4. In Vitro cloning

Question 1

list the features of in vitro cloning

Answer 1

• outside an organism
• uses PCR
• makes millions of fragments in a shorter time frame
• relies on sequencing to see which genes have been amplified

Question 2

define PCR

Answer 2

the process of using primers to amplify a specific strand of DNA, to complete the artificial replication of a short sequence of DNA

Question 3

define amplification

Answer 3

to make multiple copies of a specific DNA

Question 4

in order to complete a PCR, what must the DNA be placed in

Answer 4

a buffer solution containing:
1. DNA nucleotides
2. Taq DNA
3. primers complimentary to the DNA

Question 5

list the stages needed to heat and seperate DNA strands

Answer 5

1. Denaturing
2. Annealing
3. Elongation

Question 6

what is the rough temperature for Denaturing

Answer 6

95 degrees

Question 7

what is the ideal temperature for annealing

Answer 7

55-60 degrees

Question 9

what is the ideal temperature for elongation

Answer 9

72 degrees (optimum temperature for Taq polymerase)

Question 10

outline denaturation

Answer 10

• mixture is heated
• hydrolyses the hydrogen bonds between the nucleotides, forming single strands of DNA
• separating the double strand of DNA

Question 11

outline annealing

Answer 11

• mixture temp is lowered
• allows primers to bond to the complimentary ends of DNA via hydrogen bonds

Question 12

outline elongation

Answer 12

• optimum temp for Taq polymerase
• catalyse the free floating DNA nucleotides which join together via complimentary base pairing
• this forms a double strand of DNA

Question 13

what do PCR tests show?

Answer 13

exponential increase of DNA fragments

Question 14

what does a curve on the PCR graph suggest?

Answer 14

• it increases as DNA doubles at each cycle

Question 15

why would a PCR graph plateau off?

Answer 15

• as there are limited primers available within the solution, which will limit how many DNA fragments can be made