NCJ Number
229267
Journal
Journal of Forensic Sciences Volume: 54 Issue: 6 Dated: November 2009 Pages: 1287-1296
Date Published
November 2009
Length
10 pages
Annotation
This paper reports for the first time the successful amplification of full DNA profiles that consist of either 9 or 15 short tandem repeat (STR) loci and amelogenine in both tube reactions and 16-sample microfluidic biochip reactions in approximately 17 minutes.
Abstract
The protocols and instrumentation presented enable significant reductions in the time required to generate full profiles that satisfy interpretation guidelines for STR analysis. The time required for STR amplification is determined by the temperature ramp rates of the thermal cycler, the components of the reaction mix, and the properties of the reaction vessel. Multiplex amplifications in microfluidic biochip-based and conventional tube-based thermal cyclers have been demonstrated in 17.3 and 19 minutes, respectively. Optimized 28-cycle amplification protocols generated alleles with signal strengths above calling thresholds, heterozygous peak height ratios of greater than 0.65, and incomplete nontemplate nucleotide addition and stutter of less than 15 percent. Full CODIS-compatible profiles were generated using the Profiler PlusID, COfiler and Identifiler primer sets, PCR peresis system, Genebench-FXTM. This fast multiplex PCR approach has the potential to reduce process time and cost for STR analysis and enables development of a fully integrated microfluidic forensic DNA analysis system. The descriptions of methods and materials address the custom thermal cycler and microfluidic biochip, instrumentation and temperature profiles, reaction mix component and cycling conditions for PCR, multiplex PCR with other STR typing kits, reproducibility, sensitivity, STR separation and detection instrumentation, and data analysis. 2 tables, 7 figures, and 35 references