Genotyping single nucleotide polymorphisms using intact polymerase chain reaction products by electrospray quadrupole mass spectrometry

Both single nucleotide polymorphisms (SNPs) and mutations are commonly obse rved in the gene encoding the tumor suppressor protein, p53. SNPs occur at specific locations within genes whereas mutations may be distributed across large regions of genes. When determining nucleotide differences, mass spec trometry is the only method other than Sanger sequencing which offers direc t structural information. Electxospray ionization (ESI) quadrupole mass spe ctrometry (MS) analysis of intact polymerase chain reaction (PCR) products was performed following a simple purification and on-line heating to limit ion adduction. The PCR products were amplified directly from genomic DNA ra ther than plasmids, as in our previous work. Two known polymorphisms of the p53 gene were genotyped. A cytosine (C) or guanine (G) transversion, desig nated C <---->G (G <---->C on the opposite strand), were each detected by a 40.0 Da change upon ESI quadrupole MS analysis. Using known PCR products a s standards, the genotypes determined for 10 human samples corresponded wit h restriction fragment length polymorphism (RFLP) analysis. Cytosine/thymin e (T) transitions, designated C <---->T (G <---->A on the opposite strand), were also genotyped by ESI-MS. This SNP is discriminated by a 15.0 Da chan ge on one strand (C <---->T) and a 16.0 Da change on the other (G <---->A). Appropriate sample preparation and instrumental configuration (including h eated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products. Copyrigh t (C) 2001 John Wiley & Sons, Ltd.