Molecular typing of HLA-A, -B, and DRB using a high throughput micro arrayformat

The goal of this study was to develop a DNA micro array procedure for molec ular human leukocyte antigen (HLA) typing of a large number of samples. DNA was isolated from peripheral blood samples and polymerase chain reaction ( PCR) amplified for HLA-A, -B, and -DRB. Amplified DNA samples were spotted on silane-treated glass slides using a micro array spotter. The spotter was capable of spotting multiple slides with up to 9216 samples per slide or 2 304 samples in quadruplicate. The allele specific oligo nucleotide probes f or HLA-A, -B, and -DRB were labeled with the fluorescent dye Cy3, while a c ontrol probe, to quantitate the total amount of PCR product in a sample, wa s labeled with Cy5. Each slide was hybridized with a mixture of an allele s pecific Cy3 probe plus the control Cy5 probe. Following hybridization and w ash, the amount of probe hybridizing to each DNA sample on the slide was me asured with a micro array scanner. A computer program was used for image an alysis, to calculate the average Cy3/Cy5 ratios and to identify the positiv e and negative samples. In turn, this information was used to determine the HLA phenotype of each sample. There was very good concordance between the results obtained for all three loci using Cy-labeled probes as compared wit h those previously obtained by chemiluminescent detection of alkaline phosp hatase labeled probes. This methodology has the potential of greatly simpli fying HLA molecular typing of large number of samples. Human Immunology 62, 850-857 (2001), (C) American Society for Histocompatibility and Immunogene tics, 2001. Published by Elsevier Science Inc.