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

Abstract

Peptide nucleic acid (PNA) is a novel class of DNA analogues in which the entire sugar-phosphate backbone is replaced by a pseudopeptide counterpart. Owing to its neutral character and the consequent lack of electrostatic repulsion, PNA exhibits very stable heteroduplex formation with complementary nucleic acid that is essentially ionic strength independent and enables hybridization under minimum salt conditions. This feature as well as its superior ion stability and easy ionization compared to DNA renders PNA very attractive for hybridization-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) applications. We have developed an approach to DNA characterization that takes advantage of multiplexed PNA hybridizations analyzed by MALDI-TOFMS. Our motivation was the further development of oligonucleotide fingerprinting, an efficient technique for cDNA and genomic DNA library characterization. Through positive 'charge-tagging' of PNA the efficiency of detection in MALDI-TOFMS was considerably enhanced permitting an unparalleled degree of multiplexing. Results from the simultaneous hybridization of 21 charge-tagged PNA hexamer oligonucleotides showed that genomic DNA and cDNA clones are successfully characterized on the basis of their hybridization profiles. The degree of multiplexing achieved may render a significant increase in throughput and hence efficiency of oligonucleotide fingerprinting possible.

Typing of single nucleotide polymorphisms by MALDI mass spectrometry: Principles and diagnostic applications

“…Using DNA modification chemistry termed ''charge-tagging'' [29,30] in the molecular biological procedure for generating products of SNPs, purification could be circumvented by a simplified, one-pot reaction method called the GOOD assay [31 -34]. Applying ''charge-tag'' chemistry, the detection sensitivity is increased¨100-fold compared to unmodified DNA and DNA products as well as peptide nucleic acids [35] are rendered insensitive to impurities in MALDI by conditioning products to carry either a single fixed positive or a single fixed negative charge.…”

LNA-Modified Oligodeoxynucleotide Hybridization with DNA Microarrays Printed on Nanoporous Membrane Slides

“…The dsDNA target sequences matched the cosmid fragment at the positions 100248-101341 (dsDNA-1), 131072-132072 (dsDNA-2), 100143-101405 (dsDNA-3), 130867-132210 (dsDNA-4), 130350-132010 (dsDNA-5), 101189-102447 (dsDNA-6), 130879-132115 (dsDNA-7) and 100850-101936 (dsDNA-8), respectively. Plasmid inserts were amplified using particular clones, as described previously [13]. Concentrations of the ethanol-precipitated PCR products were measured using a Bio-Photometer (Eppendorf, Hamburg, Germany) and then adjusted to the desired concentration range (from 0.1 to 0.4 M).…”

Genome Projects and the Functional-Genomic Era

“…OFP has proven to be a powerful procedure for cDNA and genomic DNA library characterisation and normalisation. The method can be performed using either radioactive [36], fluorescence [37] or, recently, MALDI mass spectrometric detection [38]. While the fluorescence-based approach allows for experimentation in miniaturised formats using induced fluorescence resonance energy transfer (iFRET) detection [39], currently under development, we have shown that highly parallel analysis can be performed using the MALDI approach.…”