A SIGNIFICANT METHOD TO EXPLORE DNA DAMAGE IN LYMPHOCYTES
Manoj A.*, K. Ramachndra Rao, B. Vishnu Bhat, C. Venkatesh and Z. Bobby
ABSTRACT
Deoxyribo Nucleic Acid (DNA) is the genetic material providing instructions for growth, development, functioning and reproduction of all living things. When it exposed to exogenous and endogenous causative factors leads to damage and alteration of the chemical structure, break in a strand and base missing from backbone. DNA damage can be explored by Single Cell Gel Electrophoresis (SCGE) or Comet Assay is a simple and sensitive technique used to measure Single Strand Break (SSB) and Double Strand Break (DSB) of DNA leucocytes. It includes the following steps Sample collection, Preparation of slides in normal (0.75%) and low (0.50%) melting point agarose, Lysis in fresh solution for one hour inorder expose the nucleus and dissolving the cell membrane and cytoplasm. DNA unwinding and detection of Alkali labile sites by staying 20 minutes in submarine gel buffers, by applying Electrophoresis in order to migrate the damage DNA towards the anode, Neutralization for 5 minutes, Fixing for 1 hour to an overnight, Drying, Staining with silver nitrate, Scanning of Comet in Fluorescent Microscope and Comet Quantification.
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