Techniques used to define human MHC antigens: polymerase chain reaction and oligonucleotide probes.
The various HLA class II specificities, identifiable by serological or cellular typing reagents, reflect underlying polymorphisms of the constituent alpha and beta chains, encoded at several loci within the HLA-D region. Variation in these genes is concentrated in allelic hypervariable regions but can also be found elsewhere. Sequence-specific oligonucleotides (SSO) can be used to detect individual alleles with a high degree of accuracy by probing dot-blotted DNA, amplified to high copy number by the polymerase chain reaction (PCR). The classic DR specificities (DR1-DRw14) can be identified with certainty by sequential hybridization to a series of 14 SSO probes. In addition, fine resolution of specificities such as the subtypes of DR4 is possible using a combination of SSO probes and group-specific amplification in which PCR primers designed to amplify only DR4 alleles are employed. Similar methods can be applied to the typing of DQ and DP alleles even on whole blood samples which have been stored at -20 degrees C for up to 8 years. A full HLA class II type may be obtained on a large number of samples simultaneously without the need to separate and store viable lymphocytes. Thus, the sensitivity and robustiness of this technique give it major applications in the analysis of histocompatibility and disease associations, particularly in circumstances where facilities for the initial preparation and storage of samples may be limited.
Wordsworth
P
P
Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, U.K.
D000483:Alleles; D006681:HLA-D Antigens / Q000032:analysis* / Q000235:genetics; D006650:Histocompatibility Testing / Q000379:methods; D006801:Humans; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic
D006681:HLA-D Antigens; D015345:Oligonucleotide Probes