SNP detection via RecA-mediated ligation and rolling circle amplification

通过 RecA 介导的连接和滚环扩增进行 SNP 检测

• 批准号: 7480163
• 负责人: ROBERT E WAGNER
• 金额: $ 13.6万
• 依托单位: GENE CHECK, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7480163
• 关键词: Alleles; Animal Diseases; Area; Biological Assay; DNA; Detection; Diagnosis; Disease; Epidemiology; Escherichia coli; Flow Cytometry; Forensic Medicine; Genes; Genetic Recombination; Genomics; Genotype; Goals; Human; Immobilization; Individual; Ligation; Maps; Mediating; Methods; Microspheres; Numbers; Oligonucleotides; Polymerase Chain Reaction; Predisposition; Public Health; Rec A Recombinases; SNP genotyping; Sampling; Sheep; Single Nucleotide Polymorphism; Spottings; System; Tube; adduct; cost; design; gene discovery

DESCRIPTION (provided by applicant): Gene Check has developed a method of SNP detection, RecA Mediated Ligation (RML), that combines the extremely precise homology searching ability of the E. coli recombination protein, RecA, with the proven SNP detection method, oligonucleotide ligation. RML is an extremely powerful, yet simple and low cost method of SNP detection. It is the goal of this project to adapt the RML method to a microarray format and to combine it with Rolling Circle Amplification (RCA) to allow direct detection of SNPs from genomic DNA. RML products will be immobilized by hybridization to sequence specific oligonucleotides on individual microarray spots. The immobilization oligonucleotides will be designed with short regions of complementarity to each of the RML oligonucleotides to allow immobilization only of RML products and not of the component RML oligonucleotides. Following hybridization to an array, RML products will be detected by means of allele specific RCA. Allele specific RML oligonucleotides will be prepared with "universal" RCA primer sequences additions. Two RCA circles, with sequences complementary to these primer sequences will be used to extend the RML products and to create sequences for decorator probe annealing. By using different decorator probe sequences on the two RCA circles, it will be possible to genotype any number of two-allele SNPs on a single array. Four well-characterized SNPs from sheep genomic DNA will be genotyped individually and multiplexed. The RML-RCA method will be the most powerful method of SNP genotyping currently available and will have immediate and wide spread application. It will allow direct genotyping from small amounts of genomic DNA (on the order of 100ng), will not require denaturation or amplification of target DNA, will be capable of extremely high order multiplexing (greater than 1000 SNPs from a single sample in a single tube), and will be able to genotype large numbers of SNPs rapidly and inexpensively.

描述（由申请人提供）：Gene Check开发了一种SNP检测方法，RecA介导的结扎（RML），该方法结合了大肠杆菌重组蛋白RecA极其精确的同源性搜索能力和已证实的SNP检测方法，寡核苷酸结扎。RML是一种非常强大、简单、低成本的SNP检测方法。该项目的目标是使RML方法适应微阵列格式，并将其与滚动圈扩增（RCA）相结合，从而可以直接检测基因组DNA中的snp。RML产物将通过杂交固定，在单个微阵列点上对特定的寡核苷酸进行测序。固定寡核苷酸将被设计成与每个RML寡核苷酸互补的短区域，以允许只固定RML产物而不固定RML寡核苷酸成分。杂交到阵列后，RML产物将通过等位基因特异性RCA检测。等位基因特异性RML寡核苷酸将通过添加“通用”RCA引物序列制备。两个RCA圈，与这些引物序列互补的序列将用于扩展RML产品并创建装饰探针退火的序列。通过在两个RCA环上使用不同的修饰探针序列，可以在单个阵列上对任意数量的双等位基因snp进行基因分型。将对来自绵羊基因组DNA的四个具有良好特征的snp进行基因分型和多重分型。RML-RCA方法将是目前最强大的SNP基因分型方法，具有直接和广泛的应用前景。它将允许从少量基因组DNA（约100ng）中直接进行基因分型，不需要对目标DNA进行变性或扩增，将能够进行高阶多路复用（在单个试管中从单个样品中获得超过1000个snp），并且能够快速且廉价地对大量snp进行基因分型。