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IUB BIOL-L 211 - Molecular Biology Techniques I

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BIOL-L211 Lecture 36 Outline of Last Lecture I. Protein PurificationOutline of Current Lecture I. Techniques to examine DNA-protein interactionsII. EMSAIII. DNase footprinting assayIV. Chain-terminating methodCurrent LectureMolecular Biology Techniques I*Recall sequencing from prior lectures*I. Techniques to examine DNA-protein interactionsA. Electrophoretic mobility shift assay (EMSA)B. DNase footprinting assayC. Chromatin immunoprecipitation (ChIP)D. In Vitro: experiments done outside of living organisms 1. Example: PCR (Polymerase chain reaction)2. Precise cellular conditions can never be perfectly replicatedE. In Vivo: experiments performed in living organisms1. Example: clinical trials, Drosophila studies, glowing puppiesII. EMSAA. Electrophoretic mobility-shift assay1. In vitro experimental procedure used to examine protein-DNA interactionsThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.2. DNA probe: radioactively labeled dsDNA fragment with a potential DNA-binding site3. A purified protein of interest is mixed with the DNA probe4. Gel electrophoresis is performedB. Left lane: Control reaction (DNA is not mixed with the protein)1. Appears as a single band on the gel (corresponding to free DNA only)C. Right lane: Excess DNA mixed with protein of interest1. Protein does NOT bind all of DNA (DNA is in excess); some free DNA remains2. Protein-DNA complex migrates slower in the gel3. A shift in the location of the band in the right lane means that the protein has bound the DNA4. No protein-DNA interactions would yield only one band at the same location as that of just free DNAIII. DNase footprinting assayA. Used to tell where specifically a protein binds on a strand of DNA1. Many copies of a radioactive probe are produced2. The protein of interest is purified3. Control Reaction: no protein is added4. Experiment: protein is mixed with probe5. In both cases, DNA is digested with DNasea. DNase: endonuclease that cleaves the phosphodiester bond in DNAb. DNA cut into smaller fragmentsc. Protein binding prevents digestion6. Gel electrophoresis is performed on both7. Left Lane: only completely digested DNA fragments (based on size)8. Right Lane: digested DNA fragments separated by size, but with a "footprint"a. Footprint: absence of bands on a DNase footprinting assay shows where protein bindsb. Use a chain-terminating gelIV. Chain-terminating methodA. "Normal" nucleotide: Has 3' –OHB. "Chain-terminating" nucleotide: 3' –HC. A, G, T, and C can all be made into chain-terminating nucleotidesD. DNA synthesis joins the 3' –OH of one nucleotide to a 5' phosphate of anotherE. A chain-terminating nucleotide stops DNA synthesisF. Reaction ingredients (each chain-terminating nucleotide requires a separate reaction)1. Single-stranded DNA to be sequenced ("template strand")2. Primer: Anneals to template (similar to PCR)3. DNA polymerase4. All four "normal" nucleotides5. One chain-terminating nucleotide per reaction (either A, G, T, or C)G. Example: Reaction with chain-terminating G1. Some normal G's added2. Elongating chain stops when chain-terminating nucleotide is added3. Fragments of various lengths are generated4. Fragments can be separated on


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