Cui X, Li H
Coriell Institute for Medical Research, 401 Haddon Avenue, Camden, NJ 08103, USA.
Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10791-6. doi: 10.1073/pnas.95.18.10791.
To determine human Ig heavy chain variable region (VH) gene segment organization on individual homologous chromosomes, an efficient approach has been developed. Single spermatozoa were used as subjects for the study. Upon sperm lysis, VH regions in each sperm were randomly sheared into fragments by the random Brownian force. The fragments were separated from each other by aliquoting the lysate into a certain number of tubes. The gene segments in the VH1 and VH4 families in each tube were identified by denaturing gradient gel electrophoresis after PCR amplification. The polymorphic VH sequences were used to determine the parental origins of the analyzed sperm. VH segment organization in the parental haplotypes was determined by aligning the overlapping fragments from the spermatozoa with the corresponding haplotypes. Based on this comparison between the resulting haplotype maps and the composite map reported previously, the VH region on chromosome 14 could be subdivided into four portions. The numbers and compositions of the VH gene segments differ considerably among the maps in two portions, but are highly conserved in the other two. The data also indicate that the VH region on chromosome 15 may contain a large duplicated block with copy number varying among haplotypes. The approach used in the present study may be used to construct high-resolution haplotype maps without molecular cloning.
为了确定个体同源染色体上人类免疫球蛋白重链可变区（VH）基因片段的组织方式，已开发出一种有效的方法。研究以单个精子作为研究对象。精子裂解后，每个精子中的VH区域通过随机布朗力随机剪切为片段。通过将裂解物分装到一定数量的试管中，使片段彼此分离。在PCR扩增后，通过变性梯度凝胶电泳鉴定每个试管中VH1和VH4家族的基因片段。利用多态性VH序列确定所分析精子的亲本来源。通过将精子中的重叠片段与相应单倍型进行比对，确定亲本单倍型中的VH片段组织方式。基于所得单倍型图谱与先前报道的复合图谱之间的这种比较，14号染色体上的VH区域可细分为四个部分。在其中两个部分的图谱中，VH基因片段的数量和组成差异很大，但在另外两个部分中高度保守。数据还表明，15号染色体上的VH区域可能包含一个大的重复片段，其拷贝数在单倍型之间有所不同。本研究中使用的方法可用于构建无需分子克隆的高分辨率单倍型图谱。
