A Rapid Molecular Approach for Chromosomal Phasing
A Rapid Molecular Approach for Chromosomal Phasing
复制标题
DOI：
10.1371/journal.pone.0118270
复制
发表时间：
2015-03-04
期刊：
影响因子：
3.7
通讯作者：
McCarroll, Steven A.
中科院分区：
文献类型：
--
作者：
Regan, John F.;Kamitaki, Nolan;McCarroll, Steven A.
确定序列变异对的染色体相位-特定等位基因作为单倍型的排列-是分子遗传学中的常规挑战。在这里，我们描述了Drop-阶段，一种用于快速确定DNA序列变体对（相隔1-200 kb）的阶段而无需克隆或手动单分子稀释的分子方法。在每个液滴阶段反应中，基因组DNA片段与等位基因特异性荧光探针一起在单个反应孔中以数万纳升大小的液滴分离。物理连锁的等位基因分配到相同的液滴中，在荧光团跨液滴的共分布中揭示它们的染色体相。我们通过对三人组成员进行定相（揭示与遗传信息的100%一致性）证明了该方法的准确性，并通过对囊性纤维化患者基因组中11-116 kb基因组距离处的CFTR等位基因进行定相证明了常见的临床应用。Drop-Phase快速（需要不到4小时），可扩展（数百个样品），并且在长基因组距离（200 kb）下有效。
Determining the chromosomal phase of pairs of sequence variants - the arrangement of specific alleles as haplotypes - is a routine challenge in molecular genetics. Here we describe Drop-Phase, a molecular method for quickly ascertaining the phase of pairs of DNA sequence variants (separated by 1-200 kb) without cloning or manual single-molecule dilution. In each Drop-Phase reaction, genomic DNA segments are isolated in tens of thousands of nanoliter-sized droplets together with allele-specific fluorescence probes, in a single reaction well. Physically linked alleles partition into the same droplets, revealing their chromosomal phase in the co-distribution of fluorophores across droplets. We demonstrated the accuracy of this method by phasing members of trios (revealing 100% concordance with inheritance information), and demonstrate a common clinical application by phasing CFTR alleles at genomic distances of 11-116 kb in the genomes of cystic fibrosis patients. Drop-Phase is rapid (requiring less than 4 hours), scalable (to hundreds of samples), and effective at long genomic distances (200 kb).