Rapid cloning of HLA-A,B cDNA by using the polymerase chain reaction: frequency and nature of errors produced in amplification.
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.
Ennis
P D
PD
Department of Cell Biology, Stanford University, CA 94305.
Zemmour
J
J
Salter
R D
RD
Parham
P
P
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002472:Cell Transformation, Viral; D003001:Cloning, Molecular / Q000379:methods*; D005091:Exons; D005805:Genes, MHC Class I; D015234:HLA-A Antigens / Q000235:genetics*; D015235:HLA-B Antigens / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012689:Sequence Homology, Nucleic Acid
D015234:HLA-A Antigens; D015235:HLA-B Antigens; C103707:HLA-B57 antigen