the overall number of genotypes by over 45,000, significantly improving the completeness of the experimental data. This analysis suggests that the use of multiple genotype calling algorithms may be advisable in high-throughput SNP genotyping experiments. The software is written in Perl and is available from the corresponding author.     

New resources for genetic studies in Populus nigra: genome‐wide SNP discovery and development of a 12k Infinium array

Whole genome resequencing of 51 Populus nigra (L.) individuals from across Western Europe was performed using Illumina platforms. A total number of 1 878 727 SNPs distributed along the P. nigra reference sequence were identified. The SNP calling accuracy was validated with Sanger sequencing. SNPs were selected within 14 previously identified QTL regions, 2916 expressional candidate genes related to rust resistance, wood properties, water‐use efficiency and bud phenology and 1732 genes randomly spread across the genome. Over 10 000 SNPs were selected for the construction of a 12k Infinium Bead‐Chip array dedicated to association mapping. The SNP genotyping assay was performed with 888 P. nigra individuals. The genotyping success rate was 91%. Our high success rate was due to the discovery panel design and the stringent parameters applied for SNP calling and selection. In the same set of P. nigra genotypes, linkage disequilibrium throughout the genome decayed on average within 5–7 kb to half of its maximum value. As an application test, ADMIXTURE analysis was performed with a selection of 600 SNPs spread throughout the genome and 706 individuals collected along 12 river basins. The admixture pattern was consistent with genetic diversity revealed by neutral markers and the geographical distribution of the populations. These newly developed SNP resources and genotyping array provide a valuable tool for population genetic studies and identification of QTLs through natural‐population based genetic association studies in P. nigra.     

Conversion of array‐based single nucleotide polymorphic markers for use in targeted genotyping by sequencing in hexaploid wheat (Triticum aestivum)

Wheat breeders and academics alike use single nucleotide polymorphisms (SNP s) as molecular markers to characterize regions of interest within the hexaploid wheat genome. A number of SNP ‐based genotyping platforms are available, and their utility depends upon factors such as the available technologies, number of data points required, budgets and the technical expertise required. Unfortunately, markers can rarely be exchanged between existing and newly developed platforms, meaning that previously generated data cannot be compared, or combined, with more recently generated data sets. We predict that genotyping by sequencing will become the predominant genotyping technology within the next 5–10 years. With this in mind, to ensure that data generated from current genotyping platforms continues to be of use, we have designed and utilized SNP ‐based capture probes from several thousand existing and publicly available probes from Axiom® and KASP ? genotyping platforms. We have validated our capture probes in a targeted genotyping by sequencing protocol using 31 previously genotyped UK elite hexaploid wheat accessions. Data comparisons between targeted genotyping by sequencing, Axiom® array genotyping and KASP ? genotyping assays, identified a set of 3256 probes which reliably bring together targeted genotyping by sequencing data with the previously available marker data set. As such, these probes are likely to be of considerable value to the wheat community. The probe details, full probe sequences and a custom built analysis pipeline may be freely downloaded from the CerealsDB website (http://www.cerealsdb.uk.net/cerealgenomics/CerealsDB /sequence_capture.php).     

Evaluation of genotyping concordance for commercial bovine SNP arrays using quality‐assurance samples

SNP arrays are widely used in genetic research and agricultural genomics applications, and the quality of SNP genotyping data is of paramount importance. In the present study, SNP genotyping concordance and discordance were evaluated for commercial bovine SNP arrays based on two types of quality assurance (QA) samples provided by Neogen GeneSeek. The genotyping discordance rates (GDRs) between chips were on average between 0.06% and 0.37% based on the QA type I data and between 0.05% and 0.15% based on the QA type II data. The average genotyping error rate (GER) pertaining to single SNP chips, based on the QA type II data, varied between 0.02% and 0.08% per SNP and between 0.01% and 0.06% per sample. These results indicate that genotyping concordance rate was high (i.e. from 99.63% to 99.99%). Nevertheless, mitochondrial and Y chromosome SNPs had considerably elevated GDRs and GERs compared to the SNPs on the 29 autosomes and X chromosome. The majority of genotyping errors resulted from single allotyping errors, which also included the opposite instances for allele ‘dropout’ (i.e. from AB to AA or BB). Simultaneous allotyping errors on both alleles (e.g. mistaking AA for BB or vice versa) were relatively rare. Finally, a list of SNPs with a GER greater than 1% is provided. Interpretation of association effects of these SNPs, for example in genome‐wide association studies, needs to be taken with caution. The genotyping concordance information needs to be considered in the optimal design of future bovine SNP arrays.     

Identification of the genetic basis for complex disorders by use of pooling-based genomewide single-nucleotide-polymorphism association studies

We report the development and validation of experimental methods, study designs, and analysis software for pooling-based genomewide association (GWA) studies that use high-throughput single-nucleotide-polymorphism (SNP) genotyping microarrays. We first describe a theoretical framework for establishing the effectiveness of pooling genomic DNA as a low-cost alternative to individually genotyping thousands of samples on high-density SNP microarrays. Next, we describe software called "GenePool," which directly analyzes SNP microarray probe intensity data and ranks SNPs by increased likelihood of being genetically associated with a trait or disorder. Finally, we apply these methods to experimental case-control data and demonstrate successful identification of published genetic susceptibility loci for a rare monogenic disease (sudden infant death with dysgenesis of the testes syndrome), a rare complex disease (progressive supranuclear palsy), and a common complex disease (Alzheimer disease) across multiple SNP genotyping platforms. On the basis of these theoretical calculations and their experimental validation, our results suggest that pooling-based GWA studies are a logical first step for determining whether major genetic associations exist in diseases with high heritability.     

SNPMeta: SNP annotation and SNP metadata collection without a reference genome

The increase in availability of resequencing data is greatly accelerating SNP discovery and has facilitated the development of SNP genotyping assays. This, in turn, is increasing interest in annotation of individual SNPs. Currently, these data are only available through curation, or comparison to a reference genome. Many species lack a reference genome, but are still important genetic models or are significant species in agricultural production or natural ecosystems. For these species, it is possible to annotate SNPs through comparison with cDNA, or data from well‐annotated genes in public repositories. We present SNPMeta, a tool which gathers information about SNPs by comparison with sequences present in GenBank databases. SNPMeta is able to annotate SNPs from contextual sequence in SNP assay designs, and SNPs discovered through genotyping by sequencing (GBS) approaches. However, SNPs discovered through GBS occur throughout the genome, rather than only in gene space, and therefore do not annotate at high rates. SNPMeta can therefore be used to annotate SNPs in nonmodel species or species that lack a reference genome. Annotations generated by SNPMeta are highly concordant with annotations that would be obtained from a reference genome.     

水稻单核苷酸多态性及其应用现状

单核苷酸多态性（single nucleotide polymorphisms, SNPs）在水稻中数量多,分布密度高,遗传稳定性高。水稻SNPs的发现方法主要有对样本DNA的PCR产物直接测序、从SSR区段检测SNPs和从基因组序列直接搜索等。目前已有多种基因分型技术运用到了水稻SNPs检测,SNPs检测的高度自动化使水稻SNPs基因分型非常方便。单核苷酸多态性在水稻遗传图谱的构建、基因克隆和功能基因组学研究、标记辅助选择育种、遗传资源分类及物种进化等方面的应用具有巨大潜力。