7.4
TITLE: SINGLE NUCLEOTIDE EXTENSION (SNE) WITH MULTIPLEXED POLYSTYRENE BEADS: A ROBUST, HIGH–THROUGHPUT METHOD FOR HLA–A LOCUS TYPING

M. Han, Y. Zhang, C. Giang, Y. Tan, P. Stastny

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX

DNA typing for HLA alleles has been performed by a variety of techniques of which the most frequently used are sequence specific priming (SSP), hybridization with sequence specific probes (SSOP) and sequence–based typing (SBT). In the present report we describe a new method developed by combining single nucleotide extension (SNE) with flow cytometric analysis using multiplexed polystyrene beads (MPB). We have found this method to be robust, simple, very powerful and susceptible to automation. Application to an intermediate–level typing for alleles of HLA–A will be described. In this method we have used PCR with locus–specific primers that amplify exons 2 and 3. SNE reactions were performed with 32 HLA–A locus–specific primers. Flow cytometric analysis of the microspheres simultaneously identified both the microsphere type and the fluorescent red signal associated with the nucleotide incorporated by the SNE reaction, defining the polymorphism at the site tested. Because SNE allows specific detection of single nucleotide polymorphisms, sequence variations of the alleles can be analyzed with high specificity in the same reaction well. We have typed for HLA–A, using this method, more than 100 samples randomly selected from our laboratory panel. With the 32 HLA–A primers we were able to accurately resolve the 228 known HLA–A alleles as 108 distinct groups of alleles. Few ambiguities were observed and these could be distinguished using additional SNE primers. Therefore, we anticipate that high resolution HLA–A typing can be achieved by simply adding more primers to analyze additional polymorphisms, in the same reaction well. Comparing to the labor–intensive PCR–SSOP, PCR–SBT and low resolution PCR–SSP typing techniques, the SNE–MPB method was found to be rapid, simple, accurate and easy to perform. It is expected that this method will become a powerful HLA typing tool applicable to single patient samples and to high throughput population studies.