Analysis of DNA Damage in Forensic Blood Evidence Exposed to
Swimming Pool Water Using Neutral and Alkaline Agarose Gel
Electrophoresis
Joseph Batte, Master of Forensic Science, Molecular biology concentration
Mentor: Monika Jost Ph.D. Drexel University College of Medicine. Graduate School of Biomedical Sciences and Professional Studies
Introduction
Materials and Methods
Discussion and Conclusions
200µL of EDTA-anticoagulated bovine whole blood or blood
stains were exposed to 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1mL,
1.2mL, 1.4mL, 1.6mL, 1.8mL or 2mL of swimming pool water,
or Millipore water at room temperature for 24hrs. DNA was
extracted using phenol-chloroform, followed by ethanol
precipitation, and rehydrated in TE buffer. Aliquots were
analyzed using neutral or alkaline agarose gel electrophoresis
to detect double and single strand DNA breaks, respectively.
DNA digested with Alu I restriction enzyme, and bovine whole
blood exposed to 100% (v/v) bleach were used as positive,
and severe DNA damage controls respectively. Resulting gel
images were visualized using a UVB Bio Doc-It Imaging
System.
We observed dsDNA and ssDNA breaks in blood treated with
pool water, indicating DNA damage. This could potentially result
in decreased quality or failure of subsequent DNA analysis and
inconclusive DNA profiles derived from blood or other tissue
evidence. The observed prominent DNA breaks, at least the
dsDNA breaks, are probably a result of trace amounts of
disinfectants and DBPs present in swimming pool water. Our
detection method is not sensitive enough to detect differences in
ssDNA breaks between pool water-treated samples and
controls. Swimming pool water used in this study was collected
from an indoor swimming pool, with an automatic chemical
dispenser, indicating that chemical levels were well regulated.
However, the effect of pool water on DNA evidence could vary,
for example in a home pool, where the disinfecting procedure is
typically manually maintained and not performed as regularly.
The resulting irregularities in trace amounts of disinfectant and
DBPs, in conjunction with other damaging factors, such as UV
light, in outdoor pools, potentially enhance DNA fragmentation,
severely affecting forensic DNA typing. Future studies will
further characterize and quantify the effects of individual
compounds on DNA integrity and address the impact of these
damaging agents on DNA typing results.
Investigation of forensic cases in which the victim and/or evidence were exposed to
swimming pool water can be challenging. This is in part due to inconclusive forensic DNA
typing results, obtained from DNA being fragmented or damaged in another way. Swimming
pools are usually disinfected using chlorine or bromine derivatives, which are chemicals
known to hydrolyze, oxidize, destroy and/or modify DNA bases. Often, insufficient amounts
of disinfectants are added into pools, resulting in incomplete binding and inactivating of
contaminants, such as ammonia excreted by swimmers. This results in formation of
disinfectant byproducts, (DBPs), for example trihalomethane, which are genotoxic,
carcinogenic and mutagenic, altering and/or damaging DNA. Given the effects of
disinfectants and DBPs on DNA, we hypothesize that trace amounts of chlorine, bromine and
their corresponding DBPs in swimming pool water, could interfere with forensic DNA analysis,
by inducing double and/or single strand DNA breaks. We subjected bovine blood and blood
stains to swimming pool or control purified water and assessed dsDNA and ssDNA breaks
by neutral and alkaline electrophoresis, respectively.
Results
As shown in figure 1, all samples display dsDNA breaks. Compared to A)
controls, we observed more prominent DNA double strand breaks in pool
water-treated blood, with most samples displaying laddering, consistent with
cellular apoptosis. However, the laddering pattern observed in pool watertreated samples disappears in samples to which larger volumes of pool
water were added, indicating that resulting DNA fragmentation is possibly
not by induced cellular apoptosis, but as a result of chemicals in pool water
interacting directly with DNA.
B)
Figure 2: Detection of dsDNA breaks by neutral agarose gel electrophoresis (A) and ssDNA breaks by alkaline
electrophoresis (B). DNA extracted from whole blood treated as indicated was separated by neutral AGE, as in figure 1,
or by alkaline 0.8% agarose gels in alkaline running buffer.
Figure 1: Detection of dsDNA breaks by neutral agarose gel electrophoresis. DNA from anticoagulated bovine
whole blood exposed to indicated amounts of water, was separated on 0.8% agarose gels in TAE buffer. 1kb
ladder was loaded as size marker.
To directly compare strand breaks, selected samples exposed to equal volumes
of Pool or Millipore water were run on the same, neutral and alkaline gels (figure
2). Consistent with results shown in figure 1, pool water induces more intense
dsDNA breaks. In alkaline electrophoresis, only a weak distinction between
bovine whole blood exposed to swimming pool water or Millipore water is visible,
in that pool water-treated blood displayed thicker bands within the smear. These
thick bands most likely represent denatured apoptotic DNA fragments.
Overall, the observed dsDNA breaks in pool-water exposed samples
support our hypothesis that chemicals in pool water damage DNA.
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Acknowledgments
Dr. Monika Jost, my mentor for her support and endless assistance. Drexel Aquatics
Department for authorized water samples, assistance by Tori Mayer and Tara
VanDoren. Gabrielle Stine, Rebecca Witmer, and colleagues for inspiration,
motivation and encouragement. Master of Forensic Science program for an
opportunity to conduct research, Graduate School of Biomedical Sciences and
Professional Studies, and Drexel University College of Medicine.
Poster produced by Faculty & Curriculum Support (FACS), Georgetown University School of Medicine