Amplifying DNA fragments Flashcards
What are the two ways to amplify a DNA fragment?
In vivo cloning
In vitro cloning
What is in vivo cloning?
Gene copies are made inside of a living organism
What is in vitro cloning?
Gene copies are made outside of a living organism
What are the three steps in in vivo cloning?
1) Make recombinant DNA
2) Transforming cells
3) Identifying transformed cells
In the first step of in vivo cloning, what does the vector’s DNA need to be? (vivo)
Isolated
What are 2 examples of a vector? (vivo)
Bacteriophage
Plasmid
What is a vector? (vivo)
A molecule which transfers genetic material from one organism to another
Why is the same restriction endonuclease enzyme used as was when the fragment was created? (vivo)
To produce sticky ends in the vector’s DNA which are complimentary to the sticky ends of the DNA fragment which contains the target allele
What are the DNA fragments and vector’s DNA mixed with? (vivo)
DNA ligase
What is the role of DNA ligase? (vivo)
Ligation
To anneal the sticky ends of the DNA fragment with the vector
What name is given to the DNA fragment and the vector together? (vivo)
Recombinant DNA
When is a host cell considered transformed? (vivo)
When they have taken up the vector containing the target gene
What two conditions are needed for the bacteria to uptake the vector and why? (vivo)
Ice cold CaCl2 - Makes the membranes more permeable
Heat shock to 42 degrees - to encourage uptake
What is a host cell? (vivo)
The cell which the vector containing the recombinant DNA transfers the target gene into
What is a marker gene? (vivo)
It tells you whether a cell has been transformed
Where and when is a marker gene added? (vivo)
Into the vector at the same time as the target gene is added
What are three examples of marker genes? (vivo)
Antibiotic resistance
Fluorescence
Radioactivity
How can transformed cells be identified? (vivo)
Fluorescence - They will glow under UV light
Antibiotic resistance - They will survive in the presence of antibiotics
Radioactive - Show in an X-ray/on film
Why is it important to be able to identify transformed cells? (vivo)
So transformed cells can go on to divide and produce more copies of the target gene
What is another name for in vitro cloning?
Polymerase chain reaction
Why is PCR good? (vitro)
It makes millions of copies of DNA outside the body quickly
In the first step of PCR, a mixture is created. What is in this mixture? (Vitro)
Double stranded DNA sample
Primers
DNA polymerase
Free DNA nucleotides
What temperature is the mixture heated to first and why? (vitro)
92 degrees
To break the hydrogen bonds between the double stranded DNA to produce two single strands
What type of DNA polymerase is used and why? (vitro)
Thermostable
It does not denature at 92 degrees as it is collected from hot springs
What is the second temperature the mixture is heated to and why? (vitro)
Between 50 and 65
To allow the primers to anneal to the single strands
What are primers? (vitro)
Single stranded molecule with complimentary nucleotides to the DNA template strand
Why are primers required? (vitro)
- To show the enzyme where to start adding nucleotides
- So the strand of DNA is double stranded so the DNA polymerase has a starting strand
What is the third temperature that the mixture is heated to and why?
72 degrees
Optimum for the DNA polymerase to work
What is the role of DNA polymerase? (vitro)
Add free complimentary DNA nucleotides to the template strands
How many new copies of the DNA fragments are made every PCR cycle? (vitro)
2
In in vivo cloning, what do you need to make sure is added and why? (vivo)
Promoter and terminator regions
To produce the protein which is being coded for
What happens if there are no promoter regions? (vivo)
The DNA fragment wont be transcribed by the host cell and the protein wont be made
