Genotyping by PCR-ELISA of a complex polymorphic region that contains one to four copies of six highly homologous human VH3 genes.
The Humhv3005 human VH gene is located in an intricate locus that encompasses for each haplotype a combination of one to four copies of six highly homologous VH3 genes. To assess the complexity of this region, we developed a polymerase chain reaction-enzyme-derived immunosorbent assay (PCR-ELISA) method capable of detecting each of the VH3 genes. The method consisted of amplification of selected germline VH3 genes with a biotinylated primer, covalent capture of the amplicons onto streptavidin-coated wells, and quantitative typing of the bound VH3 genes with diagnostic oligonucleotides. Pilot studies of two DNA samples with known presence or absence of hv3005 [according to a characteristic BamH1 restriction fragment-length polymorphism (RFLP)] yielded the expected results. Subsequent analysis of 100 additional DNA samples with the known EcoR1 RFLP of hv3005 showed a complete match between the absence of the 9.4-kb hybridizing band and lack of hv3005-like genes, as determined by PCR-ELISA. Importantly, the PCR-ELISA analyses of these 102 genomic DNA samples revealed two new haplotypes in the complex hv3005 region. Combined, these data demonstrate the usefulness and efficiency of this new technique to ascertain the presence or absence of six highly homologous genes in an unusually heterogeneous duplication-insertion-deletion region. In the future, a similar strategy may be used to dissect other similarly complex VH genetic loci.
Cho
C S
CS
Department of Medicine, University of California, San Diego, La Jolla 92093-0663, USA.
Wang
X
X
Zhao
Y
Y
Carson
D A
DA
Chen
P P
PP
D000595:Amino Acid Sequence; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018628:Gene Dosage; D005803:Genes, Immunoglobulin; D005838:Genotype; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region