Gene Expression II
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| Name the cloning vector requirements | Autonomous replication, indentifiable sites that can be cut by restriction enzymes, selectable markers
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| Restriction Enzymes | Have the ability to recognize Palindromic sequence. They can generate staggered or blunt ends. Staggered has advantage
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| What is the 1st step in creating recombinant DNA? | pBR322 is cleaved at ampicillin-resistance element Pst1. Outcome: Ampicillin resistance is lost and retains tetracycline resistance
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| What is the 2nd step? | Foreign DNA is ligated to cleaved pBR322 where ligation is successful, ampicllin element is disrupted. Tetracycline resistance element remains intact
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| What is the 3rd step? | E. Coli cells are transformed, then grown on ager plates containing tetracycline to select for those that have taken up plasmid
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| What is the 4th step? | Ind. colonies are transferred to matching positions on additional plates, 1 plate contains ampicllin + tetracyline and other just tetracycline
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| The cells that are in tetracycline plate have what? | Recombinant plasmids with foreign DNA
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| Describe the steps of DNA ligase | Enzyme is activated by cofactor NAD (E. Coli) or ATP. After activation, it can prime 5' end of DNA. Catalysis
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| What does functional cloning require? | Requires knowledge on function & structure of gene-ecoded protein. No prior knowledge regarding chromosome position of the gene.
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| Replica platting | Transfer of exact replica form agrose plate onto a nitrocellulose solid.
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| What happens when you treat cells with alkali? | Denature DNA and expose it
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| What does the Maxam/Gilbert method do? | To sequence DNA it relies on chemicals that are able to break DNA at specific sites
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| Sanger Method | DNA pol I can extend primer using info on template. Has diff terminator that DNA incorporates when available at right positions. Generate array of products.
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| Southern Analysis | Ascertain quality of DNA, identity and presence of gene of interest. Digest DNA with restriction enzymes, fragments separated using agarose gel, hybridized with probe of choice
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| Northern Analysis | Ascertain quality of RNA, # of diff RNA species
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| Western Analysis | Establish identity of protein under the control of mRNA. Incubate with anitbody (not probe)
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| c-myc and relation to tumors | translocation of chromosome 8 & 14 which makes it under control of enhaner corresponding to immunoglobin generes, overexpression of c-myc leads to tumorogenesis
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| PCR | Heat dsDNA, add primer, add thermostable DNA polymerase and repeat 25 times
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| Reverse transcription PCR | mRNA is reversed transcribed into cDNA, cDNA is amplified by PCR
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| Gene Silencing | Silence gene to tell you that gene has functionality based on phenotype expression.
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| siRNA (small inhibitory) | Exogeneously synthesized
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| microRNA | Endogeneously synthesized, partial base pairing: transcription repression. perfect base pairing: mRNA degradation
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