Molecular typing of human leukocyte antigen and related polymorphisms following whole genome amplification

Abstract

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. DNA-based typing often requires separate analysis of closely related alleles or genes, to resolve di-allelic ambiguities. Allele or group specific amplification or physical separation can be used to achieve allele separation for haploid analysis. As an alternative, we used peptide nucleic acids (PNAs) to achieve specific blockade of one of a pair of alleles during amplification. PNAs are hybrid synthetic molecules composed of nucleotide bases on a peptide backbone. PNAs retain the ability to hybridize to single-stranded nucleic acids, with greater affinity than complementary DNA. PNAs can specifically block amplification either by competing for primer annealing sites, or inhibiting polymerase extension by annealing between the primers. We designed PNA oligomers with exact complementarity to HLA Class I and II sequences. To maximize the diversity of potential annealing sites, the PNA sequences were directed to polymorphic exon motifs located between the amplification primers. HLA alleles were typed using standard procedures, and ambiguous samples were selected for re-amplification in the presence of a PNA complementary to one of the allele groups. The PNAcontaining reactions consisted of standard PCR reagents with the same profiles except for an added PNA annealing step. The PNA concentration was optimized empirically for each PNA and required from 0.5 to 10 uM for complete inhibition. After PNA-inhibited amplification, excess primers, PNA and nucleotides were removed, and the DNA was sequenced. We have blocked HLA-A, HLA-B and HLA-DR alleles using this technique. We conclude that PNAs can be used to achieve haploid amplification and eliminate ambiguities that result from cis/trans uncertainty in commonly...

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