DISCRIMINATION OF SINGLE-NUCLEOTIDE POLYMORPHISMS IN HUMAN DNA USING PEPTIDE NUCLEIC-ACID PROBES DETECTED BY MALDI-TOF MASS-SPECTROMETRY

Human genomic and mitochondrial DNA contain large numbers of single-nu cleotide polymorphisms (SNPs), many of which are linked to known disea ses, Rapid and accurate genetic screening for important SNPs requires a general methodology which is easily implemented. We present here an approach to SNP discrimination based on high-specificity hybridization of peptide nucleic acid (PNA) probes to PCR-amplified DNA The assay i s directly applied to polymorphisms located within hypervariable regio n 1 of the human mitochondrial genome and type 1 suballeles of the hum an leukocyte antigen DQ alpha gene, Captured, single-stranded DNA mole cules prepared by PCR amplification;are hybridized with PNA probes in an allele-specific fashion. Matrix-assisted laser desorption/ionizatio n time-of-flight mass spectrometry (MALDI-TOFMS) is then used for rapi d, precise, and unambiguous detection and identification of the hybrid ized PNA probes, Since PNA oligomers bind strongly to complementary DN A under minimal salt conditions, the use of PNA probes is compatible w ith MALDI-TOFMS. The unparalleled ability of MALDI-TOFMS analysis in t erms of molecular weight resolution and accuracy, in conjunction with the highly specific PNA hybridization afforded by this method, offers promise for development into a multiplexed, high-throughput screening technique.