A genotyping system capable of simultaneously analyzing >1000 single nucleotide polymorphisms in a haploid genome.
A genotyping system capable of simultaneously analyzing >1000 single nucleotide polymorphisms in a haploid genome.
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DOI：
10.1101/gr.2885205
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发表时间：
2005-02
期刊：
影响因子：
7
通讯作者：
Hui-Yun Wang;Minjie Luo;Irina V. Tereshchenko;Danielle M Frikker;X. Cui;James Y. Li;G. Hu;Y. Chu-Y
中科院分区：
文献类型：
--
作者：
Hui-Yun Wang;Minjie Luo;Irina V. Tereshchenko;Danielle M Frikker;X. Cui;James Y. Li;G. Hu;Y. Chu-Y
一个高通量的单核苷酸多态性（SNPs）的基因分型系统已经开发。使用该系统，可以在单个测定中分析>1000个SNP，其灵敏度允许使用单个单倍体细胞作为起始材料。在多重多态性序列扩增步骤中，使用不太可能具有非特异性和生产性相互作用的引物，而不是将通用序列连接到扩增子上。然后通过使用广泛使用的微阵列技术和简单的单碱基延伸测定来确定SNP的基因型。将三个SNP组（各自由>1000个SNP组成）并入该系统中。该系统用于分析24个人类基因组DNA样本。对5 ng的人基因组DNA，单探针平均检出率为98.22%，双探针在不同方向上的检出率为96.71%。当使用单个精子时，91.88%的SNP是可检测的，这与使用非常少的遗传标记时达到的水平相当。通过使用双探针分析，5 ng人类基因组DNA的平均基因分型准确率为99.96%，单个精子的平均基因分型准确率为99.95%。该系统可用于显著地促进大规模遗传分析，即使DNA模板的量非常有限或甚至高度降解，如从石蜡包埋的癌症标本中获得的，并且使许多不切实际的研究项目高度现实和负担得起。
A high-throughput genotyping system for scoring single nucleotide polymorphisms (SNPs) has been developed. With this system, >1000 SNPs can be analyzed in a single assay, with a sensitivity that allows the use of single haploid cells as starting material. In the multiplex polymorphic sequence amplification step, instead of attaching universal sequences to the amplicons, primers that are unlikely to have nonspecific and productive interactions are used. Genotypes of SNPs are then determined by using the widely accessible microarray technology and the simple single-base extension assay. Three SNP panels, each consisting of >1000 SNPs, were incorporated into this system. The system was used to analyze 24 human genomic DNA samples. With 5 ng of human genomic DNA, the average detection rate was 98.22% when single probes were used, and 96.71% could be detected by dual probes in different directions. When single sperm cells were used, 91.88% of the SNPs were detectable, which is comparable to the level that was reached when very few genetic markers were used. By using a dual-probe assay, the average genotyping accuracy was 99.96% for 5 ng of human genomic DNA and 99.95% for single sperm. This system may be used to significantly facilitate large-scale genetic analysis even if the amount of DNA template is very limited or even highly degraded as that obtained from paraffin-embedded cancer specimens, and to make many unpractical research projects highly realistic and affordable.