Sequence-specific priming and exonuclease-released fluorescence assay for rapid and reliable HLA-A molecular typing

Background: The rapid and sensitive method described for low-resolution DNA typing of alleles at the HLA-A locus is based on a sequence-specific primer polymerase chain reaction approach that eliminates post-polymerase chain reaction gel electrophoresis to analyze the results. Methods and Results: This method takes advantage of the 5' --> 3' exonuclease activity of the Tag polymerase normally present during polymerase chain reaction. This sequence-specific priming and exonuclease-released fluorescence assay was conducted on 40 DNA samples derived from homozygous cell lines and peripheral blood lymphocytes that represented the majority of the HLA-A alleles. There was 100% concordance with the results obtained by serologic typing of these same samples. Conclusions: The advantages of this system are that assays can be performed in 2 hours, the results are easy to interpret, and no gel electrophoresis is required. The same strategy can be used for high-resolution DNA typing once appropriate primer pairs are added to the panel used for first-level discrimination.