Rapid cloning of HLA-A,B cDNA by using the polymerase chain reaction: frequency and nature of errors produced in amplification

Proc Natl Acad Sci U S A. 1990 Apr;87(7):2833-7. doi: 10.1073/pnas.87.7.2833.

Abstract

A method for cloning full-length HLA-A,B cDNA (1.1 kilobases) by using the polymerase chain reaction (PCR) is described. Six HLA-A,B alleles (HLA-A2, -A25, -B7, -B37, -B51, and -B57) were cloned, and their structures were determined. Multiple PCR clones for each allele were sequenced to obtain both an accurate consensus sequence and an "authentic" clone having that sequence. Sequences from 50 clones encoding five different alleles permit assessment of the frequency and nature of PCR-produced errors. These include recombinations, deletions, and insertions in addition to point substitutions. Authentic clones were obtained at a frequency of between 30% and 70%, and analysis of three or four clones generally should be sufficient for characterization of an allele.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alleles
  • Amino Acid Sequence
  • Base Sequence
  • Cell Line
  • Cell Transformation, Viral
  • Cloning, Molecular / methods*
  • Exons
  • Genes, MHC Class I*
  • HLA-A Antigens / genetics*
  • HLA-B Antigens / genetics*
  • Humans
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods
  • Sequence Homology, Nucleic Acid

Substances

  • HLA-A Antigens
  • HLA-B Antigens
  • HLA-B57 antigen

Associated data

  • GENBANK/M32317
  • GENBANK/M32318
  • GENBANK/M32319
  • GENBANK/M32320
  • GENBANK/M32321
  • GENBANK/M32322