Cancer has been linked to mutations within specific codons in genes that code for critical biomolecules such as tumor suppressor proteins (e.g., p53). Activated metabolites like benzo [a]pyrenediol epoxide act on preferred nucleotide sequences of DNA, and such mutations have been identified in cancers. DNA reaction site identification depends on accurate analysis of oligonucleotide fragment sizes produced by strand breakage at the damaged sites. Herein, we report a new method for DNA fragment sizing using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Absolute sizing accuracy and speed are achieved by utilizing a CE-LIF array with two-color fluorescence detection. Accuracy depends on correcting results with commercial standards by referring them to primary standards with the same sequences and identical labels as sample fragments. The method is demonstrated by detection of a […GGCGCG-CAG…] G reaction site for styrene oxide on an oligonucleotide representing the CYP1B1 gene.This approach avoids the need for radioactive isotopes and is less labor intensive and faster than the alternative PAGE with 32 P end labeling.Cytochromes P450 in oxidative liver metabolism convert foreign hydrophobic substances into hydrophilic compounds that can be more readily excreted by the kidneys. 1 However, some metabolites may be chemically reactive electrophiles. These reactive metabolites may damage DNA, proteins, and other biomolecules or be inactivated by metabolic bioconjugation enzymes. 2 DNA damage often involves covalent binding of reactive metabolites to nucleobases, which may then lead to apoptosis (programmed cell death) or to neoplastic transformations.Cancer has been linked to specific changes within the amino acid sequence of certain proteins that control the movement of cells through the cell cycle, e.g., the p53 tumor suppressor protein. 3,4 These amino acid changes result from mutations in specific codons in the genes. The majority of human cancers are found to have mutations in the p53 gene. [5][6][7] These mutations do not occur at random within the gene but occur at specific positions with , adduct formation at the first guanine of codon 248 of the p53 gene causes G→T transversions. 3 This results in a point mutation in the p53 tumor suppressor protein, the substitution of an arginine for leucine at residue 248. The loss of the tumor suppressive capabilities of this mutated protein may contribute to uncontrolled cell growth or cancer. 11,12 Mapping the nucleobase sequence specificity for reaction of a reactive metabolite with DNA can be used as a predictive tool for carcinogenicity. There are two methods for detecting the sequence specificity of a DNA-damaging compound. The first involves isotopic 15 N or 13 C labeling of specific nucleobases within an oligonucleotide and determining the amount of damage at the site by mass spectrometry. [13][14][15][16] This method is rapid and also identifies the type of nucleobase adduct formed at the specific site. However, isotope-labele...
Removal of nucleobases from the DNA backbone leads to the formation of abasic sites. The rate of abasic site formation is significantly increased for chemically damaged nucleobases. Thus, abasic sites serve as general biomarkers for the quantification of DNA damage. Herein, we show that capillary electrophoresis with laser-induced fluorescence (CE-LIF) can be used to detect the amount of abasic sites with very high sensitivity. For proof of concept, DNA was incubated with methylmethane sulfonate (MMS) and the damaged bases were removed by incubation at 80 degrees C. The resulting abasic sites were then tagged with a fluorescent aldehyde-reactive probe (FARP). The DNA was precipitated with ethanol, and then analyzed by CE-LIF. CE-LIF and HPLC analysis shows that the fluorescently tagged DNA (DNA-FARP) had a peak area directly proportional to the amount of N-7 methyl guanines. The CE-LIF method had a detection limit of 1.2 abasic sites per 1,000,000 bases or ca. 20 attomoles of abasic sites. This provides a general method for detecting DNA damage that is not only faster but also has comparable or better sensitivity than the alternative ELISA-like method.
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