Effect of Hydrogen Peroxide Treatment on Ability to Perform Alu Polymerase Chain Reaction in Hair...
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Effect of Hydrogen Peroxide Treatment on Ability to Perform Alu Polymerase Chain Reaction in Hair Samples By: Dominic Flaim Department of Biological Sciences, York College of Pennsylvania Introduction • DNA analysis has become increasingly useful for forensic scientists attempting to solve various crimes. One commonly encountered piece of evidence for the forensic scientist is human hair. • One of the most common methods used to examine DNA from a crime scene is using amplification of DNA by polymerase chain reaction (Reynolds 1991) and later analysis via electrophoresis. • DNA, although a relatively stable substance, can be damaged chemically. One commonly encountered molecule which damages DNA is hydrogen peroxide. It has been shown that hydrogen peroxide can damage DNA in very minute concentrations (Imlay et. al 1988). • Hydrogen peroxide is present in relatively high concentrations in common commercial hair dyes. The concentration of peroxide in these dyes is much higher than is necessary to cause strand breakage of DNA. • The objective of this study was to determine if use of hair treatments containing hydrogen peroxide have an effect on ability to use the hair for PCR and analysis of the Alu sequence. This could provide data to determine if treatment with hydrogen peroxide dyes has an effect on forensic analysis. Questions Asked 1.Does treatment with hydrogen peroxide based dye affect the ability to amplify DNA and detect in via electrophoresis for forensic purposes? 2.Does length of time hair has been treated have any affect on ability to detect DNA? http://www.nexxus.com/images/page_images/_ex_b_hair_anatomy.gif Methods Results Percentage ofSuccessfulA lu PC R Sam ples Cheek ntreated Hai Treated Hair 0 10 20 30 40 50 60 70 80 90 100 r 2 =.4370 G roups P ercen t o f S u ccessfu l S am ples Figure 2. Graph of successfully run samples in all groups. One way ANOVA was performed and the r 2 value was found to be 0.4370, with a P value of 0.4224. Figure 1. 2 percent agarose gel containing 100 bp ladder and cheek and hair samples run at 200 volts for 20 minutes. Wells 2, 5, 6, 8, 9, and 10 showed positive results (bands). Conclusions •Treatment with hydrogen peroxide based dye does not affect the ability of a sample to be amplified and analyzed via electrophoresis for forensic purposes. •Amount of time hair has been being treated has no affect on ability to amplify the DNA from the sample, as samples which had been treated more recently and samples which had not been treated for long periods of time amplified with equal likelihood. Acknowledgements: Thank you to Dr. Kaltreider, who was a great help in the process from start to finish and who put up with me throughout the project. Also, thank you to Dr. Rehnberg for inspiring me to look into research in biology. A very special thanks also to all those who let me pluck hair for this project. Pluck Hair (3-5 per sample) and Obtain Cheek Swabs Split Into Cheek, Treated (along with time since last treatment), and Untreated (2 Subgroups each) Place samples in Chelex (chelating agent) PCR On 1 uL With Alu Primers Run 10 uL of All Samples on 2% Agarose Gel in 35mL 1X SB Buffer (200 Volts for 20 Minutes) View For Bands at 100 (no insertion) and/or 400 bp (with insertion) Statistical Analysis (One-Way ANOVA) 400 bp band 100 bp band Literature Cited •Imlay, James A., Chin, Sherman M., Linn, Stuart. 1988. Toxic DNA Damage by Hydrogen Peroxide Through the Fenton Reaction in Vivo and in Vitro. Science [serial online] 240: 640-642. Available from: http://www.jstor.org/view/00368075/ap003583/00a00270/0 •Yakes, F. M. and Van Houten, B. 1997. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proceedings of the National Academy of Sciences of the United States of America [serial online] 94: 514-519. Available from: http://www.pnas.org/cgi/reprint/94/2/514 Yakes and Van Houten 1997 100 bp ladder Cheek Samples Future Direction • Possible future studies inclued analysis of treated and untreated hair for mitochondrial DNA and analysis of nuclear and mitochondrial DNA at different loci in treated and untreated hair. Treated Hair
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Transcript of Effect of Hydrogen Peroxide Treatment on Ability to Perform Alu Polymerase Chain Reaction in Hair...
Effect of Hydrogen Peroxide Treatment on Ability to Perform Alu Polymerase Chain Reaction in Hair Samples
By: Dominic FlaimDepartment of Biological Sciences, York College of Pennsylvania
Introduction• DNA analysis has become increasingly useful for forensic scientists attempting to solve various crimes. One commonly encountered piece of evidence for the forensic scientist is human hair.
• One of the most common methods used to examine DNA from a crime scene is using amplification of DNA by polymerase chain reaction (Reynolds 1991) and later analysis via electrophoresis.
• DNA, although a relatively stable substance, can be damaged chemically. One commonly encountered molecule which damages DNA is hydrogen peroxide. It has been shown that hydrogen peroxide can damage DNA in very minute concentrations (Imlay et. al 1988).
• Hydrogen peroxide is present in relatively high concentrations in common commercial hair dyes. The concentration of peroxide in these dyes is much higher than is necessary to cause strand breakage of DNA.
• The objective of this study was to determine if use of hair treatments containing hydrogen peroxide have an effect on ability to use the hair for PCR and analysis of the Alu sequence. This could provide data to determine if treatment with hydrogen peroxide dyes has an effect on forensic analysis.
Questions Asked1.Does treatment with hydrogen peroxide based dye affect the ability
to amplify DNA and detect in via electrophoresis for forensic purposes?
2.Does length of time hair has been treated have any affect on ability to detect DNA?
http://www.nexxus.com/images/page_images/_ex_b_hair_anatomy.gif
Methods Results
Percentage of Successful Alu PCR Samples
Cheek Untreated Hair Treated Hair0
102030405060708090
100 r2=.4370
Groups
Pe
rce
nt
of
Su
cc
ess
ful
Sam
ple
s
Figure 2. Graph of successfully run samples in all groups. One way ANOVA was performed and the r2 value was found to be 0.4370, with a P value of 0.4224.
Figure 1. 2 percent agarose gel containing 100 bp ladder and cheek and hair samples run at 200 volts for 20 minutes. Wells 2, 5, 6, 8, 9, and 10 showed positive results (bands).
Conclusions•Treatment with hydrogen peroxide based dye does not affect the ability of a sample to be amplified and analyzed via electrophoresis for forensic purposes.
•Amount of time hair has been being treated has no affect on ability to amplify the DNA from the sample, as samples which had been treated more recently and samples which had not been treated for long periods of time amplified with equal likelihood.
Acknowledgements:
Thank you to Dr. Kaltreider, who was a great help in the process from start to finish and who put up with me throughout the project. Also, thank you to Dr. Rehnberg for inspiring me to look into research in biology. A very special thanks also to all those who let me pluck hair for this project.
Pluck Hair (3-5 per sample) and Obtain Cheek Swabs
Split Into Cheek, Treated (along with time since last treatment), and Untreated
(2 Subgroups each)
Place samples in Chelex (chelating agent)
PCR On 1 uL With Alu Primers
Run 10 uL of All Samples on 2% Agarose Gel in
35mL 1X SB Buffer (200 Volts for 20 Minutes)
View For Bands at 100 (no insertion)
and/or 400 bp (with insertion)
Statistical Analysis (One-Way ANOVA)
400 bp band
100 bp band
Literature Cited
•Imlay, James A., Chin, Sherman M., Linn, Stuart. 1988. Toxic DNA Damage by Hydrogen Peroxide Through the Fenton Reaction in Vivo and in Vitro. Science [serial online] 240: 640-642. Available from: http://www.jstor.org/view/00368075/ap003583/00a00270/0
•Yakes, F. M. and Van Houten, B. 1997. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proceedings of the National Academy of Sciences of the United States of America [serial online] 94: 514-519. Available from: http://www.pnas.org/cgi/reprint/94/2/514
Yakes and Van Houten 1997
100 bp ladder
Cheek Samples
Future Direction
• Possible future studies inclued analysis of treated and untreated hair for mitochondrial DNA and analysis of nuclear and mitochondrial DNA at different loci in treated and untreated hair.
Treated Hair
