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Gene Expression II G

Gene Expression II

Name the cloning vector requirements Autonomous replication, indentifiable sites that can be cut by restriction enzymes, selectable markers
Restriction Enzymes Have the ability to recognize Palindromic sequence. They can generate staggered or blunt ends. Staggered has advantage
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
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
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
What is the 4th step? Ind. colonies are transferred to matching positions on additional plates, 1 plate contains ampicllin + tetracyline and other just tetracycline
The cells that are in tetracycline plate have what? Recombinant plasmids with foreign DNA
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
What does functional cloning require? Requires knowledge on function & structure of gene-ecoded protein. No prior knowledge regarding chromosome position of the gene.
Replica platting Transfer of exact replica form agrose plate onto a nitrocellulose solid.
What happens when you treat cells with alkali? Denature DNA and expose it
What does the Maxam/Gilbert method do? To sequence DNA it relies on chemicals that are able to break DNA at specific sites
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.
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
Northern Analysis Ascertain quality of RNA, # of diff RNA species
Western Analysis Establish identity of protein under the control of mRNA. Incubate with anitbody (not probe)
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
PCR Heat dsDNA, add primer, add thermostable DNA polymerase and repeat 25 times
Reverse transcription PCR mRNA is reversed transcribed into cDNA, cDNA is amplified by PCR
Gene Silencing Silence gene to tell you that gene has functionality based on phenotype expression.
siRNA (small inhibitory) Exogeneously synthesized
microRNA Endogeneously synthesized, partial base pairing: transcription repression. perfect base pairing: mRNA degradation