Rapid direct PCR for forensic genotyping in under 25 min

In this paper, a rapid thermal cycling procedure is combined with a direct amplification from a paper punch, permitting a high‐speed amplification of a 7‐locus multiplex that requires no extraction step. When coupled with a short 1.8 cm microfluidic electrophoresis system, the entire procedure from paper punch to genotype can be completed in under 25 min. The paper describes selection and optimization of enzyme, direct amplification conditions, the reproducibility of the procedure, and concordance with standard forensic genotyping methods. The procedure utilizes a small high‐speed thermal cycler and microfluidic device along with a small laptop and is highly portable. Overall, this technique should provide a useful and reliable procedure for rapid determination of identity of individuals retained at checkpoints as well as a quick method for preliminary identification of individuals at remote locations following mass disasters.

[1]  O. Feugeas,et al.  Direct PCR from whole blood, without DNA extraction. , 1990, Nucleic acids research.

[2]  R A Mathies,et al.  Capillary electrophoresis chips with integrated electrochemical detection. , 1998, Analytical chemistry.

[3]  Bruce R McCord,et al.  A Study of PCR Inhibition Mechanisms Using Real Time PCR *,† , 2010, Journal of forensic sciences.

[4]  Suzanne Kennedy,et al.  PCR troubleshooting and optimization : the essential guide , 2011 .

[5]  J. Butler,et al.  Demonstration of rapid multiplex PCR amplification involving 16 genetic loci. , 2008, Forensic science international. Genetics.

[6]  James P Landers,et al.  Forensic DNA Analysis on Microfluidic Devices: A Review , 2007, Journal of forensic sciences.

[7]  M. Sola-Penna,et al.  Stabilization against thermal inactivation promoted by sugars on enzyme structure and function: why is trehalose more effective than other sugars? , 1998, Archives of biochemistry and biophysics.

[8]  Lihua Zhang,et al.  Rapid Microvolume PCR of DNA Confirmed by Microchip Electrophoresis , 2005, Analytical sciences : the international journal of the Japan Society for Analytical Chemistry.

[9]  Dennis Y. Wang,et al.  Development and Validation of the AmpFℓSTR® Identifiler® Direct PCR Amplification Kit: A Multiplex Assay for the Direct Amplification of Single‐Source Samples *,† , 2011, Journal of forensic sciences.

[10]  M. A. Northrup,et al.  Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device. , 1996, Analytical chemistry.

[11]  Peng Liu,et al.  Integrated DNA purification, PCR, sample cleanup, and capillary electrophoresis microchip for forensic human identification. , 2011, Lab on a chip.

[12]  N. Sato,et al.  Additive effects of bovine serum albumin, dithiothreitol and glycerolon PCR , 1998, Biochemistry and molecular biology international.

[13]  L. Blanco,et al.  Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs , 2010, Proceedings of the National Academy of Sciences.

[14]  U J Balis,et al.  The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. , 1997, BioTechniques.

[15]  High‐resolution single‐stranded DNA analysis on 4.5 cm plastic electrophoretic microchannels , 2003, Electrophoresis.

[16]  Christine A. Hara,et al.  Under-three minute PCR: probing the limits of fast amplification. , 2011, The Analyst.

[17]  A. Woolley,et al.  High-speed DNA genotyping using microfabricated capillary array electrophoresis chips. , 1997, Analytical chemistry.

[18]  Robert Burger,et al.  High‐speed analysis of multiplexed short tandem repeats with an electrophoretic microdevice , 2002, Electrophoresis.

[19]  P. B. Vander Horn,et al.  A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. , 2004, Nucleic acids research.

[20]  Jazelyn M. Salvador,et al.  Isolation of DNA from saliva of betel quid chewers using treated cards. , 2003, Journal of forensic sciences.

[21]  D. Garling,et al.  Rapid cycle DNA amplification: time and temperature optimization. , 1991, BioTechniques.

[22]  F. Perler,et al.  Thermostable DNA polymerases. , 1996, Advances in protein chemistry.

[23]  Titia Sijen,et al.  A protocol for direct and rapid multiplex PCR amplification on forensically relevant samples. , 2012, Forensic science international. Genetics.

[24]  Peng Liu,et al.  Integrated portable polymerase chain reaction-capillary electrophoresis microsystem for rapid forensic short tandem repeat typing. , 2007, Analytical chemistry.

[25]  Z. Shihabi Capillary electrophoresis of double-stranded DNA in an untreated capillary. , 1999, Journal of chromatography. A.

[26]  C. Shackleton,et al.  MASS SPECTROMETRIC METHODS FOR THE DETECTION AND IDENTIFICATION OF ANABOLIC STEROID DRUGS , 2005 .

[27]  P. Neužil,et al.  Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes , 2006, Nucleic acids research.

[28]  D. Fygenson,et al.  DNA polymerase fidelity: from genetics toward a biochemical understanding. , 1998, Genetics.

[29]  R. Abramson Thermostable DNA polymerases: An update , 1999 .

[30]  A Manz,et al.  Chemical amplification: continuous-flow PCR on a chip. , 1998, Science.

[31]  Reza Alaeddini Forensic implications of PCR inhibition--A review. , 2012, Forensic science international. Genetics.

[32]  Igor L. Medintz,et al.  Single-molecule DNA amplification and analysis in an integrated microfluidic device. , 2001, Analytical chemistry.

[33]  Gillian Tully,et al.  Integrated microfluidic system for rapid forensic DNA analysis: sample collection to DNA profile. , 2010, Analytical chemistry.

[34]  Su Jeong Park,et al.  Direct STR Amplification from Whole Blood and Blood‐ or Saliva‐Spotted FTA® without DNA Purification * , 2008, Journal of forensic sciences.

[35]  Paul C. H. Li Microfluidic Lab-On-A-Chip for Chemical and Biological Analysis and Discovery , 2005 .

[36]  Young-Han Song,et al.  A novel buffer system, AnyDirect, can improve polymerase chain reaction from whole blood without DNA isolation. , 2007, Clinica chimica acta; international journal of clinical chemistry.

[37]  Benjamin E. Krenke,et al.  Developmental validation of the PowerPlex 16 HS System: an improved 16-locus fluorescent STR multiplex. , 2010, Forensic science international. Genetics.