HPLC as a method for purifying and identifying PNAs

Peptide nucleic acids, PNAs are now widely used in DNA typing. This typing method represents a powerful alternative and improvement to all traditional DNA typing methods. During the PNA synthesis procedure, byproducts are produced which will interfere with the typing. HPLC is a separation technique used to purify PNAs. In addition to purification, information about retention time and its relationship to sequence and the size of the PNA was also obtained. This study has used allele specific PNA probes with HPLC detection for DNA typing. We have applied this method to HLA-DQalpha genotyping for detection of DQalpha1, DQalpha2, DQalpha3 and DQalpha4, the four major alleles. During the process of the assay, PNAs were used in the water solution. In this case, they always formed a dimer. The size of the dimer was about 25mer to 34mer, which was too big to be separated by the HPLC. The appearance of the dimer was confirmed by the MALDI-TOF MS. It was also found that PNA2 was not very stable under the condition for the assay. PNA has a peptide-like backbone. This peptide-like backbone is not stable at low pH. Under the acidic condition, the backbone could be cleaved. The pH of the HPLC solvent system is about 2, so the peptide-like backbone would be chopped off especially for PNA2, which has three Ts at the C-terminus end. This three C-terminus end Ts made PNA2 more unstable than other PNAs under the acidic conditions. Due to the problem of the dimerization of PNAs in water solution, the RP-HPLC can not be the only detection method used for the PNA affinity capture assay. It has to be combined with other detection technique for the DNA identification.