MCB 121 Lecture 6

MCB 121 Lecture 6 - MCB 121 - Lecture 6 Outline: - cDNA...

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MCB 121 - Lecture 6 Outline: - cDNA libraries - PCR - Infounatics ------------------------------------------------------------------------------------------------------------------------- mRNA - largely includes the protein encoding sequences - distilled protein coding regions. - indicate what parts of the genome are expressed in cell. - to make an library of mRNA => cDNA library: complementary DNA - complement means the complement to the mRNA - HOW? 1. Take mRNA 2. apply "reverse transcriptase" (make DNA from RNA template) => produce single stranded cDNA (= a single stranded copy of mRNA) 3. produce double stranded cDNA (the process isn't mentioned here. ..... ) => take ds cDNA to clone in order to make library * the library would be the clones derived from the mRNA => results a set of clones represent the original member of mRNA population Cannot express genomic clone for cDNA, because all the introns are removed. But cDNA can be expressed in bacteria. make cDNA library expression vector: - this can be screened with nucleic acid hybridization with the method that detects the proteins with antibody (plate out the Make filter replicas Apply radioactive
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cDNA library) bind proteins antibody against the protein X each spot on the filter has expose X-ray protein from the Ecoli cell. film, we can see which would include the one which colony express being transcribed by the protein X expression vector then go back to the cDNA library plate and pick that colony and has cDNA clone to clone that protein X. PCR (Polymerase Chain Reaction) - purpose: copy particular region of DNA - need synthetic primers that frame the region of interest we want to amplify * the top primer is identical to top strand, complementary to the bottom strand * the bottom primer is complementary to the top strand, identical to the bottom strand. Step 1. Denature dsDNA at 95C. - a lot of primers are flowing around - they will find the complementary sequences and bind to Step 2: annealing at 60-65C - primers are hybridized and base paired onto DNA Step 3: use a heat-stable enzyme Tag Polymerase to 72C
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- extension of synthesized DNA occurs - now we have two copies of the original cDNA Step 4: repeat the same procedure 20 - 40 times - end product: amplified region begins with one primer and ends with another primer - the end products are >10^9 folds amplified Use PCR to change the sequence: - add sites to the ends of the sequence => To clone a plasmid that has Eco and Hind sites. * primer contains Eco R1 site and another primer contains Hind 3 site
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MCB 121 Lecture 6 - MCB 121 - Lecture 6 Outline: - cDNA...

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