Detection of abasic sites with capillary electrophoresis with laser induced flourescence

• Main Authors: Fundador, Erwin Oliver V., Rusling, James
• Format: text
• Published: Animo Repository 2006
• Subjects: DNA damage; Capillary electrophoresis; Biochemistry; Chemistry
• Online Access: https://animorepository.dlsu.edu.ph/faculty_research/5519
• Institution: De La Salle University

Damaged bases and abasic sites are two of the major DNA lesions caused by free radicals. These damaged bases could further be removed by specific enzymes leaving an abasic site in the DNA making the site a very good marker for detection of specific DNA damage. Right now the most widely used method for abasic site detection is an ELISA like method in which the DNA with abasic sites is tagged with biotin, adsorbed on a surface and then attached with streptavidin-HRP. This method is susceptible to non specific binding, uses more sample than our method (150 µl vs 15 µl) and takes 1 or 2 days to get results. A more rapid method which we are developing is Capillary Electrophoresis with Laser Induced Flourescence (CE-LIF) which takes only about 5 hrs. Our method uses a Fluorescent Aldehyde Reactive Probe to tag the abasic sites. The tagged DNA was then separated by capillary electrophoresis from the excess probe that was not completely removed by ethanol precipitation. Our results show that CE-LIF could be used as an alternative to the ELISA like method of detecting abasic sites.