DNA Typing of Human Leukocyte Antigen Sequence Polymorphisms by Peptide Nucleic Acid Probes and MALDI-TOF Mass Spectrometry

Abstract

A novel analytical method using PNA probes detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied to type sequence polymorphisms within the human leukocyte antigen (HLA), DQA locus. Streptavidin-coated magnetic beads were used to immobilize biotinylated DNA. PNA probes representing possible alleles were then prepared for the immobilized DNA hybridization. The nonspecific PNA probes were removed with stringent washes. The PNA/DNA/beads conjugate was analyzed by MALDI-TOFMS. The genotype of the DNA was determined by the detected molecular masses of the released PNA probes. Reproducible and accurate genotyping was achieved by this analytical method.

Multiplexed hybridizations of positively charge‐tagged peptide nucleic acids detected by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

“…The feasibility and degree of multiplexing of PNA hybridizations detected by MALDI‐TOFMS was investigated. In previous reports on the use of PNA in MALDI‐TOFMS based DNA genotyping,21–23 hybridizations were restricted to maximally four PNA oligonucleotides applied in parallel. Beside strong sequence‐dependent variations in PNA hybridization stability and specificity the lack of satisfactory PNA probe signal intensities and resolution in simultaneous analyses prevented proper multiplexing 21,23.…”

“…Both phenomena account for reduced sensitivity and signal resolution. With regard to PNA, MALDI‐TOFMS has been successfully applied to the characterization of mixed‐base PNA oligomers20 as well as to DNA genotyping 21–23. Hybridizations in these studies, however, were restricted to maximally four PNA oligonucleotides applied in parallel.…”

BioMEMS using electrophoresis for the analysis of genetic mutations

“…MALDI has been used to detect different sizes of excised endonuclease restriction fragments [41], products of primer extension reactions (with and without mass tagged ddNTPs) [42,43] and peptide nucleic acid (PNA) probes used for the analysis of mutated DNA [44,45]. MALDI has been used to detect different sizes of excised endonuclease restriction fragments [41], products of primer extension reactions (with and without mass tagged ddNTPs) [42,43] and peptide nucleic acid (PNA) probes used for the analysis of mutated DNA [44,45].…”

Peptide nucleic acid probes with charged photocleavable mass markers

“…2 PNA hybridization probes have been used in combination with MALDI-TOF mass spectrometry for the detection of sequence polymorphisms in PCR-amplified DNA. [3][4][5][6] Photocleavable linkages, with or without mass tags, have also been used in conjunction with DNA probes for genetic analysis using MALDI techniques. 7-10 PNA shows excellent discrimination between wild-type and mutant DNA targets i.e., under appropriate conditions, it binds selectively to its fully complementary DNA target in the presence of an equivalent PNA sequence containing a single mismatched base.…”