TYPING FOR ALL KNOWN HLA-DR ALLELES BY GROUP-SPECIFIC PCR AND FLOW CYTOMETRY-BASED MULTIPLEXED SINGLE NUCLEOTIDE EXTENSION.

4.1
#63-OR
TYPING FOR ALL KNOWN HLA-DR ALLELES BY GROUP-SPECIFIC PCR AND FLOW CYTOMETRY-BASED MULTIPLEXED SINGLE NUCLEOTIDE EXTENSION.
Yanzheng Zhang M.D. , Charlie Costin B.A. , Christopher Giang B.S. , Robert Vorhaben B.S. and Peter Stastny M.D. . Dallas TX, University of Texas Southwestern Medical Center, 75390-8886, Internal Medicine .

We have developed a rapid, high-throughput method for allele-level HLA-DR genotyping by using multiplexed single nucleotide extension (MSNE) and flow cytometric analysis of an array of fluorescent microspheres. This technique employs PCR-derived target DNA containing all the polymorphic sites of HLA-DR, synthetic complementary primers, fluorescent reporter molecules (streptavidin-phycoerythrin), and a thermophilic DNA polymerase. Genomic DNA was amplified by HLA-DR group-specific primers, based on the first hypervariable region and position 366-346 of exon 2, which gave 10 groups of specific amplifications. An additional 3 groups, consisting of DRB3, DRB4 and DRB5, were included as controls. MSNE reactions were carried out in thermosequenase buffer in the presence of single nucleotide extension primers, one biotin-labeled ddNTP, three non-labeled ddNTPs and the group specific amplified DNA template(s). An array of fluorescent microspheres was hybridized to the specific MSNE reaction products and sequestered them for flow cytometric analysis. The MSNE reactions were used to assay polymorphisms of the HLA-DRB1 genes in a 96-well format. 60 HLA homozygous B cell lines and UCLA exchange DNA representing 41 HLA-DR alleles were used as reference material. The concordance between MSNE typing and SBT analysis performed by the 13^th IHWC and our laboratory was 99%. The reproducibility was 100% in 60 samples typed on two separate occasions. No false-positive or false-negative typing results were obtained in the homozygous cell lines or in the heterozygous individuals investigated. The methodology described offers a powerful new approach to high resolution DNA typing for HLA-DR and for other HLA genes.