Analysis of single nucleotide polymorphisms by primer extension and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

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Abstract
A method for typing single nucleotide polymorphisms (SNPs) by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) is described, in which a mass‐tagged dideoxynucleoside triphosphate is employed in a primer extension reaction in place of an unmodified dideoxynucleoside triphosphate (ddNTP). The increased mass difference due to the presence of the mass‐tag greatly facilitates the accurate identification of the added nucleotide, and is particularly useful for typing heterozygous samples. Twenty commercially available mass‐tagged dideoxynucleoside triphosphates were screened for amenability to incorporation by AmpliTaq FS and ThermoSequenase DNA polymerases in single nucleotide primer extension (SNuPE) reactions. Several sample preparation and purification methods were also examined and compared. Float dialysis was found to be a simple, versatile, and effective method for purification of the extension products. High specificity and sensitivity were obtained, and all six possible biallelic SNP heterozygotes were determined accurately using a 44‐mer synthetic oligonucleotide target DNA as a model system. Further validation of the method was demonstrated in the analysis of five single‐base mutations in exon IV of the human tyrosinase gene. Single nucleotide variations within 182‐bp PCR amplicons amplified from three plasmid and three human genomic DNA samples were genotyped at five variable positions, with results in 100% concordance with conventional sequencing. Genotypes were determined accurately at five sequence‐tagged sites (STSs). Copyright © 2000 John Wiley & Sons, Ltd.