Optimizing PCR conditions for HLA class II SSO typing.
The polymerase chain reaction (PCR) has made the technique of sequence-specific oligonucleotide (SSO) typing fast, accurate and very sensitive. These combined techniques are an ideal tool for analysing the complex patterns of polymorphism seen throughout the HLA complex. The success of the technique relies heavily on accurate and specific amplification of the DNA under study. This paper considers the principles behind the PCR amplification technique and discusses the factors which lead to optimal amplification. Primer design is discussed and a variety of sources of target DNA considered. Precautions designed to prevent contamination are discussed. Reaction components are considered both in isolation and as part of the complete reaction. Finally, a complete PCR protocol is suggested. The paper is illustrated with examples of HLA class II amplification.
Ivinson
A J
AJ
North West Regional Tissue Typing Laboratory, St Mary's Hospital, Manchester, U.K.
D004247:DNA / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D004340:Drug Contamination; D006681:HLA-D Antigens / Q000145:classification / Q000235:genetics*; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
D006681:HLA-D Antigens; D004247:DNA; D004259:DNA-Directed DNA Polymerase