Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Genome Res. 2009 Nov;19(11):2081-9. doi: 10.1101/gr.093856.109. Epub 2009 Oct 1.
Manipulating the mouse genome is a widespread technology with important applications in many biological fields ranging from cancer research to developmental biology. Likewise, correlations between copy number variations (CNVs) and human diseases are emerging. We have developed the reference-query pyrosequencing (RQPS) method, which is based on quantitative pyrosequencing and uniquely designed probes containing single nucleotide variations (SNVs), to offer a simple and affordable genotyping solution capable of identifying homologous recombinants independent of the homology arm size, determining the micro-amplification status of endogenous human loci, and quantifying virus/transgene copy number in experimental or commercial species. In addition, we also present a simple pyrosequencing-based protocol that could be used for the enrichment of homologous recombinant embryonic stem (ES) cells.
操纵小鼠基因组是一种广泛应用于从癌症研究到发育生物学等多个生物学领域的重要技术。同样，拷贝数变异（CNVs）与人类疾病之间的相关性也在不断显现。我们开发了参考-查询焦磷酸测序（RQPS）方法，该方法基于定量焦磷酸测序和独特设计的包含单核苷酸变异（SNVs）的探针，提供了一种简单且经济实惠的基因分型解决方案，能够独立于同源臂大小鉴定同源重组体，确定内源性人基因座的微扩增状态，并定量实验或商业物种中的病毒/转基因拷贝数。此外，我们还提出了一种简单的基于焦磷酸测序的方案，可用于同源重组胚胎干细胞（ES）的富集。
