Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus.

TLDR

The polymerase chain reaction can enzymatically amplify single-copy genomic sequences, enabling mutation screening and linking amplification to automated sequencing. Using unequal primer concentrations, a single‑step PCR produces excess single‑stranded DNA of a chosen strand for direct sequencing or probe use, and allele‑specific oligonucleotides allow direct sequencing of individual alleles in heterozygotes. Application of these methods revealed eight HLA‑DQA alleles and three additional haplotypes and their association with serologically defined HLA‑DR and ‑DQ types.

Abstract

Single-copy sequences can be enzymatically amplified from genomic DNA by the polymerase chain reaction. By using unequal molar amounts of the two amplification primers, it is possible in a single step to amplify a single-copy gene and produce an excess of single-stranded DNA of a chosen strand for direct sequencing or for use as a hybridization probe. Further, individual alleles in a heterozygote can be sequenced directly by using allele-specific oligonucleotides either in the amplification reaction or as sequencing primers. By using these methods, we have studied the allelic diversity at the HLA-DQA locus and its association with the serologically defined HLA-DR and -DQ types. This analysis has revealed a total of eight alleles and three additional haplotypes. This procedure has wide applications in screening for mutations in human genes and facilitates the linking of enzymatic amplification of genes to automated sequencing.