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SELECTIVE AMPLIFICATION BLOCKADE OF HLA ALLELES WITH PEPTIDE NUCLEIC ACIDS.
Philip Paul, Ph.D., Ray Jurcago, B.S., Edward J. Ball, Ph.D. and Daniel J. Cook, Ph.D.. Cleveland OH, USA, The Cleveland Clinic Foundation, 44195, Allogen Laboratories.

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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 PNA-containing 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 encountered diploid amplifications.