SNP detection via RecA-mediated ligation and rolling circle amplification

描述(申请人提供)：基因检测已经开发出一种检测SNP的方法，RecA介导的连接(RML)，它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成熟的SNP检测方法，即寡核苷酸连接。RML是一种功能极其强大、操作简单、成本低廉的SNP检测方法。本项目的目标是将RML方法应用于微阵列格式，并将其与滚环扩增(RCA)相结合，以实现从基因组DNA中直接检测SNPs。RML产物将通过杂交固定，以在单个微阵列斑点上对特定的寡核苷酸进行测序。固定化寡核苷酸将被设计成与每个RML寡核苷酸具有短的互补区，以允许仅固定化RML产物，而不固定化组成RML的寡核苷酸。在与阵列杂交后，RML产物将通过等位基因特异性RCA进行检测。等位基因特异的RML寡核苷酸将与“通用的”RCA引物序列一起制备。两个RCA环，具有与这些引物序列互补的序列，将用于扩展RML产物并创建用于装饰物探针退火的序列。通过在两个RCA环上使用不同的修饰探针序列，将有可能在单个阵列上对任意数量的双等位基因SNPs进行基因分型。来自绵羊基因组DNA的四个特征良好的SNPs将分别进行基因分型和多重分析。RML-RCA方法将是目前最有效的SNP基因分型方法，并将立即得到广泛的应用。它将允许从少量基因组DNA(约100 ng)中直接进行基因分型，将不需要变性或扩增目标DNA，将能够进行极高顺序的多重(在单个试管中的单个样本中超过1000个SNP)，并将能够快速且廉价地对大量SNP进行基因分型。

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.