Ann M. Lins, Cynthia J. Sprecher and James W. Schumm
Promega Corporation, 2800 Woods Hollow Road, Madison, WI
Multiplex PCR amplification systems offer a non-isotopic method for the rapid, simple, and accurate analysis of short tandem repeat (STR) loci. Two multiplex STR systems were developed for fluorescence detection using well characterized, polymorphic STR loci. The first multiplex contains the CSF1PO, TPOX, TH01, vWA loci and the second includes the F13A01, FESFPS, F13B, LPL loci. The combined matching probability with these eight loci exceeds 1 in 17,000,000 for each racial or ethnic group tested to date. The same eight loci were previously developed for silver stain detection, with six of them present in two triplex systems (CSF1PO, TPOX, TH01 and F13A01, FESFPS, vWA).
In this report, the sensitivity of the fluorescent quadriplexes and silver stained triplexes has been analyzed. Fully configured commercially available multiplex primer sets (GenePrint™ STR Systems) were used to amplify from 0.5ng to 250ng of K562 DNA. The resulting products were separated by denaturing polyacrylamide gel electrophoresis, and visualized using silver stain or fluorescence detection. The fluorescently labeled amplification products were evaluated using the Hitachi FMBIO fluorescent scanner, Molecular Dynamics FluorImager SI scanner, ABI 373 DNA Sequencer, and ABI Prism 377 DNA Sequencer. As little as 0.5ng of K562 DNA can be detected with any of the amplification and detection formats used.
Allelic ladders with each system simplified the rapid and precise allele determinations which did not vary with the amplification or detection systems used. Discrimination of a single base difference in alleles such as the TH01 allele 10 and allele 9.3 was easily obtained with each approach. These high throughput methods for DNA identification have valuable applications in forensic analysis, paternity determination, tissue culture strain identification, and bone marrow transplantation studies.
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