Introduction Using DNA evidence in criminal cases has become increasingly accepted over the past twenty years. When one thinks of using DNA to solve cases, one usually assumes the DNA is human and the cases pertain to such things as homicide. However, there are other types of cases and scenarios that can also be solved by means of DNA evidence. Cases such as protection of endangered animals (Hsieh, et. al.), detection of mislabeled meat products (Lin, Hwang), as well as determination of the origin of forensic samples are all examples of how DNA can be used in cases that do not directly pertain to humans. There are many different types of DNA that can be used in identification, some are better than others. Mitochondrial DNA is a great choice when the comparison of species is desired. Mitochondrial DNA has a high rate of base substitution, which means that it mutates rapidly (Lin, Hwang). Also, it is inherited only from the mother and does not recombine making the DNA much simpler than genomic DNA which can recombine with the DNA from another source, like the father (Lin, Hwang). Another plus when working with mitochondrial DNA is the high number of copies found in each cell (Bravi, et. al.). Cytochrome b is a specific gene found within the mitochondrial DNA, and has been used extensively to identify species as well as in inheritance and evolutionary studies (Lin, Hwang). Polymerase chain reaction, or PCR, has been used extensively to create copies of a specific region of DNA, such as the cytochrome b gene. PCR uses primers, which are short, single stranded chains of nucleotide bases with a specific sequence that will bind to a specific sequence of the DNA, to replicate that region of the DNA. Restriction enzymes are also used with PCR to help identify a sample. Restriction enzymes are enzymes that recognize short DNA sequences and bind to them. After they bind, they cut the DNA at the sight at which it is bound, leaving the DNA in pieces. This allows the differentiation of samples, because different species generally have different arrangements in the sequence of their DNA which is reflected in different size fragments from restriction enzyme digestion. In this experiment species will attempt to be differentiated by means of PCR and restriction enzyme digests. It is hypothesized that the species will be differentiated by the bands they produce on an agarose gel. Methods DNA extractions DNA samples were collected from living species by way of oral swabs. Tissue samples were collected from dead species. These samples were: human, fox, cat, rat, rabbit, raccoon, possum, guinea pig, pig, turkey, mole, horse, goat, chinchilla, ferret, mouse, cow, deer, and coyote. DNA was extracted from these samples by digestion with TN buffer, then a series of phenol/chloroform extractions, and finally a wash with ethanol followed by a twenty four hour incubation period to precipitate the DNA as described in Molecular Lab Manual (Brown). The DNA was centrifuged and the supernatant removed the DNA was then allowed to air dry and then resuspended with TE buffer (Brown). PCR PCR was carried out using the primer pair L14816 (5’- CCATCCAACATCTCAGCATGATGAAA-3’) and H15173 (5’- CCCCTCAGAATGATATTTGTCCTCA-3’) (Bravi, et. al.). The PCR reactions were carried out on the product of DNA extraction. Each PCR reaction consisted of 1 (or 5) μl DNA, 10 μl 5x TaqMaster buffer, 5 μl 10x Taq buffer with Mg 2+, 1 μl L14816, 1 μl H15173, 1μl dNTP mixture, and 0.5 μl Taq polymerase brought up to a final volume of 50 μl with sterile ultra pure water (Brown). The samples were then loaded into the thermocycler and ran according to the temperature profile described in Bravi, et. al. Electrophoresis Electrophoresis of the PCR product was run on a 1.2% agarose gel with 20 μl PCR product and 4 μl loading buffer. Restriction enzyme digests Restriction enzyme digests were then carried out on the PCR products for the samples that had bands present using HinfI and AluI. The digests consisted of 20 μl DNA, 0.25 μl BSA, 1.75 μl distilled ultra pure water, 0.5 μl restriction enzyme, 2.5 μl buffer. The digests were incubated at 37ْ C for one to two hours. Electrophoresis of restriction enzyme digests Once the digests were complete they were electrophoresed on a 1.2% agarose gel. Twenty μl DNA was mixed with 4 μl loading buffer and loaded onto the gel. The size marker θX174 digested with HaeIII was run on each gel for later size comparisons. The samples were run at 10 volts overnight. Bands were then measured for size comparisons. Results Electrophoresis of PCR product Of the twenty three sample run, there were six that had visible bands of the PCR product. Those six are pictured in lanes 3, 4, and 10 of Figure 1, and lanes 3, 6, and 9 of Figure 2. Figure 1 Results of electrophoresis of PCR products for mouse, fox, coyote, deer, cow, goat, pig, turkey, mole, and rabbit. Bands are present for turkey, pig, and mouse. Figure 2 Results of electrophoresis of the PCR products for fox, raccoon, horse, guinea pig, ferret, rat, hawk, human, possum, and rabbit. Bands are shown for human, ferret, and raccoon. Electrophoresis of restriction enzyme digest Of the six sample that gave bands, all were visible on the HinfI digest, and two were visible on the AluI digest. Figure 3 results of electrophoresis of HinfI digest on raccoon, human, ferret, mouse, pig, and turkey DNA. Figure 4 Using the equation from this graph, the number of base pairs for each band can be determined by plugging in the band length for x. Figure 5 results of electrophoresis of AluI digest on raccoon, human, ferret, mouse, pig, and turkey DNA. Figure 6 Tables 1 and 2 show the base pair sizes for bands that were visible on the gel. Works cited Bravi, Claudio M., Lirón, Juan P., Mirol, Patricia M., Ripoli, María V., Peral-García, Pilar, Giovambattista, Guillermo A simple method for domestic animal identification in Argentina using PCR-RFLP analysis of cytochrome b gene Legal Medicine 6: Brown, D Molecular biology lab manual. Marietta College, Marietta, OH: Hsieh, Hsing-Mei, Chiang, Hsiao-Ling, Tsai, Li-Chin, Lai, Shu-Ya, Huang, Nu-En, Linacre, Adrian, Lee, James Chun-I Cytochrome b gene for species identification of the conservation animals Forensic Science International 122: Lin, Wen-Feng, Hwang, Deng-Fwu Application of PCR-RFLP analysis on species identification of canned tuna Food Control 18: Acknowledgements I would like to say thank you to the Marietta College Biology Department for funding my research, Dr. Brown for all of the help trouble shooting my procedures, Dr. Tschunko for being my personal advisor, Dr. Hogan, capstone instructor, Dr. Mcshaffery for teaching me how to use Photoshop, my fellow students for all their help and advice, and my husband for putting up with me through everything. Conclusion Every band that was present was different from each of the other bands; however, a conclusion can not be reached because no duplicate samples were tested of a certain species to ensure that all individuals of a particular species give a band of the same size. It is possible that the banding patterns seen are different for each individual rather than each species. To ensure this is not the case additional samples from the same species are needed. A few reasons why there were not bands for every sample are: no DNA was present in the sample, the DNA in the sample was degraded, there was a problem in the extraction procedure, or not enough of the extraction was used in PCR and there was no, or very little, DNA due to the very small quantities used. In the future, it would be wise to run a large number of samples for each species as well as a larger variety of species. In the future, it would be wise to run a large number of samples for each species as well as a larger variety of species. Table 1: HinfI digest Species# base pairs Turkey110 Pig486 Mouse439 Ferret392 Human63 Raccoon345 Table 2: AluI digest Species# base pairs Turkey352 Pig250 Alicia CecilCapstone 2007