Accurate DNA Fragment Sizing by Capillary Electrophoresis with Laser-Induced Fluorescence Array for Detection of Sequence Specificity of DNA Damage

Abstract

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...

Use of an automated capillary DNA sequencer to investigate the interaction of cisplatin with telomeric DNA sequences

“…In addition, the use of capillary electrophoresis permits increased sensitivity and hence more accurate intensity determination of damage bands compared with slab gels. There have been previous attempts to utilize automated DNA sequencers to examine the sequence selectivity of DNA binding agents in slab gels (Hardman and Murray, ; Yindeeyoungyeon and Schell, ; Bando et al , ) and nonautomatic capillary gels (Fundador et al , ) with varying degrees of success.…”

“…In contrast, using a primer with a fluorescent tag such as FAM, VIC, PET or NED for linear amplification eliminates radiation hazard risk (Shimizu et al , ). Replacing slab gels with capillary gel electrophoresis with laser‐induced fluorescence (CE‐LIF) also reduces the time needed to analyse samples, and allows for more accurate data quantification (Deforce et al , ; Fundador et al , ).…”

Ribonucleotide and ribonucleoside determination by ambient pressure ion mobility spectrometry

“…Unambiguous identification of these DNA reaction sites requires the implementation of suitable methods for the accurate analysis of oligonucleotide fragments derived from strand breakage that may occur at specific sites. A capillary electrophoresis laser induced fluorescence (CE-LID) assay was reported for analysis of DNA fragment sizing [9]. A CE-LID assay has also been developed for effective DNA extraction, reflecting the increased need for miniaturization and automation applied to genetic analysis [10].…”

Characterisation of the DNA sequence specificity, cellular toxicity and cross-linking properties of novel bispyridine-based dinuclear platinum complexes

“…However, more recently cisplatin’s DNA sequence specificity has effectively been assessed by the LAR procedure, through the incorporation of a fluorescently-labelled primer, which is subsequently analysed by capillary electrophoresis with laser-induced fluorescence (CE-LIF) [ 40 ]. Furthermore, the use of CE-LIF, in place of the conventional gel electrophoresis, is a faster method and the data can be quantified more accurately [ 41 , 42 ].…”