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.
ARC Epidemiology Research Unit, Manchester University, United Kingdom.
L J
LJ
Kennedy
K V
KV
Poulton
P A
PA
Dyer
W E
WE
Ollier
W
W
Thomson
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