Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of peptide nucleic acids for DNA typing

DNA typing (identification) has broad applications in forensic analysis, diagnostic medicine and plant and animal sciences. Current DNA typing procedures are based on gel electrophoresis methods which are slow and labor intensive. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has many desirable attributes in analysis of protein, peptides and DNA, namely, speed, sensitivity, accuracy and ability to be automated. However, direct analysis of DNA by MALDI for DNA typing (sequencing and sizing) is limited by the pre-analysis preparation of samples which are free of alkali and alkaline earth metal salts and other impurities. Peptide nucleic acids are a new class of biomolecules. They are DNA mimics, which are capable of hybridizing to complementary DNA with the added benefits of higher specificity, stronger affinity and facile analysis by MALDI. The analysis of DNA for the presence of a particular mutation or polymorphisms can be accomplished by differential hybridization of the DNA with an allele-specific (sequence-specific) PNA probe that can be detected by MALDI. In the present work, a novel analytical method was developed to type DNA sequence polymorphisms by using PNA probes which were detected by MALDI. In this method, streptavidin-coated magnetic beads were used to immobilize biotinylated DNA. PNA probes representing possible alleles were then mixed with the immobilized DNA and allowed to hybridize. Nonspecific PNA probes were removed with stringent washes. The PNA/DNA/beads conjugate was analyzed by MALDI. The genotype of the DNA was determined by the detected molecular masses of PNA probes. This assay has been applied to HLA-DQA genotyping. Reproducible and accurate genotyping was achieved by this analytical method.