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Transcript
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 DIG Structure of the DIG-dUTP nucleotideDIGbase: DIG DNA containing DIG label Second: Hybridize your DIG-labeled probe to genomic DNA in your blot DIG DIG DIG DIG DIG DIG Third: Detect DIG using an anti-DIG antibody… DIG DIG DIG DIG DIG DIG That is covalently linked to an alkaline phosphatase (AP) moiety = AP Fourth, and lastly, the AP is detected using a substrate, CSPD, that generates light Spontaneous CSPD substrate =Light! Unstable intermediate! 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 CSPD Light! DIG Source for CSPD: Roche Applied Sciences, DIG High Primer DNA labeling and detection starter kit II
