Improving genetic profiling techniques for low copy number DNA / by Shana Hayter.

The extraction of DNA from ancient samples presents many obstacles to the analyst. These samples are often subjected to many years of harsh environmental conditions and many forms of damage resulting in low copy number and highly fragmented DNA. Furthermore, due to their degraded nature, these samples may only be present in small quantities and thus limit the analysis to only one or two extractions. Therefore strategic methodological approaches must be designed to accommodate these limiting factors, while maximizing the results that may be achieved. The initial stages of analysis are the most crucial, since they are responsible for isolating the DNA. Therefore, the assessment of different decontamination, sample preparation and extraction techniques to determine their ability to yield high quality DNA was conducted. Three types of tissue, 50 bone extractions, 49 teeth extractions and 10 soft tissue extractions were evaluated using two sample preparation methods and four extraction methods. Homogenization or pulverization of the sample increased the overall surface area of the sample, and resulted in a higher success of retrieving DNA for all three tissue types. Both bone and teeth were found to be reliable sources for DNA, however the success of the extraction method was dependent upon the preservation of the sample. Proteinase K and Guanidinium Thiocyanate were determined to be the most reliable methods for aneient samples from all three types of tissue with 45% and 36% success respectively. A novel technique. Pressure Cycling Technology was found to have promising implications for modem samples, with 100% detection. mtDNA sequence analysis concluded these aDNA samples were of low copy number and highly fragmented

Postmortem Miscoding Lesions In Sequence Analysis Of Human Ancient Mitochondrial Dna

Genetic miscoding lesions can cause inaccuracies during the interpretation of ancient DNA sequence data. In this study, genetic miscoding lesions were identified and assessed by cloning and direct sequencing of degraded, amplified mitochondrial DNA (mtDNA) extracted from human remains. Forty-two individuals, comprising nine collections from five geographic locations, were analyzed for the presence of DNA damage that can affect the generation of a correct mtDNA profile. In agreement with previous studies, high levels (56.5% of all damage sites) of proposed hydrolytic damage products were observed. Among these, type 2 transitions (cytosine → thymine or guanine → adenine), which are highly indicative of hydrolytic deamination, were observed in 50% of all misincorporations that occurred. In addition to hydrolytic damage products, oxidative damage products were also observed in this study and were responsible for approximately 43.5% of all misincorporations. This level of misincorporation is in contrast to previous studies characterizing miscoding lesions from the analysis of bone and teeth, where few to no oxidative damage products were observed. Of all the oxidative damage products found in this study, type 2 transversions (cytosine → adenine/guanine → thymine or cytosine → guanine/guanine → cytosine), which are commonly formed through the generation of 8-hydroxyguanine, accounted for 30.3% of all genetic miscoding lesions observed. This study identifies the previously unreported presence of oxidative DNA damage and proposes that damage to degraded DNA templates is highly specific in type, correlating with the geographic location and the taphonomic conditions of the depositional environment from which the remains are recovered. © 2008 Springer Science+Business Media, LLC