1. DIG Bio210 Fall, 2005 Detection by chemiluminescence: a complicated chain of events First: Incorporate “DIG” (Digoxigenin) into your probe DNA Structure of DIG: DIG-dUTP is used as a dTTP analog, gets incorporated by DNA polymerase in place of T bases Structure of the DIG-dUTP nucleotide base: DNA containing DIG label

2. DIG Second: Hybridize your DIG-labeled probe to genomic DNA in your blot Third: Detect DIG using an anti-DIG antibody… DIG That is covalently linked to an alkaline phosphatase (AP) moiety = AP

3. Unstable intermediate! =Light! Fourth, and lastly, the AP is detected using a substrate, CSPD, that generates light CSPD substrate DIG Shown together, the DIG label is used, via an antibody via AP, to produce light in the location on the blot where DNA matching your probe resides CSPDLight! Spontaneous Source for CSPD: Roche Applied Sciences, DIG High Primer DNA labeling and detection starter kit II