High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification.
High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification.
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DOI：
10.1186/1471-2164-2-4
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发表时间：
2001
期刊：
影响因子：
4.4
通讯作者：
Lasken RS
中科院分区：
文献类型：
--
作者：
Faruqi AF;Hosono S;Driscoll MD;Dean FB;Alsmadi O;Bandaru R;Kumar G;Grimwade B;Zong Q;Sun Z;Du Y;Kingsmore S;Knott T;Lasken RS
单核苷酸多态性（SNPs）是复杂性状和药物基因组学分析的基础。然而，大型SNP数据库的可用性强调了对相应简单、稳健性和可扩展性的廉价SNP基因分型方法的需求。我们描述了一种基于溶液的人类基因组DNA SNP基因分型的微滴板方法。该方法基于等位基因的识别，通过连接开环探针，然后使用荧光引物对信号进行滚圈放大。只有与SNP互补的3'碱基探针通过结扎循环化。通过结扎进行SNP评分，对与SNP不匹配的探针进行100,000倍的区分。该方法用于从192个基因组DNA样本中以高通量格式对10个snp进行基因型分析。直接从基因组DNA进行分析消除了像许多其他基因分型方法那样需要预先扩增目标的需要。从1 ng基因组DNA中进行基因分型，证明了该方法的敏感性。我们证明了该方法可以检测到圆形探针的单个分子。与同质格式的兼容性和分析少量基因组DNA的能力满足自动化，高通量SNP评分的严格要求。
Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation. SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe. Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.