The use of modified primer competitors to enhance yields and specificity of HLA class I amplification by polymerase chain reaction (PCR).
Various PCR based techniques have been developed for genomic HLA typing. The fidelity of these techniques is highly dependent upon the specificity of the primers for the given HLA locus. Due to the high degree of homology between HLA class I loci, few primer sites that selectively amplify genes at a given HLA class I locus may be identified. To avoid coamplification of homologous loci, we designed and applied primer competitors for PCR amplification of HLA-A, -B and -C loci. Primer competitors identical to the 3' end of the specific primers and completely degenerate in the 5' end were designed and titrated into the respective HLA-locus PCR mixtures. We found that inclusion of primer competitors in the PCR reaction increased the specificity and yields of HLA class I amplifications, in particular when crude DNA preparation was used as template. This was particularly true for DNA preparations of low quality. The method described here may be useful for various protocols for downstream genomic typing of HLA-A, -B and -C alleles. In particular the method is useful when DNA is in scarce supply (i.e., for extensive PCR based allelic typing) or when high yields and locus specificity of amplicons are needed (i.e., sequencing-based typing).
Etokebe
G E
GE
Institute of Immunology, National University Hospital, Oslo, Norway. g.e.etokebe@labmed.uio.no
Wiencke
K
K
Haug
T
T
Spurkland
A
A
D000483:Alleles; D001483:Base Sequence; D001667:Binding, Competitive; D017931:DNA Primers / Q000235:genetics*; D005805:Genes, MHC Class I; D006680:HLA Antigens / Q000235:genetics*; D015234:HLA-A Antigens / Q000235:genetics; D015235:HLA-B Antigens / Q000235:genetics; D015236:HLA-C Antigens / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D006680:HLA Antigens; D015234:HLA-A Antigens; D015235:HLA-B Antigens; D015236:HLA-C Antigens