Technical Note - Forensic Science Communications - January 2008

Technical Note - Forensic Science Communications - January 2008

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January 2008 - Volume 10 - Number 1

Contents

Technical Note

DNA Recovery from Semen Swabs with the DNA IQ System

María Fernanda Lazzarino
Graduate Student
Medical Molecular Biology
University of Buenos Aires
Buenos Aires, Argentina

Andrea Colussi
DNA Expert
Laboratory of Comparative DNA Analysis
Supreme Court of Justice
La Plata, Argentina

María Mercedes Lojo
DNA Expert
Laboratory of Comparative DNA Analysis
Supreme Court of Justice
La Plata, Argentina

Introduction | Materials and Methods | Results and Discussions | Conclusions | Acknowledgments | References

Introduction

Because of the saccharic composition of their membranes, sperm cells stick to a solid support, especially cotton, thus affecting male DNA recovery. Male DNA yield can be increased using a differential extraction procedure if the supporting matrix is carried through all steps of the process. However, after extraction using the DNA IQ System (Promega, Madison, Wisconsin) following the suggested protocol for sperm-containing samples (Promega 2006) with slight modifications, a fraction of the sperm cells are retained in the solid support. Our experimental results indicate that the incubation in the lysis buffer at 95 ºC for 30 minutes prior to resin extraction improves DNA yield.

Materials and Methods

A cotton swab was embedded with 100 µL of a dilution of a semen sample from a volunteer donor (3.0 x 105 sperm cells/mL) placed onto a Petri dish (Figure 1). Half of the swab was processed according to the protocols recommended for sperm-containing samples, with slight modifications: the matrix was maintained during the whole process and the dithiothreitol (DTT) concentration in the lysis buffer was raised threefold (3 µL DTT 1.0 M per 100 µL of buffer). The remaining cotton support was then extracted with 200 µL of Chelex 100 (Bio-Rad, Hercules, California) added to 1 µL of proteinase K (20 mg/mL) and 7 µL DTT (1.0 M), as described by Budowle et al. (2000) (Figure 1). DNA samples were typed with the AmpFℓSTR Profiler Plus kit (Applied Biosystems, Foster City, California) and analyzed on an ABI Prism 310 Genetic Analyzer (Applied Biosystems).

Figure 1: Schematic showing male DNA isolation from semen swabs

Results and Discussion

Amplicon signals in the GeneScan Analysis Software Version 3.1 (Applied Biosystems) data display show that after the extraction, almost 20 to 30 percent of the male profile remained attached to the solid matrix, as indicated by the observed differences in the mean peak area (Figure 2 and Table 1). The incubation step in the lysis buffer at 95 ºC for 30 minutes in the presence of the solid support helps release male DNA, improving DNA yield.

When 30-minute incubation at 95 ºC was added to the protocol (see Figure 1), a high signal was observed in the electropherogram corresponding to the sperm-cell fraction (Figure 3A), and no signal was detected in the cotton swab (Figure 3B). The increase in DNA yield seems to reflect the more efficient recovery of the cells from the solid matrix (Figure 4 and Table 1). Results shown herewith were selected from three different assays performed with each of three different semen samples, which rendered reproducible results.

Figure 2: AmpFℓSTR Profiler Plus GeneScan displays corresponding to (A) DNA extraction performed with the sperm-containing samples protocol (with slight modifications) and (B) Chelex extraction from the cotton support

Figure 3: AmpFℓSTR Profiler Plus GeneScan displays corresponding to (A) DNA extraction performed with the incubation step in the lysis buffer at 95 ºC for 30 minutes and (B) Chelex extraction from the cotton support

Figure 4: AmpFℓSTR Profiler Plus GeneScan displays corresponding to (A) DNA extraction performed with the incubation step in the lysis buffer at 95 ºC for 30 minutes and (B) DNA extraction performed with the sperm-containing samples protocol (with slight modifications)

Table 1: Mean Peak Area in the GeneScan Display ± SD


Mean Peak Area*


Classic Protocol

Modified Protocol

IQ

5040 ± 1322.5

9820 ± 2268.8

Cotton Swab

2240 ± 1462.7

--

Total

7280

9820

*Relative fluorescent units (RFUs)

Conclusions

This report demonstrates that 30-minute incubation at 95 ºC with the lysis buffer in the presence of the solid matrix increases DNA yield in DNA IQ System extraction from sperm swabs. Since the implementation of the protocol described in our analyses of sexual assault cases, we have improved the effectiveness in male DNA recovery, even when dealing with low-copy-number DNA samples.

Acknowledgments

The authors thank Lisandro Laborde for his technical assistance and the reviewers for their useful suggestions.

References

Budowle, B., Smith, J., Moretti, T., and DiZinno, J. DNA Typing Protocols: Molecular Biology and Forensic Analysis. BioTechniques Books-Eaton Publishing, Natick, Massachusetts, 2000, pp. 36–37.

Promega Corporation. DNA IQSystem—Small Sample Casework Protocol. Technical Bulletin 296 [Online]. (April 2006). Available: http://www.promega.com/tbs/tb296/tb296.pdf.