CMMB 403 Lecture Notes - Lecture 1: In Situ Hybridization, Digoxigenin, Enzyme
8 Jan 2019
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A method to tell where in an embryo a given gene is transcribed (expressed) First clue to function: find out where a gene is expressed during development. In situ hybridization because it involves a probe. Shows you where a gene is being actively transcribed. Finding where a certain rna transcript is located. Add rna probe that is complementary to the interest mrna. D is a molecule known as dig (digoxigenin) Add anti-dig antibody that is conjugated to an enzyme (alkaline-phosphatase) Enzyme: cleaves substrate and converts it into a blue dye. Example: find staining in eye and tail because enzyme is present and has found a transcript of interest. Complementary rna probe detects transcribed sequences (mrna) in embryos. Make dig-labelled rna probe in test tube by in vitro transcription. Carry out transcription in test tube with necessary components. Add 4 nucleotides to test tube, and u is labeled with dig molecule. Rna polymerase to carry out synthesis reaction.
