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

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

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

DNA Extraction

Bisulfite Modification

High Resolution Melt

Methodology7

Bisulfite modification 8

Unmethylated - Predominantly T=A bound

Methylated - Predominantly GΞC bounds

T. Azhikina, et al. Biochem. (Moscow) 70 (2005) 722-730

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

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

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

Thank you!

Questions?