Direct detection of insertion/deletion polymorphisms in an autosomal region by analyzing high-density markers in individual spermatozoa.
Direct detection of insertion/deletion polymorphisms in an autosomal region by analyzing high-density markers in individual spermatozoa.
复制标题
通过分析单个精子中的高密度标记，直接检测常染色体区域中的插入/缺失多态性。
DOI：
10.1086/344713
复制
发表时间：
2002
影响因子：
9.8
通讯作者：
Li,Honghua
中科院分区：
文献类型：
--
作者：
Pramanik,Sreemanta;Li,Honghua
Direct polymerase chain reaction (PCR) detection of insertion/deletion (indel) polymorphisms requires sample homozygosity. For the indel polymorphisms that have the deletion allele with a relatively low frequency in the autosomal regions, direct PCR detection becomes difficult or impossible. The present study is, to our knowledge, the first designed to directly detect indel polymorphisms in a human autosomal region (i.e., the immunoglobulin VHregion), through use of single haploid sperm cells as subjects. Unique marker sequences (n=32), spaced at ∼5-kb intervals, were selected near the 3′ end of the VHregion. A two-round multiplex PCR protocol was used to amplify these sequences from single sperm samples from nine unrelated healthy donors. The parental haplotypes of the donors were determined by examining the presence or absence of these markers. Seven clustered markers in 6 of the 18 haplotypes were missing and likely represented a 35–40-kb indel polymorphism. The genotypes of the donors, with respect to this polymorphism, perfectly matched the expectation under Hardy-Weinberg equilibrium. Three VHgene segments, of which two are functional, are affected by this polymorphism. According to these results, >10% of individuals in the human population may not have these gene segments in their genome, and ∼44% may have only one copy of these gene segments. The biological impact of this polymorphism would be very interesting to study. The approach used in the present study could be applied to understand the physical structure and diversity of all other autosomal regions.
