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Optimization of ultrahigh-speed multiplex PCR for forensic analysis

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Abstract

In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl2, and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims.

Rapid amplification of forensic DNA using high speed thermal cycling followed by capillary or microfluidic electrophoresis

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Abbreviations

FTA®:

Fast technology for analysis of nucleic acids

RFU:

Relative fluorescence units

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Acknowledgments

The authors would like to thank Streck Inc. and Agilent Technologies for their technical assistance and support. We also would like to thank Marcus Gassmann and Maurice Aboud for their technical support. This project was supported by Award No. 2015-IJ-CXK038 of the National Institute of Justice, Office of Justice programs, U.S. Department of Justice. Points of view in the document are those of the authors and do not necessarily represent the official view of the U.S. Department of Justice.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry and International Forensic Research Institute, Florida International University, University Park, Miami, FL, 33199, USA

    Georgiana Gibson-Daw, Karin Crenshaw & Bruce McCord

Authors
  1. Georgiana Gibson-Daw
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  2. Karin Crenshaw
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  3. Bruce McCord
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Corresponding author

Correspondence to Bruce McCord.

Ethics declarations

Sampling of individuals was done with their informed consent, via signed consent forms, and in accordance with the guidelines and regulations set in place by the FIU IRB and approved under reference number 101831.

Conflict of interest

Technical assistance and support for this project were provided by Agilent Technologies and Streck. The authors declare no other conflict of interest.

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Gibson-Daw, G., Crenshaw, K. & McCord, B. Optimization of ultrahigh-speed multiplex PCR for forensic analysis. Anal Bioanal Chem 410, 235–245 (2018). https://doi.org/10.1007/s00216-017-0715-x

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  • DOI: https://doi.org/10.1007/s00216-017-0715-x

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