7.4
TITLE: ESTABLISHING HLA HOMOZYGOSITY WITH REAL–TIME PCR

Lee Ann Baxter–Lowe, Denice Z. Kong

Department of Surgery, University of California San Francicso, San Francisco, CA

Most laboratories establish HLA homozygosity using HLA typing of family members to create a pedigree that establishes MHC haplotypes. This approach is expensive and may be precluded by lack of typing for essential family members. This investigation takes advantage of the quantitative aspects of real–time PCR to determine homozygosity without HLA typing of related individuals. The copy number of the target sequence was determined using real–time PCR with sequence–specific primers for the HLA–A*02. PCR products were initially detected using SYBR™ Green staining, with specificity evaluated using Tm and copy number extrapolated from a standard curve. This approach was sufficiently accurate to determine homozygosity for approximately 60% of samples. In order to eliminate variation attributable to amplification efficiency, an internal control (cyclophilin B) was included in each tube. PCR products from each HLA gene and the internal control were distinguished using TaqMan™ probes labeled with different combinations of quencher and reporter dye molecules. A relative threshold cycle was determined for each sample (difference between the threshold cycle of the HLA target and the internal control). Experimental variation in the relative threshold cycles of homozygous and heterozygous controls was determined and used to establish a range that accurately distinguishes homozygosity and heterozygosity in all specimens (22 homozygous and 28 heterozygous). This investigation demonstrates that real–time PCR can be used to reliably determine homozygosity only if an internal control is included in each reaction tube.