Development of a fast PCR protocol enabling rapid generation of AmpFlSTR(R) Identifiler(R)profiles for genotyping of human DNA

Amanda Foster,Nancy Laurin

doi:10.1186/2041-2223-3-6

Abstract

BackgroundTraditional PCR methods for forensic STR genotyping require approximately 2.5 to 4 hours to complete, contributing a significant portion of the time required to process forensic DNA samples. The purpose of this study was to develop and validate a fast PCR protocol that enabled amplification of the 16 loci targeted by the AmpFℓSTR® Identifiler® primer set, allowing decreased cycling times.MethodsFast PCR conditions were achieved by substituting the traditional Taq polymerase for SpeedSTAR™ HS DNA polymerase which is designed for fast PCR, by upgrading to a thermal cycler with faster temperature ramping rates and by modifying cycling parameters (less time at each temperature) and adopting a two-step PCR approach.ResultsThe total time required for the optimized protocol is 26 min. A total of 147 forensically relevant DNA samples were amplified using the fast PCR protocol for Identifiler. Heterozygote peak height ratios were not affected by fast PCR conditions, and full profiles were generated for single-source DNA amounts between 0.125 ng and 2.0 ng. Individual loci in profiles produced with the fast PCR protocol exhibited average n-4 stutter percentages ranging from 2.5 ± 0.9% (THO1) to 9.9 ± 2.7% (D2S1338). No increase in non-adenylation or other amplification artefacts was observed. Minor contributor alleles in two-person DNA mixtures were reliably discerned. Low level cross-reactivity (monomorphic peaks) was observed with some domestic animal DNA.ConclusionsThe fast PCR protocol presented offers a feasible alternative to current amplification methods and could aid in reducing the overall time in STR profile production or could be incorporated into a fast STR genotyping procedure for time-sensitive situations.

Highlights

Traditional polymerase chain reaction (PCR) methods for forensic short tandem repeats (STR) genotyping require approximately 2.5 to 4 hours to complete, contributing a significant portion of the time required to process forensic DNA samples
The strategy employed in order to reduce time allotted to PCR amplification included the replacement of AmpliTaq Gold® with SpeedSTARTM DNA polymerase, the use of a thermal cycler with greater efficiency (Bio-Rad C1000TM, maximum ramp rate of 5°C/s) [14] and modification of the cycling parameters including the adoption of a two-step PCR approach and decreased hold times at all steps
The concentration of specific PCR reagents, such as deoxyribonucleotide triphosphate (dNTP) and DNA polymerase, and annealing temperatures were varied in order to determine the best conditions for Identifiler reactions under fast PCR conditions

Summary

Introduction

Traditional PCR methods for forensic STR genotyping require approximately 2.5 to 4 hours to complete, contributing a significant portion of the time required to process forensic DNA samples. 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. 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. A growing selection of advanced thermal cyclers (for example, Bio-Rad C1000TM, Eppendorf Mastercycler®, Finnzymes Piko®) with improved ramp rates and temperature control, and ‘fast’ enzymes (for example, SpeedSTARTM HS from Takara, PyroSTARTTM from Fermentas, Phusion® Flash from Finnzymes) with enhanced performance are becoming available, which offer new opportunities to reduce overall PCR time

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