Human immunoglobulin VH4 sequences resolved by population-based analysis after enzymatic amplification and denaturing...

Exhaustive gene identification followed by assignment of the genes identified to corresponding loci is a key step in elucidating the physical structure of a multigene family. However, problems occur in this process because genes in a multigene family usually share a high degree of sequence identity and are highly polymorphic. To address these problems, an efficient population-based approach was developed. Using this approach, sequences in the human immunoglobulin VH4 family were amplified by PCR with family-specific primers. Denaturing gradient gel electrophoresis (DGGE) was used to separate the resulting sequences with either very similar or identical sizes and differing by as little as 1 base pair (bp). Eighteen distinct bands and 21 banding patterns were observed in the samples collected from 41 unrelated individuals. Of the 18 bands, 12 were polymorphic. No sample had all 18 bands. The estimated frequencies for the alleles represented by the 18 bands ranged from 1.2 to 100%. The 18 sequences differed from each other by 1-19 bases (0.7 to 13%) within the 145-146-bp amplified region. Sequences in eight bands (44%) were not reported previously. These results were used to assign the majority (14 out of 18) of the VH4 sequences to 10 loci. This PCR-DGGE method, in conjunction with a population-based assay, may also be used to study other multigene families including those involved in the development of the immune system.