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

A method for typing single nucleotide polymorphisms (SNPs) by matrix-assist ed laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFM S) is described, in which a mass-tagged dideoxynucleoside triphosphate is e mployed in a primer extension reaction in place of an unmodified dideoxynuc leoside triphosphate (ddNTP). The increased mass difference due to the pres ence of the mass-tag greatly facilitates the accurate identification of the added nucleotide, and is particularly useful for typing heterozygous sampl es. Twenty commercially available mass-tagged dideoxynucleoside triphosphat es were screened for amenability to incorporation by AmpliTaq FS and Thermo Sequenase DNA polymerases In single nucleotide primer extension (SNuPE) rea ctions. Several sample preparation and purification methods were also exami ned and compared. Float dialysis was found to be a simple, versatile, and e ffective method for purification of the extension products. High specificit y and sensitivity were obtained, and all six possible biallelic SNP heteroz ygotes were determined accurately using a 44-mer synthetic oligonucleotide target DNA as a model system. Further validation of the method was demonstr ated in the analysis of five single-base mutations in exon IV of the human tyrosinase gene. Single nucleotide variations within 182-bp PCR amplicons a mplified from three plasmid and three human genomic DNA samples were genoty ped at five variable positions, with results in 100% concordance with conve ntional sequencing. Genotypes were determined accurately at five sequence-t agged sites (STSs). Copyright (C) 2000 John Wiley & Sons, Ltd.