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

The goal of this study was to develop a DNA micro array procedure for molecular 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 2304 samples in quadruplicate. The allele specific oligo nucleotide probes for HLA-A, -B, and -DRB were labeled with the fluorescent dye Cy3, while a control probe, to quantitate the total amount of PCR product in a sample, was labeled with Cy5. Each slide was hybridized with a mixture of an allele specific Cy3 probe plus the control Cy5 probe. Following hybridization and wash, the amount of probe hybridizing to each DNA sample on the slide was measured with a micro array scanner. A computer program was used for image analysis, to calculate the average Cy3/Cy5 ratios and to identify the positive 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 with those previously obtained by chemiluminescent detection of alkaline phosphatase labeled probes. This methodology has the potential of greatly simplifying HLA molecular typing of large number of samples.