Reliable genotyping of short tandem repeat loci without an allelic ladder using time-of-flight mass spectrometry

DNA separations which traditionally have been performed by slab gel or capi llary electrophoresis, may now be conducted via time-of-flight mass spectro metry (TOF-MS). The advantages of using a mass spectrometry approach for sh ort tandem repeat (STR) characterization include a dramatic increase in bot h the speed of analysis and the accuracy of mass measurements. We report he re typing of the STR loci TH01, TPOX, and CSF1PO as well as the sex-typing marker amelogenin using TOF-MS Allelic ladders, which are typically used wi th electrophoretic separation systems to correct for mobility differences o f DNA fragments under various conditions, are not needed for accurate genot yping with TOF-MS. A mass precision of 0.1% RSD, which corresponds to appro ximately 0.1 nucleotide, was routinely observed. Mass accuracies were bette r than a fraction of a single nucleotide when a daily mass calibration was used. STR microvariants, such as the TH01 allele 9.3, could be detected and resolved from alleles which differ by as little as a single base. In addit ion, the smaller PCR product sizes (55-125 bp) examined in this study have the potential advantage of being more successful when amplifying forensic s amples with degraded DNA.