1. Introduction The use of forensic science has had a significant impact within the criminal justice system. The tools and knowledge provided by utilizing criminalistics can help solve cases and implicate or disprove an individual’s guilt. The unique distinctive ability of fingerprints has been known for over a century (Saferstein 2001). The individualizing properties of DNA have generated a momentous role in forensic science in the past 20 years. Because of this role, the majority of new crime scene techniques are tested against the impact on DNA analysis. For example, a recent technique has been developed to detect and analyze fingerprints made in blood. The most common method is a methanol based amido black solution, which reacts with the proteins in the blood and turns the print a blue-black color. This produces a fairly accurate and detailed fingerprint. However, several health risks are associated with the methanol solution. It is toxic, flammable, and cannot be used outside of the laboratory setting (Sears and Prizeman 2000). Therefore samples must be gathered in the field and brought back to the lab. Studies have also suggested that the methanol-based solution may ‘set’ the blood or even degrade the DNA (Roux et al. 1999). Because of this, recent experiments have attempted to replace the methanol with a safer ethanol solution. Preliminary studies have concluded that this solution is as effective as the methanol in isolating fingerprints in blood (Sears and Prizeman 2000). However, no studies have tested the effect this solution may have on subsequent DNA analysis. Therefore, the goal of this study was to determine the effect of an ethanol-based amido black solution on the PCR analysis of a bloodstained fingerprint. The Effects of Ethanol-Based Amido Black Solution on DNA Objectives: To determine if the ethanol-based amido- black solution has any negative effects on the PCR analysis of the DNA in the bloodstain Hypothesis: Does the newly developed ethanol-based amido black solution degrade DNA? The ethanol based solution will degrade the DNA There will be no difference between the ethanol and methanol based solutions Abstract A recent technique has been developed to enhance the detection of fingerprints made in blood. Older methanol-based solutions were found to hinder or degrade the blood so that PCR analysis could not be completed. This study looked at the effects of an ethanol-based solution on the PCR analysis of the bloodstain. It was concluded that the ethanol solution also prevented the PCR analysis from being conducted. Laura Beyer Advisors: Dr. Ronald Kaltreider and Professor Sherry Brown Methods Bloody fingerprints were made on three separate linoleum tiles: 1.Control sample 2.Ethanol sample 3.Methanol sample A check cell sample was also taken for use as a positive control. The ethanol and methanol solutions were prepared using the same protocol. Fixing solution -5g 5-sulfosalicylic acid -250 ml distilled water -stirred with magnetic stirrer until powder dissolved Staining solution -0.25g acid black -62.5 ml ethanol/methanol -12.5 ml glacial acetic acid -175 ml distilled water -stirred with magnetic stirrer until powder dissolved Destaining solution -62.5 ml ethanol/methanol -12.5 ml glacial acetic acid -175 ml distilled water -stirred with magnetic stirrer for 5 minutes Each solution was sprayed on the fingerprints and allowed to stand for 5 minutes. Once dry, the process was repeated for the next solution. DNA samples were obtained from the 3 bloodstains using the following protocol: -blood was scraped into a PCR tube using a sterile razor -100 µl of Chelex was added -5 min. heat block and 1 min. ice -solution was centrifuged for 1 min. -5µg of DNA and 20µg of VNTR primer were added to a PCR tube containing Pure Taq Ready-to-Go PCR bead The samples were placed in the thermacycler under the VNTR PCR analysis and ran for 30 cycles. A 1.5% agarose gel was prepared using 1 µl of ethidium bromide. Another 1.5% agarose gel was prepared using 1 µl of Gel Star as a substitute for the ethidium bromide. Once the thermacycle was complete, the samples had 2 µl of loading dye added and were pipetted into the gel. The ethidium bromide gel was run for 1 hour at 100 volts. The Gel Star gel was run for 45 minutes at 100 volts. Results Cheek cell control produced the expected banding pattern around 400 basepairs Control sample of blood taken from fingerprint made in blood did present a faint banding pattern at the same location A double exposure of the photo made the band easier to view Only primers were detected in the lanes containing the ethanol and methanol tainted DNA No bands appeared in either of these lanes around the same location It was then determined that no other bands were present Figure 5. Agarose gel (1.5%) run for 1 hour using ethidium bromide. Double exposure of the picture was used to enhance the visibility of the faint band created by the control bloodstain sample found at 400 bp. Figure 6. Agarose gel (1.5%) run for 45 minutes using Gel Star substituted for the ethidium bromide. Gel Star is more sensitive and allows for the viewing of even smaller fragments than the ethidium bromide. The photo was also double exposed in order to see the banding more clearly. The band was also located around 400 bp. Literature Cited Roux, C., Gill, K., Sutton, J., Lennard, C. (1999). A further study to investigate the effect of fingerprint enhancement techniques on the DNA analysis of bloodstains. Journal of Forensic Identification, 49 (4), 357-377. Saferstein, Richard. Criminalistics: An Introduction to Forensic Science. 7th ed. New Jersey: Prentice Hall, 2001. Sear, V., and Prizeman, T. (2000). Enhancement of fingerprints in blood – part 1: The optimization of amido black. Journal of Forensic Identification, 50 (5), 470-481. Acknowledgements The author would like to thank Dr. Kaltreider, Professor Brown, and Dr. Thompson for their continued support and expertise. Conclusions Hypothesis was correct *Neither the methanol nor the ethanol displayed banding patterns* * Ethanol also degraded the DNA so that no banding appeared* It was determined that the ethanol solution produced the same effect as the methanol solution in regard to the degradation of DNA. if the ethanol solution was used to enhance the fingerprint ridges, VNTR PCR DNA analysis would not be able to be completed DNA profiles could not be obtained from the bloodstain Advantages to replacing the methanol solution with the ethanol solution include: Better development of ridge detail and fingerprint characteristics Can take ethanol to the crime scene Less toxic to humans Non-flammable Less corrosive to surfaces being tested Figure 2. Bloodstained fingerprint on linoleum tile sprayed with ethanol-based amido black solution. Figure 3. Bloodstain fingerprint on linoleum tile treated with the methanol-based amido black solution. Figure 4. Agarose gel (1.5%) run for 1 hour using ethidium bromide. The band is located at 400 bp and is the control cheek cell sample of DNA. Figure 1. Bloodstained fingerprint left untreated for 24 hour period on a linoleum tile.