Analysis of polymerase chain reaction products by on-line liquid chromatography-mass spectrometry for genotyping of polymorphic short tandem repeat loci

Capillary ion-pair reversed-phase high-performance liquid chromatography (I P-RP-HPLC) was used to separate and purify DNA fragments amplified by the p olymerase chain reaction (PCR) prior to their characterization by electrosp ray ionization mass spectrometry (ESI-MS). The investigation by ESI-MS of s ingle- or double stranded species could be effortlessly selected by chromat ography of the nucleic acids under either nondenaturing or denaturing condi tions, which were realized by proper adjustment of the column temperature. ESI-MS detection sensitivity was improved by a factor of 10 upon replacemen t of 25 mM triethylammonium bicarbonate as ion-pair reagent by 25 mM butyld imethylammonium bicarbonate because of the applicability of higher acetonit rile concentrations to elute the DNA from the monolithic, poly(styrene/divi nylbenzene)-based capillary columns. For fragments ranging in size from 67 to 84 base pairs, the mass accuracies and mass reproducibilities were typic ally better than 0.02 and 0.008%, respectively, which enabled the character ization and identification of the PCR products with high confidence. The hy phenated method was applied to the genotyping of polymorphic short tandem r epeat (STR) loci fi-om the human tyrosine hydroxylase gene (humTH01). The d ifferent alleles both in homo- and heterozygotes were identified on the bas is of the masses of the single-stranded amplicons and were in full accordan ce with the alleles identified by conventional capillary electrophoretic si zing.