Novel approaches to enhance sample throughput and reduce turnaround time for the processing of casework and database samples are of high interest to the forensic community. Moreover, in some circumstances, the need for rapid human identification might be critical. Significant efforts are thus being devoted to the development of methods enabling rapid generation of STR profiles. Fast, or rapid, PCR [1–5], direct profiling circumventing DNA extraction [2, 4, 6], and microdevices with portable modules for on-site sample processing [3, 7–9] are emerging alternatives to traditional approaches. Some protocols are already being offered to investigators for specific situations requiring quick actions and for rapid screening of stains .
Generation of STR profiles generally involves DNA extraction and quantification, PCR amplification and detection and analysis of STR products in a process that can take from 8 to 10 hours to several days. Of this time, 2.5 to 4 hours are attributed to the traditional PCR methodologies used to amplify STRs. The length of this time-block is dominated by the properties of the thermal cycling instrument (ramp rates and temperature profiles) and DNA polymerase (processivity and extension rates) generally employed, which restrict any significant time reduction. However, a growing selection of advanced thermal cyclers (for example, Bio-Rad C1000™, Eppendorf Mastercycler®, Finnzymes Piko®) with improved ramp rates and temperature control, and 'fast' enzymes (for example, SpeedSTAR™ HS from Takara, PyroSTART™ from Fermentas, Phusion® Flash from Finnzymes) with enhanced performance are becoming available, which offer new opportunities to reduce overall PCR time.
A fast PCR protocol could provide a valuable alternative to current amplification methods. In combination with other accelerated analytical steps, such as a reduced lysis step (for example, a 30-min lysis step validated by Frégeau and De Moors ) and quick DNA extraction techniques (for example, < 30 min for 16 samples using the Maxwell® 16 from Promega ), a fast protocol could easily be developed enabling rapid generation of STR profiles for human identification at low cost with all the advantages of a basic laboratory infrastructure. Significant time savings can also be foreseen from protocols bypassing the DNA extraction steps and amplifying directly from the biological material [2, 4, 6].
The AmpFℓSTR® Identifiler® kit (Identifiler, Applied Biosytems, Foster City, CA, USA) primer set was chosen for the development of a fast PCR protocol. The loci amplified by this kit includes the 13 Combined DNA Index System (CODIS) core STRs loci, two additional widely used STRs (D2S1338 and D19S433) and the sex-marker Amelogenin. Fast PCR protocols have been established by other research groups for multiplex amplification of STRs, including Identifiler; however, each group experienced their own set of challenges [2–5].
A fast protocol able to produce high quality profiles in 26 min using the SpeedSTAR® HS DNA polymerase (Takara Bio Inc., Madison, WI, USA) and a Bio-Rad C1000™ thermal cycler (BioRad, Mississauga, ON, Canada) was developed by our group using AmpFℓSTR® Profiler Plus® (Profiler Plus) . The purpose of this study was to adapt this fast PCR protocol for the Identifiler primer set and to validate this new protocol for the amplification of the 16 loci targeted using Identifiler without compromising profile quality. This posed a unique challenge due to the multiplex nature of the Identifiler assay, with multiple pairs of primer tightly optimized to produce efficient and simultaneous amplification of target DNA fragments within specific PCR conditions . To ensure the profiles produced were of high quality and would satisfy criteria set by the forensic community, peak height intensity, inter-loci peak height balance, heterozygote peak height ratios, stutter percentages, non-templated nucleotide addition and assay specificity and sensitivity were assessed using a wide collection of samples, including mixtures.