High fidelity polymerase chain reaction (hifi-PCR) DNA amplification has been developed and used in combination with denaturing gradient gel electrophoresis (DGGE) as a means to observe mutational spectra in human cells. DGGE is used to separate mutant sequences from the wild type sequences. The mutant sequences are then amplified using high fidelity polymerase chain reaction and analyzed by a second DGGE to observe mutational spectra. The project aims at: (1) improving the conditions of T4 DNA polymerase in the PCR process to increase its efficiency and its fidelity to carry out PCR. This will be carried out by using proteins associated with T4 DNA polymerase to synthesize DNA in vitro including: (a) the protein P32 which has the property of dissociating the double strand target DNA. (b) accessory proteins which increase the processivity and fidelity of T4 DNA polymerase. (c) an antimutator T4 DNA polymerase. (2) improving the conditions for this new tool to observe mutational spectra without the need for phenotypic selection. This will be carried out by using specific probe to hybridize with the target sequences investigated to increase their efficiency of hybridization and subsequently their separation and isolation from the wild type with high resolution. (3) apply the means developed to study unselected mutational spectra in human cells for chromium compounds. (4) which are major contaminants arising from tanneries in the history of the Aberjona Watershed.
Showing the most recent 10 out of 80 publications
