Recombinant DNA Technology Flashcards
Outline the steps required to clone DNA
- isolate donor and vector DNA
- cut donor and vector DNA with same restriction enzyme
- ligate donor and vector DNA together
- amplify recombinant DNA molecules in bacteria
- Select for bacteria that contains plasmid DNA
- Screen for bacteria containing a recombinant DNA molecule
Describe restriction enzymes
naturally occurring enzymes in bacteria that are used to cut both strands of the DNA sugar-phosphate backbone into two linear pieces
How do restriction enzymes work?
They scan along DNA until they reach the restriction sequence (GAATTC) and bind to DNA to break the covalent bond between GA on both strands
Describe vector
plasmid - accessory chromosomes that are not part of the bacterial chromosome
aka cloning vector
What is the purpose of the vector?
it is used to amplify the donor DNA in bacterial cell
What are 2 ways a restriction enzyme can cut the DNA strands?
Leaving single stranded overhangs by cutting at different locations on the strands = Make staggered cuts and leave ‘sticky’ ends
Leaving blunt ends by cutting at the same location on both strands
How do restriction enzymes work?
They scan along DNA until they reach the restriction sequence (ex. GAATTC for EcoRI) and bind to DNA to break the covalent bond between GA on both strands
Describe vector
AKA plasmid cloning vector = accessory chromosomes that are not part of the bacterial chromosome
What does ‘sticky’ end mean?
A result of how some restriction enzymes (ex. EcoRI) cut the DNA
they cut in a staggered way that leaves single stranded regions that are complimentary and when nearby, they can hydrogen bond to ‘stick’ back together
Describe genomic DNA
DNA obtained directly from chromosomes of the organism of interest
When is genomic DNA used in studies?
if the researcher wants to collect fragments of DNA that represent the entire genome of the organism
What does the vector contain that allows for the replication of DNA in bacteria?
origin of replication
What expression does the Amp^R gene have?
a selectable marker gene that gives bacteria ampicillin resistance
What is the function of the lacZ gene?
codes for an enzyme to break down Xgal (sugar) for the bacteria to digest
What is the multiple cloning site (MCS)?
a piece of DNA that contains a lot of restriction enzyme recognition sequences and is only found once in the plasmid
Where is the MCS located?
in the lacZ gene of the plasmid
What is the multiple cloning site (MCS)?
a piece of DNA that contains a lot of restriction enzyme recognition sequences and is only found once in the plasmid (THE oNLY PLACE ENZYMES WILL CUT)
What 4 things make up the vector/plasmid?
origin of replication (ori)
bla which produces Amp^R
lacZ which contains MCS
MCS
T or F: the donor and vector DNA are cut with different enzymes?
False.
They are cut with the same restriction enzyme
How are the donor and vector DNA cut with an enzyme?
by micropipetting
How many times will the plasmid be cut? Where will it be cut?
Once
Only where the MCS is
How many times will the donor DNA be cut? Where will it be cut?
thousands of times into tiny fragments of single stranded regions
cut wherever the restriction sequence occurs
How do you ligate the donor and vector DNA together?
pipette some liquid from the donor DNA tube and some from the vector/plasmid DNA tube and combine into a third tube
Ideally they will find complementary single stranded regions and DNA ligase will form covalent bonds between the strands
What enzyme will help in ligating the donor and vector DNA?
DNA ligase
What is the most common method to get a recombinant DNA molecule into a bacterial host cell? why?
transformation
most bacteria are able to take up small fragments of DNA from surrounding environment
In order for transformation to occur, what state do the cells need to be in?
they need to be competent
How do you make cells competent?
adding salts and heating up will increase chances of the cells picking up the DNA from the environment
What are the four different results of transformation?
Some bacteria will only take up the donor DNA
Some bacteria will only take up the vector/plasmid DNA
Some bacteria will take up no DNA
Some bacteria will take up the recombinant DNA (Both plasmid + donor DNA)
What is the ideal result of transformation?
The bacterial cells taking up the recombinant DNA (donor + vector DNA)
What is the ideal result of transformation?
The bacterial cells taking up the recombinant DNA (donor + vector DNA)
Describe how DNA is amplified?
After transformation is successful,
replication will replicate a cells own genome + plasmid = large colony of bacteria
After amplification, which bacterial cells are selected for? how?
bacteria with plasmid
Ampicillin is added to the medium to prevent the growth of the bacteria that do not have the plasmid
only bacteria with plasmid will have the Amp^R gene and be able to grow
this results in bacteria that took up only plasmid and bacteria that took up both
How do you screen for bacteria containing a recombinant DNA molecule?
lacZ in the plasmid allows you to identify plasmids containing donor DNA (= recombinant DNA molecules)
How does lacZ help identify recombinant DNA molecules?
lacZ gene produces enzyme that breaks down X-gal (sugar)
If lacZ gene is disrupted, there is donor DNA in the bacteria and it will not be able to break down X-gal (Recombinant)
How can a researcher use lacZ to tell which bacteria are recombinant?
If lacZ is functioning, the bacterial colonies will be able to breakdown Xgal and the colonies will be blue
If lacZ is not functioning and was disrupted by donor DNA, it will not be able to breakdown Xgal and the colonies will be white
What colour will the colonies be if the bacteria has recombinant DNA?
white
Why do you have to screen for bacteria with recombinant DNA after selecting for AmpR?
Because 2 colonies will grow even after the ampicillin is added
One will be recombinant and one will be just carrying the plasmid
screening with lacZ will show which colonies ALSO have the donor DNA
