In-Vitro Bromodeoxyuridine BrdU Assay

Theoretical Background / Context

• In genetics, genotoxicity testing describes the property of chemical agents that damages the genetic information within a cell causing mutations, which may lead to cancer. 
• While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic, whereas not all genotoxic substances are mutagenic. 
• The alteration can have direct or indirect effects on the DNA: the induction of mutations mistimed event activation, and direct DNA damage leading to mutations. 
• The permanent, heritable changes can affect either somatic cells of the organism or germ cells to be passed on to future generations. 
• Cells prevent the expression of the genotoxic mutation by either DNA repair or apoptosis; however, the damage may not always be fixed leading to mutagenesis.

Brdu Assay Principle of Work

• 5-Bromo- 2’ -deoxyuridine (Bromodeoxyuridine (BrdU)) is a thymidine analogue that differs from thymidine in its substitution of bromine for a methyl group. 
• BrdU competes with thymidine for incorporation into nuclear DNA during the S-phase of the cell cycle. 
• Therefore, BrdU serves as a marker of DNA synthesis in the BrdU proliferation assay, and separate means such as counting mitotic figures may be employed to ensure accuracy with regard to cell division.

• After BrdU assay is incorporated into nuclear DNA, samples are fixed and incubated with anti-BrdU monoclonal antibodies and nucleases (or they are exposed to heat or other conditions which cause DNA denaturation) according to the BrdU cell proliferation assay principle.
• This denaturation is necessary to allow anti-BrdU monoclonal antibodies to gain access to the incorporated BrdU in single-stranded DNA. 
• The sample is subsequently incubated with a fluorescent secondary antibody against the anti-BrdU antibody and visualized by a fluorescence microscope.