Recombinant DNA Flashcards
Need
The desired protein sequence and the correct promotor for the organism you are doing the replicating in.
E.g insulin = human insulin gene and bacterial promotor.
Step 1
Extract plasmid DNA
Step 2 - Cut plasmid DNA
Restriction enzymes: recognise specific sequences of DNA and remove a section of the plasmid. NOTE: bacterial genome is not cut as it is protected by methylation (enzymes cant recognise bases).
Enzymes cut phosphodiester bonds -> leave 5’ overhang -> sticky ends -> able to bind to complementary sequence.
Restriction sites
Have to be unique -> only found once in the plasmid -> dont want multiple sections removed,
Step 3 - Make copies of DNA sequence of desired protein
DNA -> needs sticky ends to bind to plasmid -> primers are incorporated into the final strand so they are used to add additional sequences to the DNA.
PCR
Then restriction enzymes are used to cut the PCR product -> sticky ends.
Step 4 - Ligation
Ligase forms phosphodiester bond between DNA + plasmid (5’ overhangs are complementary).
Step 5 - Add promotor
Promotor inserted in same process as adding DNA sequence.
Step 6 - Transformation
Putting plasmid in host
E.coli: able to take up DNA from their environment -> needs to be competent and then plasmid + heat/electricity added
Only a few cells are transformed
Cells plated individually onto an agar plate -> grow into a colony (bacteria in a colony are identical).
Step 7 - Identifying Transformants
Plasmid needs to have a selectable marker (genetic sequence which produces an easily observable physical characteristic.
E.g antibiotic resistance.
Identifying Transformants - Antibiotic Resistance
amp^R gene encodes the enzyme beta-lactamase -> breaks down ampicillin (anitbiotic) -> placed in plasmid
When the bacteria is plated out on agar containing ampicillin the non-transformants will die and transformants will survive.
Step 8 - Distinguishing between bacteria with recombinant + non-recombinant DNA
During ligation step cut plasmid can stick to DNA or the section of plasmid that was removed by the restriction enzyme.
Plasmid + DNA = recombinant
Plasmid = non-recombinant
E.g lac Z gene
lac Z gene + identifying recombinant and non-recombinant DNA
Blue/white screening
lac Z gene encodes beta-glactosidase -> breaks down X-gal and coverts it into a blue compound.
Recombinant plasmid: will not be able to produce beta-glactosidase -> bacterial colonies will be white
Non-recombinant Plasmid: will be able to produce beta-glactosidase -> bacterial colonies will be blue.
Other methods for identifying recombinant DNA
Colony PCR
Restriction Digestion
DNA sequencing
Colony PCR
Replica plate + then rest of the bacteria in a test tube
Do PCR -> primers will anneal to the plasmid either side of the multiple cloning site
If recombinant -> strand will be much larger + thus will not travel as far along the electrophoresis gel.
Restriction Digestion
Replica plate + then rest of the bacteria in a test tube -> grow a large culture
Purify plasmid + then run PCR
Will have 2 strands: backbone + gene
