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PMID: 2320591 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 87 ·No. 7 ·1990-04-00 ·Pages 2833-7

Ennis PD, Zemmour J, Salter RD, Parham P

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.

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
Chemicals
HLA-A Antigens HLA-B Antigens HLA-B57 antigen
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Ennis P D
Department of Cell Biology, Stanford University, CA 94305.
Zemmour J
Salter R D
Parham P
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24 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.87.7.2833
Published
1990-04-00
Pages
2833-7
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC53785
Subset
IM
Grants
NIAID NIH HHS · AI24258 · United States
Databases
GENBANK
M32317, M32318, M32319, M32320, M32321, M32322
External Links
PubMed source DOI: 10.1073/pnas.87.7.2833 PMC Full Text View journal details
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