5.1
TITLE: ALLELE SPECIFIC FLUORESCENT PROBES AND REAL TIME PCR FOR HLA TYPING

Alison Castley, S. L. Castley, David C. Sayer, Frank T. Christiansen

Clinical Immunology and Biochemical Genetics, Royal Perth Hospital, Perth, Western Australia; Pathology, University of Western Australia, Nedlands, Western Australia, Australia

Fluorescent energy transfer probes (FRET) and real time PCR technology have been applied to HLA typing to detect the presence of PCR products following amplification with (many) allele or allele group specific primers. FRET probes (one or few) are designed to anneal to conserved regions within the amplicon and indicate successful PCR amplification. The development of real time PCR instruments for high throughput detection of single nucleotide polymorphisms (SNPs) present the opportunity of taking the alternative approach of using a single, generic amplification primer and many allele specific FRET probes. We have performed a preliminary study to determine the potential for HLA typing using this approach on an Applied Biosystems 7700 Sequence Detection System. We designed 15 HLA–A allele specific or allele group specific minor groove probes. We followed the recommendations of the probe manufacturer for probe design. HLA–A was amplified with locus specific primers (primers used for our HLA–A SBT procedure) followed by amplification of exons 2 and 3 in separate nested PCRs on high dilutions of the original PCR product. The second round exon specific PCRs were performed in the presence of allele specific FRET probes. SYBR green was added at the end of the PCR and melting curve analysis was performed to confirm the presence of a PCR product. Each probe was tested against a panel of 20 DNA. Thirteen probes demonstrated 100% sensitivity and specificity. One probe had zero sensitivity and specificity and one probe (specific for HLA–A*3401) was sensitive but was reactive with two DNA that did not contain HLA–A*3401. These DNA were HLA–A*03011+A*2601 and A*3101+A*0206. Furthermore, the data obtained showed quantitative differences between homozygous and heterozygous samples. Minimal optimisation was required with the dilution of the first round PCR product being the most critical factor. The data indicates that this procedure has considerable potential as a medium to high resolution and highly automated technique applicable to unrelated bone marrow registry and cadaver donor HLA typing. It is currently under evaluation for HLA–B.