Clark, Graeme Thomas (1999) A study on DNA repair. University of Southampton, Doctoral Thesis.
Abstract
With more diseases commonly becoming linked to genetic disorders, enzymes that process DNA within repair pathways need to be examined. Polyhistidine tagged proteins from the E.coli methyl-directed MutHLS mismatch repair pathway have been expressed and purified via one-step Ni2+ chromatography. The mismatch binding affinity of MutS has been demonstrated in vitro and the Kd established as 0. 126μM. Attempts to isolate the minimal mismatch binding domain of MutS were unsuccessful and initial results indicate that this may not be feasible. Attempts at synthesising a non-hydrogen bonding analogue of Thymine for investigation into potential recognition sites for MutS upon DNA were also unsuccessful.
Classical methods for DNA analysis are both costly and time consuming and previous attempts at using MALDI-TOF MS for analysis have resulted in complex sample preparation and ambiguous peak assignment. It has been shown that a novel sample preparation used in conjunction with complete enzymatic digestion of DNA with phosphodiesterases, can successfully sequence oligonucleotides up to 39 bases in length, with increased resolution, a 40 fold increase in sensitivity and in a greatly reduced time scale.
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