Demonstration of rapid multiplex PCR amplification involving 16 genetic loci.
Current forensic DNA typing is conducted in approximately 8-10h. Steps include DNA extraction, quantification, polymerase chain reaction (PCR) amplification of multiple short tandem repeat (STR) loci, capillary electrophoresis separation with fluorescence detection, data analysis and DNA profile interpretation. The PCR amplification portion of the workflow typically takes approximately 3h with standard thermal cycling protocols. Here we demonstrate a rapid cycling protocol that amplifies 15 STR loci and the sex-typing marker amelogenin from the Identifiler STR typing kit in less than 36 min. This rapid protocol employs commercially available polymerases and the widely used GeneAmp 9700 thermal cycler. Complete concordance of STR allele calls (for 60 samples) between the rapid and standard thermal cycling protocols were observed although there was incomplete adenylation at several of the loci examined and some PCR artifacts were detected. Using less than 750 pg of template DNA and 28 cycles, STR peaks for all loci were above a 150 relative fluorescent unit (RFU) detection threshold with fully adequate inter-locus balance and heterozygote peak height ratios of greater than 0.84.
Vallone
Peter M
PM
National Institute of Standards and Technology, Biochemical Science Division, 100 Bureau Drive, Mail Stop 8311, Gaithersburg, MD 20899-8311, USA. petev@nist.gov
Hill
Carolyn R
CR
Butler
John M
JM
D002874:Chromosome Mapping; D004247:DNA / Q000235:genetics*; D016172:DNA Fingerprinting / Q000379:methods*; D004259:DNA-Directed DNA Polymerase; D053803:Forensic Genetics / Q000379:methods*; D005784:Gene Amplification / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D018895:Microsatellite Repeats / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D013698:Templates, Genetic; D013816:Thermodynamics
D011933:Reagent Kits, Diagnostic; D004247:DNA; D004259:DNA-Directed DNA Polymerase