RAPID HLA TYPING USING MICROARRAY TECHNOLOGY.
BL Ray, RT Cunningham, I Balazs, Lifecodes Corporation, Stamford, CT.

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Dot blot hybridization assays have been routinely employed in simultaneously typing multiple DNA samples for various HLA classes. Likewise, reverse dot blots can be employed to type an individual DNA sample on a single blot. This process, however, is limited in its ability to simultaneously type a sample for multiple HLA classes or other additional loci.

Herein, a simple method is presented for rapidly typing an individual DNA sample for HLA A, B & DRB using microarray technology. The method involves attaching a set of HLA A, B & DRB probes to a single glass slide in a microarray format. The assay is straightforward and involves: 1) Separate PCR amplification of HLA A, B & DRB, 2) combining the three PCR products, 3) hybridizing the PCR products to the glass slide for 1-2 hrs followed by a brief washing, 4) scanning the slide to quantify the fluorescence, and 5) analyzing the data. By using differently labeled primers, loci with similar sequences, such as HLA A & B, can be simultaneously processed.

The entire method, including DNA extraction and PCR amplification, takes approximately 6 hrs. The complete set of HLA A, B & DRB probes used to type samples for the National Marrow Donor Program, including some additional probes for more specific typing, comprises 164 probes which utilize only a small fraction of the 1536 probes that we can attach to a single slide. Thus, this technology has the capacity to permit inclusion of several more tests without increasing the amount of time or labor required. In addition, typical microarray scanners can accommodate up to 4 different fluorescent dyes permitting simultaneous processing of up to four loci with similar sequences.