Recombinant DNA tech Flashcards
What are 3 methods used for isolating DNA that codes for a specific protein?
Rev. Transcriptase - mRNA into cDNA (intron free) so polymerase can form double strand
Restriction Endonuclease - cut DNA at specific recognition sequences to form sticky ends
Gene machine
How is DNA inserted into a plasmid vector? (In Vivo cloning)
- DNA + plasmid cut w/ same restriction enzyme, comp. sticky ends
- DNA inserted + joined by phosphodiester linkages DNA ligase
- recombinant plasmid formed
How are recombinant plasmids reintroduced to bacterial cells?
Mix of recombinant DNA plasmids, bacteria, calcium ions
Changes to calcium ion conc. -> bacterial cells become permeable so plasmids move through membrane
What percentage of bacteria take up plasmids?
about 1%
some plasmids don’t even contain DNA sequence
How are bacteria containing recombinant plasmid identified?
Markers;
- antibiotic resistance
- fluorescence
- enzyme action
How are antibiotic resistant genes used to identify plasmid w/ DNA fragment?
- plasmid w/ 2 genes resistant to tetracycline + ampicillin
- DNA fragment added to tet. gene disrupting it
- grow bacteria on agar, transfer to pate with ampicillin then to plate with tetracycline, see differences
How are fluorescent markers used to identify DNA in plasmid?
GFP found in jellyfish inserted into bacterial plasmid
If DNA transfer successful then recombinant plasmids will not glow green
How are enzyme markers used to identify DNA in plasmid?
Gene that produces lactase (substrate blue -> colourless) used
If DNA transfer successful then enzyme will not turn substrate blue as gene split
What are the ingredients of PCR?
DNA fragment to copy
DNA taq polymerase
Primers
Nucleotides
Thermocycler
Steps of PCR (In Vitro)
- temp of 95C breaks H bonds separating strands
- temp of 55C allows primers to anneal
- temp of 72C allows DNA polymerase to attach nucleotides forming new strand next to each template
repeat
Advantages of in vitro cloning
Rapid - valuable when only small amount available
Does not require living cells - only base DNA sequence required
No culturing techniques - easy to carry out
Advantages of in vivo cloning
No risk of contamination - cut by same enzyme so sticky ends complimentary, in vitro needs pure sample
Very accurate - few errors as mutations rare
Produces transformed bacteria - can produce proteins for commercial + medical use
How is DNA extracted for profiling?
Extracted from hair, blood, semen + skin, can be amplified by PCR
DNA cut into millions of small fragments by restriction endonuclease
Gel electrophoresis for separating DNA fragments
Separated by size
DNA negatively charged so attracted to anode (positive end of gel)
Smaller fragments move faster
How is the pattern of DNA fragments transferred to a thin nylon membrane?
Gel immersed in alkali to separate double strands
Southern blotting where membrane laid over gel with absorbent paper (capillary action)
DNA fragments then attached using UV light
