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The application of alkaline lysis and pressure cycling technology in the differential extraction of DNA from sperm and epithelial cells recovered from cotton swabs

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Abstract

This study reports the development of a two-step protocol using pressure cycling technology (PCT) and alkaline lysis for differential extraction of DNA from mixtures of sperm and vaginal epithelial cells recovered from cotton swabs. In controlled experiments, in which equal quantities of sperm and female epithelial cells were added to cotton swabs, 5 min of pressure pulsing in the presence of 0.4 M NaOH resulted in 104 ± 6 % recovery of female epithelial DNA present on the swab. Following the pressure treatment, exposing the swabs to a second 5-min alkaline treatment at 95 °C without pressure resulted in the selective recovery of 69 ± 6 % of the sperm DNA. The recovery of the vaginal epithelia and sperm DNA was optimized by examining the effect of sodium hydroxide concentration, incubation temperature, and time. Following the alkaline lysis steps, the samples were neutralized with 2 M Tris (pH 7.5) and purified with phenol-chloroform-isoamyl alcohol to permit downstream analysis. The total processing time to remove both fractions from the swab was less than 20 min. Short tandem repeat (STR) analysis of these fractions obtained from PCT treatment and alkaline lysis generated clean profiles of female epithelial DNA and male sperm DNA for 1:1 mixtures of female and male cells and predominant male profiles for mixtures up to 5:1 female to male cells. By reducing the time and increasing the recovery of DNA from cotton swabs, this new method presents a novel and potentially useful procedure for forensic differential extractions.

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Acknowledgments

Major support for this research was provided by award # 2011-NE-BX-K550 from the National Institute 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. We are thankful to Nathan Lawrence from Pressure Biosciences Inc. and George Duncan from the Broward Sheriff’s Office (BSO) crime lab for their technical expertise and the generous support of this research. We are also thankful to Margie Phipps, Vanessa Martinez, and Pero Dimsoski for the technical support.

Conflict of Interest

The authors have declared no conflict of interest.

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Authors and Affiliations

  1. Department of Chemistry CP304, Florida International University, 11200 SW 8th St, Miami, FL, 33199, USA

    Deepthi V. Nori & Bruce R. McCord

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  1. Deepthi V. Nori
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  2. Bruce R. McCord
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Correspondence to Bruce R. McCord.

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Published in the topical collection Capillary Electrophoresis of Biomolecules with guest editor Lisa Holland.

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Nori, D.V., McCord, B.R. The application of alkaline lysis and pressure cycling technology in the differential extraction of DNA from sperm and epithelial cells recovered from cotton swabs. Anal Bioanal Chem 407, 6975–6984 (2015). https://doi.org/10.1007/s00216-015-8737-8

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  • DOI: https://doi.org/10.1007/s00216-015-8737-8

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