Assessing in vitro DNA repair methods / by Ryan Lehto.

Abstract

The DNA molecule is constantly under attack from endogenous and exogenous sources leading to degradation by a variety of forms of damage. In this study current methods of in vitro DNA repair methods were evaluated for efficiency and for their relative ability to repair three different experimentally induced forms of damage strand breaks, abasic sites, modified bases and crosslinks. GCMS was used to identify and evaluate effectiveness of repair methods for 16 different damaged products or modified bases. The methods were then used on ancient samples from three sites 1) Daklah Oasis, Egypt; 2) Copan, Honduras; and 3) Çayonü Tepesi, Turkey. Success was achieved repairing these ancient samples depending on which method was used and what type of dam age was present. These results were consistent with the DNA damaged expected in each of these sets of samples predicted hypothetically from the environment in which these samples were recovered. A new helicase dependant DNA polymerase. Phi 29 had an unexpected DNA repair capability notably on hydrolytic damage while the PreCR ™ enzyme repair mix was very effective at repairing the affects of oxidative damage. The DNA repair capabilities of 8 DNA repair systems were characterised and the demonstrated successful retrieval of DNA from 6 ancient DNA samples previously shown to be non-viable for genetic analysis were successfully analyzed and amplified.