Generation of Single-stranded DNA by the Polymerase Chain Reaction and Its Application to Direct Sequencing of the HLA-DQA Locus

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1988 Oct 1

PMID 3174659

Citations 390

Authors

U B Gyllensten

H A Erlich

Affiliations

Soon will be listed here.

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.

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