Detection of damaged DNA by capillary electrophoresis with laser induced fluorescence using labelled abasic sites

• Main Authors: Fundador, Erwin Oliver V., Rusling, James
• Format: text
• Published: Animo Repository 2005
• Subjects: Capillary electrophoresis; Laser-induced fluorescence; DNA damage; Biochemistry; Chemistry
• Online Access: https://animorepository.dlsu.edu.ph/faculty_research/5520
• Institution: De La Salle University

Abasic sites in DNA can be generated by excision of damage bases. Abasic sites if left unrepaired are known to be mutagenic or lethal. These sites occur spontaneously and their rate of formation is significantly increased in the presence of DNA damage agents. A quant. measure for this lesion would therefore provide an assay of the amount of damage of DNA by various agents. Researchers have explored quantitation of abasic DNA sites by tagging them with a suitable reporter groups such as radioactive labels, biotin (via aldehyde reactive probe or ARP) or a fluoropore (fluorescent aldehyde reactive probe or FARP). These tags are detected using various methods each having its own advantages and disadvantages. The most common method used right now is an ELISA like method using biotin tag named ARP. This ARP assay is considered to be very sensitive (1 abasic sites per 100,000 bases). However, this method may be prone to high background signal due to non-specific binding and may not be fully quant. because DNA is absorbed on a surface. We are attempting to devlop capillary electrophoresis with laser induced fluorescence (CE-LIF) to measure abasic sites. In this method the abasic sites of the DNA was tagged with a fluoropore and analyzed by CE-LIF. We have shown this method to be a lot faster, require minimal amounts of sample and can detect 1 abasic site per 10,000 bases with the possibility of detecting less than 1 abasic site per 100,000 bases.