Determination of gene organization in individual haplotypes by analyzing single DNA fragments from single spermatozoa.
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.
Cui
X
X
Coriell Institute for Medical Research, 401 Haddon Avenue, Camden, NJ 08103, USA.
Li
H
H
D002883:Chromosomes, Human, Pair 14; D002884:Chromosomes, Human, Pair 15; D004247:DNA / Q000235:genetics*; D053938:DNA Fragmentation; D017931:DNA Primers; D006239:Haplotypes; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D013094:Spermatozoa / Q000378:metabolism*
D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D004247:DNA