Definition of HLA-C alleles using sequence-specific oligonucleotide probes (PCR-SSOP)

• Published in: Tissue antigens Vol. 46; no. 3 ( Pt 1); p. 187
• Main Authors: Kennedy, L J, Poulton, K V, Dyer, P A, Ollier, W E, Thomson, W
• Format: Journal Article
• Language: English
• Published: England 01.09.1995
• Subjects: Alleles; Base Sequence; Genes, MHC Class I; Histocompatibility Testing - methods; HLA-C Antigens - genetics; Humans; Molecular Sequence Data; Oligonucleotide Probes; Polymerase Chain Reaction
• ISSN: 0001-2815
• DOI: 10.1111/j.1399-0039.1995.tb03118.x

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.