Determination of DNA sequences using peptide nucleic acid in combination with chitosan particles by maldi-top mass spectrometry

Abstract

Determination of DNA sequences is significantly important for many biotechnology-related applications ranging from medical, forensic, agriculture, and food science. The concept of using a new conformationally constrained pyrrolidinyl peptide nucleic acid (acpcPNA), anion-exchange captures in combination with MALDI-TOF mass spectrometry has been recently proven as a simple and effective way for DNA sequence analysis. This research has introduced chitosan quaternized chitosan particles as anion-exchange captures that may be applicable for the same purpose. The particles were prepared by either homogeneous or heterogeneous quaternization. The success of sub-micron, spherical, and positively charged quaternized chitosan particle formation was verified by FT-IR, 1H NMR, PCS, and TEM analyses. Investigation by MALDI-TOF mass spectrometry suggested that some quaternized particles in combination with acpcPNA were capable of detecting a single mismatched base out of 9-14 base DNA sequences. The success of selective detection generally required rinsing of the particles after capturing PNADNA hybrid by 20% formamide in phosphate buffer solution. Potential application of this technique for a synthetic DNA of which sequence mimics mutant K-ras DNA, a mutated region that is associated with cancer has also been demonstrated.