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Gene Expression II G
Gene Expression II
| Question | Answer |
|---|---|
| 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 |
Created by:
Kahiranoel@gmail.com
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