ALLELE-SPECIFIC INHIBITION OF HLA SEQUENCING USING PEPTIDE NUCLEIC ACIDS (PNA).

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ALLELE-SPECIFIC INHIBITION OF HLA SEQUENCING USING PEPTIDE NUCLEIC ACIDS (PNA).
Phil Paul PhD ¹, Ray Jurcago BS ¹, Heather Schatz BA ¹, Daniel Cook PhD ¹ and Edward Ball PhD ¹. ¹ Allogen Labs, The Cleveland Clinic Foundation, Cleveland, OH, USA .

HLA genotyping often results in linkage-phase ambiguity among related alleles, requiring independent allele analysis for resolution. DNA analogs that specifically inhibit one allele are an alternative to physical separation or selective amplification methods. PNAs are DNA analogs that form complexes with DNA with greater affinity and specificity than DNA/DNA hybrids. Further, PNAs cannot be extended by DNA polymerase. We have previously used PNAs to block specific HLA alleles during PCR to achieve selective amplification of the other allele. Here we report the ability of PNA oligomers to inhibit one allele when added directly to sequencing reactions. PNAs were designed for HLA Class I loci that targeted polymorphic sequence motifs in exons 2 or 3. We successfully blocked sequence reactions of major allelic groups at HLA-A, -B, and -C loci with specific PNAs. The addition of PNAs directly to sequence reactions has the advantage that no additional PCR is required, decreasing assay complexity and turn-around time. However, a PNA that anneals anywhere between the primer sites will block PCR, whereas only sequence downstream of the PNA binding site will be inhibited during sequencing reactions. Thus, PNA targets must be chosen based on exon position as well as sequence to inhibit regions that will resolve the ambiguity. Also, sequence reactions extend only one strand (sense or anti-sense) of complementary DNA, which further limits PNA design to that template. We conclude that PNAs added to sequencing reactions can be used to achieve haploid sequence and eliminate many phase ambiguities commonly encountered with diploid sequencing. Currently, PNA synthesis modifications are underway to improve binding, solubility, and allele discrimination.