Definition of HLA-C alleles using sequence-specific oligonucleotide probes (PCR-SSOP).
Many new HLA-C locus alleles have recently been identified by DNA sequencing, and a molecular based method for their detection using PCR with sequence specific primers has been reported. However, other methods may be more appropriate for the identification of C locus alleles in larger studies. Here we describe one such system, based on PCR sequence specific oligonucleotide probes, (SSOP) for C locus typing. Advantages of SSOP typing compared to SSP are that it is easier to detect new alleles, more cost effective and less time consuming. We have developed a DNA typing method to identify the broad C locus antigens (including those not yet defined serologically) using a minimum of probes with one amplification. We use a C locus specific sense primer in exon 2 and a consensus antisense primer in exon 3, in a two-step PCR, giving a product of 710 bp. Probes were designed with similar melting temperatures (54-56 degrees C) that would identify as many alleles as possible. The method was established using DNA from B lymphoid cell lines of known C locus type, mostly 10th workshop homozygous cell lines, plus as many other sequenced cell lines as possible. The system was able to correctly identify their C locus types using only 26 probes. DNA was tested from a panel of serologically typed individuals which included many different heterozygous combinations. We found a high concordance of results, with all discrepancies being additional antigens identified by molecular typing, filling in serological blanks. We can identify all common heterozygote combinations using this method.
Kennedy
L J
LJ
ARC Epidemiology Research Unit, Manchester University, United Kingdom.
Poulton
K V
KV
Dyer
P A
PA
Ollier
W E
WE
Thomson
W
W
D000483:Alleles; D001483:Base Sequence; D005805:Genes, MHC Class I; D015236:HLA-C Antigens / Q000235:genetics*; D006650:Histocompatibility Testing / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction
D015236:HLA-C Antigens; D015345:Oligonucleotide Probes