DNA sequence analysis using PNA probe and maldi-tof mass spectrometry

Abstract

This research emphasizes on the development of a novel mass spectrometric technique for DNA sequence analysis by combining a new beta-pyrrolidinyl peptide nucleic acid called acpcPNA and an anion exchange solid support. This method relies on the selective absorption of negatively charged PNA-DNA hybrids onto a positively charged ion exchange support. The procedure involves mixing the PNA probe and the DNA sample in a buffer followed by addition of the anion exchange support. The solid support-bound PNA-DNA hybrid was then washed and directly analyzed by MALDI-TOF. The sequence of the DNA sample was determined by the detected molecular masses of the absorbed PNA probe. Since the neutral PNA could not be not adsorbed by the anion exchanger unless it hybridized with the complementary DNA, the presence of PNA on the support suggested a successful formation of the PNA-DNA hybrid, implying that the sequence of the PNA and the DNA were complementary. The technique was simple, rapid, cost-effective and required no labelling of the DNA samples. Because of an exceptional high specificity of this new PNA probe, the method is capable of detecting a single mismatched base out of 9–15 base DNA sequences. In all cases tested - including real DNA samples analysis - a clear-cut differentiation between complementary and single base mismatch DNA targets was achieved. Potential applications of this technique in genotyping of Single Nucleotide Polymorphisms (SNP), especially in multiplex format, have also been demonstrated.