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How to determine between Type I/II recombinase?
- Mutate Tyr/Ser in gene, observe effects on protein
- If mutating Tyr stops it= T1, if Ser= T2
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Process to apply mutation to hypothetical ENE gene
- 1. isolate DNA sequence coding for ENE
- -PCR to amplify
- 2. introduce mutations that disrupt tyr or ser residue
- -Use synth oligonucleotides w/ mutant sequence to create mutations
- 3. Join mutant ENE gene with DNA vector
- -restriction enzymes and DNA ligase
- 4. transform bacteria mutant ENE plasmid
- 5. induce bacteria to make protein, isolate for molecular assays
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Generate recombinant DNA bacteria
- Take gene of intrest
- Place into a plasmidm with selection gene (antibiotic resistence)
- Put plasmid into bacteria
- Now you have a recombinant bacteria with the gene of interest.
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Plasmid:
- DNA polynucleotide capable of independent regulation, contain:
- mechanism for integration
- origin of replication (ori) recognized by host machinery
- (when in research) selection gene
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Polymerase Chain Reaction (PCR)
- PCR amplifies a target DNA sequence via multiple rounds of DNA synthesis
- Only target DNA is amplified
- -Only add short polynucleotide oligonucleotides to prime a specific gene's DNA polymerization
- Use DNA polymerase from thermophylic microbe, allows DNA to be made at the high temps needed for DNA denaturement
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Three steps of PCR
- DNA denatured
- Short polynucleotides prime polymerase
- Polymerase synthesizes copies of the DNA
- Repeat
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Uses for synthetic DNA
- Produce the oligonucleotide primers for PCR
- Introduce specific/random point mutations into recombinant DNA molecules
- Possible to synthesize entire genes
- (first synthesized yeast tRNA)
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Process of synthesizing polynucleotide chains
- Attach 1st nucleotide to silica support, prep for next nucleotide (functional groups protected with DMT to prevent accidental reactions, removed after attachment)
- Next nucleotide added to 5' OH of 1st nucleotide (chemical synthesis is 5'->3')
- Repeat
- When done, remove blocking groups from bases, remove methyl groups from phosphates, cleave chain from silica support
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Molecular biology commonly uses what?
Type II restriction enzyme
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Type II restriction enzymes are:
sequence specific (generate products with 5' phosphate and 3' OH)
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Uses for Type II restriction enzymes
- DNA cloning
- Diagnose presence of mutation if it adds a new/removes restriction site
- Can be used to identify source of tissue sample
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DNA Ligases
Catalyze Phosphodiester bond formation, DNA ligase acts during replication to seal nicks in lagging strand synthesis
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Ending PCR's
Commonly use ddNTP, these lack an OH at the 3' position, so the reaction ends
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Sequencing DNA in a PCR
- Fluorescent tags added to the ddNTP
- See how long each strand is with a gel run (high resolution polyacrylamide gel electrophoresis or capillary electrophoresis
- Shortest is first, look at color, covert to flavor or ddNTP, and go from there.
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