TECHNICAL NOTE Development and validation of a rapid PCR method for the PowerPlex® 16 HS system for forensic DNA identification James White & Sheree Hughes-Stamm & David Gangitano Received: 15 July 2014 /Accepted: 29 October 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Currently, the amplification step of most forensic DNA profiling systems takes 3–4 h to complete. A decrease in the amplification time would allow for increased laboratory throughput, which may help reduce backlog when is due to limited cycling capacity. By using the SpeedSTAR™ HS Polymerase (Takara Bio, Otsu, Japan) in combination with the Veriti® (Applied Biosystems, Foster City, CA, USA) rapid thermal cycler, the amplification time for the PowerPlex® 16 HS (Promega, Madison, WI, USA) kit was reduced by 66 % (1 h). The sensitivity of this fast method was comparable to the standard system (0.13 ng). Although this rapid protocol showed an increase in average stutter ratios (2.8 %) and a decrease in average peak height ratios (7 %) across all loci when compared with standard conditions, it was able to con- sistently generate reliable DNA profiles. The results of this study indicate that the rapid protocol could be implemented in forensic laboratories with an optimal range of 0.25–2 ng of input DNA using appropriate analytical interpretation guidelines. Keywords Forensic DNA . Short tandem repeats . Rapid PCR . PowerPlex® Introduction Forensic DNA profiling is a four-step process consisting of DNA extraction, quantitation, amplification of short tandem repeats (STR) via polymerase chain reaction (PCR), and capillary elec- trophoresis, which separates and detects the amplified fragments. This process takes approximately 1 to 2 days to complete, with 10 to 12 h of laboratory time. PCR amplification requires 3 to 4 h to complete when standard Taq DNA polymerase and standard thermal cyclers and amplification protocol are used [1]. A reduc- tion in the amount of time required to perform the amplification step would allow for increased laboratory throughput and may decrease sample backlog in cases where it is due to limited cycling capacity. Fast chemistries and rapid PCR cycling instrumentation have decreased the time required for forensic DNA profiling [2]. Fast chemistry involves new polymerases with increased extension rates and decreased activation times compared to standard chemistries. This shortens the time needed for am- plification. These fast polymerases have been designed for use with rapid thermal cyclers such as the Veriti® (Applied Biosystems, Foster City, CA, USA), SpeedCycler® (Analytik Jena, Jena, Germany), and Philisa® (Streck, Omaha, NE, USA) where temperature transition times and hold times are shortened [1, 3, 4]. In order to become accepted in the forensic community, these fast PCR chemistries must yield equally robust results as the standard systems. Fast chemistries are also being integrated into newly designed kits such as GlobalFiler ™ (Life Technologies, Carlsbad, CA, USA), GlobalFiler™ Express (Life Technologies), PowerPlex® Fusion (Promega, Madison, WI), and PowerPlex® 18D (Promega) [5–8]. All of these kits offer reduced amplification times ranging from 40 to 90 min. However, the GlobalFiler ™ Express and PowerPlex® 18D kits are solely optimized for reference and single source samples, not for casework samples [6, 8]. Although reference samples do not require quantitation according to the FBI’ s Quality Assurance Standards for Forensic DNA Testing Laboratories, casework samples must be quantitat- ed [9]. Therefore, crime laboratories that prefer using one kit are unlikely to use GlobalFiler™ Express or PowerPlex® 18D un- less they solely process reference samples. The GlobalFiler™ and PowerPlex® Fusion kits are optimized for casework samples J. White : S. Hughes-Stamm : D. Gangitano (*) Department of Forensic Science, College of Criminal Justice, Sam Houston State University, Chemistry and Forensic Science Bldg., 1003 Bowers Blvd., Office 221C, Huntsville, TX 77340-2525, USA e-mail: dag006@shsu.edu Int J Legal Med DOI 10.1007/s00414-014-1102-1