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#39-OR
MOLECULAR TYPING OF HUMAN LEUKOCYTE ANTIGEN AND RELATED POLYMORPHISMS FOLLOWING WHOLE GENOME AMPLIFICATION.
Wenshuo Shao, MD, Jianming Tang, PhD, Tevfik Dorak, MD, Wei Song, MD, PhD, Elena Lobashevsky, MD, PhD, Charles S. Cobbs, MD, Margaret R. Wrench, MD and Richard A. Kaslow, MD. Birmingham AL, USA, University of Alabama at Birmingham, 35294, Department of Epidemiology; Birmingham AL, USA, University of Alabama at Birmingham, 35294, Department of Medicine; Birmingham AL, USA, University of Alabama at Birmingham, 35294, Department of Surgery and San Francisco CA, USA, University of California at San Francisco, 94143, Department of Epidemiology and Biostatistics.

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DNA samples with suboptimal quality or limited quantity often compromise reliable, high-resolution HLA typing. We tested the feasibility of molecular typing for variants at HLA and neighboring loci using whole genome amplification (WGA) strategy facilitated by the Phi29 DNA polymerase. With little (~50 ng) starting material, amplified DNA provided adequate templates for PCR-based genotyping of several HLA (A, B, C, DRB1, and DQB1) and related loci (HFE, MICA, and 10 microsatellites). The PCR amplicons ranged from 92 to 2,200 bp, and genotyping was performed successfully using several techniques including amplification size polymorphism, PCR with sequence-specific primers, restriction fragment length polymorphism, reference strand-mediated conformation analysis, and sequencing-based typing. In our analyses of 47 cell lines and 122 DNA specimens from native Africans and European Americans, a total of 321 genotypes have been confirmed by typing both original and amplified DNA. Additional genotyping using amplified (Phi29 processed) DNA alone produced results that were consistent with known patterns of allelic distribution and linkage disequilibrium observed in highly comparable populations. Thus, WGA appeared to provide a reliable and simple approach to securing ample genomic DNA for a variety of genotyping schemes.