High-Throughput STR Analysis by Time-of-Flight Mass Spectrometry

Rapid, cost-effective methods for high-throughput DNA analysis are needed to process samples currently being gathered for large criminal DNA databases around the world. Within the U.S., several states have sample backlogs of over 50,000 samples with limited funds and manpower to analyze these samples. Currently available slab gel or capillary electrophoresis instruments can handle only a few dozen samples per day. Using time-of-flight mass spectrometry coupled with parallel sample preparation on a robotic workstation, we can process thousands of samples daily with a single mass spectrometer. Analysis times on the order of 10 seconds per sample with improved accuracy for sizing short tandem repeat (STR) alleles compared to electrophoresis methods have been demonstrated. Following polymerase chain reaction (PCR) amplification of DNA templates, a proprietary solid-phase purification procedure is used to prepare the samples for mass analysis. We are currently working with over 20 tetranucleotide repeat DNA markers of forensic interest including HUMTH01, vWA, TPOX, CSF1PO and the sextyping marker amelogenin. Mass difference measurements have been shown to benefit STR genotyping by permitting analysis of nontemplated addition, repeat content identification, and repeat spread measurements in heterozygotes. In short, time-of-flight mass spectrometry offers rapid and reliable genotyping of short tandem repeat DNA markers used in human identity testing.