Recovery of DNA from Latent Blood after Identification by Fluorescein

Recovery of DNA from Latent Blood
after Identification by Fluorescein

From the Journal of Forensic Identification
Vol. 54, No. 6, November/December 2004*

by

Laurie A. Martin
Police Department, University of Alaska, Fairbanks, AK

Catherine F. Cahill
Department of Chemistry and Biochemistry and Geophysical
Institute, University of Alaska, Fairbanks, AK

Abstract: Luminol has been widely used in the field of crime scene investigations to detect latent blood; however, luminol has the tendency to destroy DNA evidence. Fluorescein, an alternative to luminol for detecting latent blood at a crime scene, does not destroy DNA evidence. This paper demonstrates the successful recovery of DNA from a blood sample treated with fluorescein. DNA was extracted from blood-containing denim substrates after fluorescein was applied to the substrates. The DNA locus, D18S51, was amplified using standard polymerase chain reaction (PCR) techniques, analyzed by electrophoresis, and used to demonstrate that DNA was successfully recovered from the samples.

Introduction

Violent crimes often go unsolved because of the lack of evidence leading to a perpetrator. Therefore, any evidence found at the crime scene is vital to solving the crime. Violent crimes often leave latent evidence, such as DNA, at the crime scene. In the United States, 13 Short Tandem Repeats (STR) loci on DNA strands are employed by the FBI for typing all felons in the National DNA Databank (CODIS) and for analyzing biospecimens found at crime scenes. The 13 core STR loci are CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, THO1, TPOX, and VWA [1]. According to the literature, it is possible to recover DNA, "typeable at all 13 core CODIS STR loci" [2].

Luminol has been widely used in the field of crime scene investigations to detect latent blood. However, conflicting studies have shown that luminol, when applied to a latent blood sample, loses valuable DNA information [2, 3], likely because of the repeated applications needed to maintain visualization. This is a major drawback to using luminol because any trace blood discovered through the use of luminol could be useless for DNA recovery.

Unlike luminol, fluorescein, a new alternative method for latent blood detection [4], has not been tested for its impacts on DNA recovery. The goal of this study was to determine whether DNA could be recovered from latent blood samples that have been treated with fluorescein. The approach used in this study was to treat denim substrates with blood, use fluorescein on the denim to identify the blood, and extract the blood from the denim using Chelex 100 Resin. After the blood was extracted, a polymerase chain reaction (PCR) was set up to amplify one of the 13 STR loci, D18S51. The D18S51 loci was chosen to provide a simple test of the feasibility of the procedure. Future analysis will include more than a single locus. Gel electrophoresis, using certified PCR agarose gels, was used to separate the DNA. This analysis showed that DNA could be recovered from the treated samples.

Experimental Methods

The substrates used for the experiment were 1.5 square-inch portions of blue denim. The experimental design evaluated the ability to recover DNA from the substrates by applying six different dilutions of blood using three different methods as shown in Table 1. The first method, (a), was a direct application of the blood dilutions to the chelex solution followed by PCR and gel electrophoresis. In the second method, (b), blood dilutions were applied to the denim substrates and dried for three days. The DNA was then extracted using the chelex solution and analyzed by PCR and gel electrophoresis. The final method, (c), followed the same steps as (b) except that after the blood dried, it was treated with fluorescein before the DNA was extracted. The fluorescein was applied in a light spray that just covered the substrate. The blood stains were checked for fluorescence after the fluorescein treatment and before the samples were extracted.

Table 1

Experimental design and results.
+ means DNA was extracted from the sample.
- means no DNA was extracted.

Sample Preparation

The blood dilutions were prepared by diluting blood with deionized water (DI) to produce a range of blood concentrations between 1: 1 to 1: 50,000 (blood : DI water) by volume (v/v). The range of dilutions are shown in Table 1. A set of each dilution of blood was kept as a control. The blood dilutions were deposited in 40 uL aliquots onto denim substrates in marked locations, approximately 4 mm in diameter, and allowed to dry for three days.

Extraction of DNA from Substrates

For the denim samples, 4 x 4 mm sections of substrate, containing the tested area, were cut out of the denim square and placed in 1.5 mL tubes. Next, 0.75 mL of DI water was added to each tube and the sample was incubated at ambient temperature for 30 minutes. The tubes were then vortexed for 5 seconds and centrifuged for 1 minute at a maximum speed of 7000 RPMS [5]. The supernate, ~500 uL, was drawn off and placed into another 1.5 mL tube.

A Chelex 100 extraction [2] with a 5% w/v (5 g Chelex to 100 mL of DI water) [6] was performed by adding 200 uL of the Chelex solution to each tube and vortexing each sample gently for 1 minute. The tubes were then incubated at 56 degrees C for 10 minutes in a water bath [7]. At the halfway point (5 min), the contents of the tube were remixed by placing them on a vortex stirrer for 15 seconds and then placing them back in the 56 C bath for the remaining 5 minutes [7]. The contents of the tubes were remixed and then placed in a boiling water bath for 6 minutes [7]. The tubes were again vortexed to resuspend the contents and then spun down for 5 minutes in a centrifuge. The Chelex binds to everything except the DNA (the supernate), allowing the supernate to be pipetted off and then used for DNA amplification.

Amplification

The extracted DNA was amplified at the D18S51 locus [8]. To perform this amplification, the DNA template, the upper supernatant phase, was transferred from the sample tube into the PCR tube in 6 uL portions. The positive DNA control (9947A [1]) and the negative DNA control (DI water) were also placed into clean PCR tubes in 6 uL portions. The PCR Master Mix was then transferred in 12 uL portions into all of the PCR tubes. The Master Mix contains dNTP (deoxynucleotide triphosphate); 10 X Reaction Buffer (Tris-HCl, KCl, MgCl2, and 0.01% gelatin); and taq polymerase.

The DNA primers (prepared by InvitrogenTM Life Technologies) were added in 3.5 uL quantities to each tube just prior to loading the samples into the thermocycler. The primer sequences were
5'-CAAACCCGACTACCAGCAAC-3' (P1) and 5'-GAGCCATGTTCATGCCACTG-3' (P2).

Mineral oil was added in ~20 uL aliquots to each sample to prevent evaporation. The reaction underwent 40 cycles (steps 1-3 below) of PCR amplification under the following conditions:

1. 1 minute @ 95 degrees C (Denature)
2. 1 minute @ 58 degrees C (Anneal)
3. 2 minutes @ 70 degrees C (Extend)
4. 10 minutes @ 72 degrees C (Final Extension-only performed once)
5. Maintain @ 4 degrees C

Preparation of Agarose Gel

For gel concentrations of 2% agarose, 1.6 g agarose was placed in a 250 mL flask and 80 mL of 1X TAE (Tris-Acetate-EDTA) buffer solution and 42 uL of ethidium bromide [0.5 ug/mL] were added. The 1X TAE buffer solution was prepared by placing 20 mL of 50X TAE buffer into a 1 L volumetric flask and filling it to 1000 mL with DI water. The solution was mixed on a stirrer hot plate until the agarose was dissolved and solution was clear. The gel solution was poured into an electrophoresis tray to a depth of about 5 mm.

PCR

To each PCR tube, 5 uL of loading dye [bromophenol blue (50 ug bromophenol blue per 100 mL buffer)] was added. The DNA ladder used was GeneRuler 50bp DNA ladder. Each well in the gel was loaded with 13 uL of PCR sample. The gel was then subjected to electrophoresis at 74V in a refrigerator (~ 4 oC) for approximately four hours. The electrophoresis run had its greatest resolution and separation under the conditions of 74V for 4 hours at T = 4 oC.

DNA Visualization

The resulting DNA was viewed under UV light using the Multi Image Light Cabinet ChemImager Ready and the Alpha Imager 2200 v. 5.5 program (Figure 1). The positive (+) results in Table 1 represent the presence of DNA bands when the electrophoresis gels were viewed in the ultraviolet cabinet. The negative (-) results correspond to no visible DNA bands on the gel.

Results and Discussion

DNA was recovered off of each treated blood sample (Figure 1). Therefore, fluorescein did not degrade the DNA in this study. This means that DNA can be recovered after the use of fluorescein to identify the latent blood evidence.

The blood used for this study was from the same individual. Therefore, it was expected that all of the DNA banks would occur with the same intensities across the ladder. However, this was not the case (Figure 1). This might be due to the annealing temperature being too low. Further experiments will test this hypothesis.

The blood to DI water ratios indicate this method's sensitivity. At any given violent crime scene, whole (neat) blood is deposited on various materials. Even when a clean-up of the crime scene is attempted, there is still generally more blood remaining in a square inch area than what was tested in this study

Figure 1

Positive results for DNA extraction post-fluorescein application.
A 2% agarose gel run at 74V and approximately 4 degrees C using method (c) in Table 1.

(1) positive DNA control, (2) 1:1000 (blood : DI water), (3) 1:10 (blood : DI water),
(4) 1:10 (blood : DI water), (5) negative DNA control, (6) 1:10 (blood : DI water),
(7) negative DNA control, (8) 1:100 (blood : DI water), (9) 1:100 (blood : DI water),
(10) 1:1 (blood : DI water), (11) 1:100 (blood : DI water), (12) 1:1000 (blood : DI water),
and (13) 1kb DNA ladder.

Conclusion

Fluorescein may be used at crime scenes to detect latent blood stains without destroying DNA evidence. This is an important advance in evidence collection and analysis.

The purpose of this study was to extract DNA post-fluorescein use. The study's goals were achieved and DNA was successfully extracted from the substrates after fluorescein was used to identify the latent blood on the substrates. The next step will be to examine the parameters of sensitivity and chemical interactions.

Future research into this technique will include optimizing the DNA sequencing, identifying the chemical reaction mechanism by which fluorescein binds to the blood while preventing damage to DNA, and determining whether fluorescein will react with any other body fluids or compounds (such as iron in urine).

This technique, when properly characterized, will provide investigators with a valuable tool for the identification, collection, and analysis of latent blood evidence.

Acknowledgments

This material is based upon research funded by the Geophysical Institute at the University of Alaska in Fairbanks, Alaska.

For further information, please contact:

Laurie A. Martin
University of Alaska Fairbanks, Police Department
612 Yukon Drive
Fairbanks, AK 99708
Laurie.Martin@uaf.edu
(907) 474-7436

References

1. Budowle, B.; Moretti T. Forensics: Analysis of Short Tandem Repeat Loci by Multiplex PCR and Real-time Fluorescence Detection During Capillary Electrophoresis. In DNA Profiling and DNA Fingerprinting; Epplen, J., Lubjuhn T., Eds.; Birkhauser: Basel, Switzerland, 1999; pp 101-116.
2. Budowle, B.; Leggitt, J. L.; Defenbaugh, D. A.; Keys, K. M.; Malkiewicz, S. F. The Presumptive Reagent Fluorescein for Detection of Dilute Bloodstains and Subsequent STR Typing of Recovered DNA. J. For. Sci. 2000, 45 (5), 1090-1092.
3. Schiro, G. Collection and Preservation of Blood Evidence from Crime Scenes. www.securityandsafetysupply.com/news/news37.htm, (accessed 6 September 2004).
4. Cheeseman, R.; Tomboc, R. Fluorescein Technique Performance Study on Bloody Foot Trails. J. For. Ident. 2001, 51 (1), 16-27.
5. Budowle, B.; Baechtel, F. S.; Comey, C., et al. Simple Protocols for Typing Forensic Biological Evidence: Chemiluminescent Detection for Human DNA Quantitation and Restriction Fragment Length Polymorphism (RFLP) Analysis and Manual Typing of Polymerase Chain Reaction (PCR) Amplified Polymorphisms. Electrophoresis 1995, 16, 1559-1167.
6. Bio-Rad Laboratories. Chelex 100 Instruction Manual (LIT200 Rev B). Hercules, CA.
7. Waye, J.; Presley, L.; Budowle, B.; Shutler, G.; Fourney, R. A Simple Method for Quantifying Human Genomic DNA in Forensic Specimen. Biotechniques 1989, 8 (7), 852-855.
8. Comey, C.; Koons, B.; Presley, K.; et al. DNA Extraction Strategies for Amplified Fragment Length Polymorphism Analysis. J. For. Sci. 1994, 39 (5), 1254-1269.

*From the Journal of Forensic Identification Vol. 54, No. 6, November/December 2004
The Official Publication of the International Association for Identification
"Reproduction of the Journal of Forensic Identification, in whole or in part, for noncommercial, educational use is permitted provided proper citation of the source is noted. Reproduction for any other use is prohibited without prior written permission. Requests for permission may be addressed to the editor (of the Journal of Forensic Identification -- jfieditor@theiai.org)."

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Article posted: January 6, 2008