Characterization of a specific single nucleotide polymorphism using limited primer extension assay and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

摘要

Many factors have been shown to influence the manifestation of cancer, but the two major classes of factors are hereditary and environmental. Unregulated cell growth is discernible through a series of acquired or inherited mutations to genomic DNA, which leads to the loss of the genetic information that define cell functions reducing or eliminating the normal control over the cell. The long term goal of Dr. Chiu's research program is to study the relationship between DNA adduction and genetic mutation. The experimental model chosen was a carcinogenic 4-aminobiphenyl-DNA (4-ABP-DNA) adduct that has been characterized and related to different types of cancer. To study the possible relationship between DNA adduction and genetic mutation, the experimental model in this study also included a single nucleotide polymorphism (SNP) on codon 72, exon 4 of the human p53 gene that has been linked to bladder cancer. To achieve the long term goal of Dr. Chiu's research program, a novel assay for determining the adduct identity and its position in the human genome was proposed. The DNA template used in this study was consistent with the sequence and condition that may be present before and/or during the development of a carcinoma in bladder tissue. The DNA template also included sites for sequence recognition by restriction endonucleases. The same restriction sites will allow the DNA fragment from genomic DNA to be isolated. As part of our efforts to develop the new assay, the selected p53 gene fragment sequence from a sample has been fully characterized at the selected SNP position in this thesis. The characterized sequence will be used in future work to prepare a standard for the development of a new assay for pinpointing the position of carcinogen-DNA adduction, which has potential to be used as both a diagnostic tool for clinicians, and a method of assessment for the safety of possible carcinogens which may be present in industry and the environment.