high-resolution melt analysis for semen discrimination
TRANSCRIPT
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High Resolution Melt analysis of DNA methylation
to discriminate semen in biological stains from crime
scenes
Joana Antunes, Ph.D candidate Biochemistry
Advisor: Dr. Bruce McCord
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This work was possible thanks to…
Dr. Balamurugan and Robin Bombardi, University of Southern Mississippi Dr. Duncan, DNA Laboratory, Broward County Sheriff’s Office Dr. Liu, Dr. Mills and Dr. Almirall, Committee members GPSC
Awards 2012-DN-BX-K0182015-R2-CX-0012
Major support provided by:
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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Why do we want to discriminate body fluids?
DNA found on child potential victim of sexual assault
DNA matches the suspectSuspect claims that he hugged the childIf DNA is from skin = innocent intent
If DNA is from vaginal epithelia/semen = criminal intent
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Current chemical, protein and microscopic tests are presumptive with varying degrees of specificity
and sensitivity
Recently suggested procedures include gene expression profiling using mRNA
Concerns with long term stability of proteins/RNA compared to DNA
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Kong, A., Zhang, D., Guangming L., A study of identical twins’ palmprints for personalLegget, B., Whitehall, V., Role of the serrated pathway in colorectal cancer pathology.2010 Gastroenterology 138 (6):2-88-2100
Same genome location in different cells have different methylation
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We can use differences in DNA methylation to distinguish semen
from other body fluids using methods and instruments
commonly available in forensic laboratories
Hypothesis
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DNA Extraction
Bisulfite Modification
High Resolution Melt
Methodology7
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Bisulfite modification 8
Unmethylated - Predominantly T=A bound
Methylated - Predominantly GΞC bounds
T. Azhikina, et al. Biochem. (Moscow) 70 (2005) 722-730
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High Resolution Melt PCR (HRM) 9
T. Azhikina, et al. Biochem. (Moscow) 70 (2005) 722-730http://jcm.asm.org/content/49/9/3132.figures-only
UnmethylatedPredominantly T=A bounds Melts at lower temperatures
MethylatedPredominantly GΞC bounds Melts at higher temperatures
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10Melt curve analysis of ZC3H12D can be used to discriminate semen
from other body fluids
Semen 75.5 ± 0.2 ºC
Blood 78.2 ± 0.4 ºC
Saliva78.1 ± 0.3 ºC
Madi, T., Balamurugan, K., Bombardi, R., Duncan, G., McCord, B. Electrophoresis 2012, 33, 1736-1745.
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Genomic DNA
Bisulfite-modified DNA
Primers are specific for bisulfite-modified DNA
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Blood Saliva Semen Blood Saliva Semen Blood Saliva Semen
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If less than 1 ng of genomic DNA is used amplification may fail
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Conclusions 14
The ZC3H12D primers designed by us are specific for bisulfite-modified DNA, preventing false results due to failure in bisulfite-modification
This method presents good sensitivity providing results with only 1 ng of genomic DNA
We were able to distinguish semen from blood and saliva employing methods and instruments commonly used in forensic laboratories
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Thank you!
Questions?