In vitro gene cloning

Question 1

What does PCR stand for

Answer 1

Polymerase chain reaction

Question 2

What does PCR require and why

Answer 2

DNA fragment - to be copied
DNA polymerase (thermostable) - to rapidly join nucleotides and withstand high temp
Primers - short sequences of nucleotides that have bases complenentary to those at ends of the 2 DNA fragments
Nucleotides
Thermocycler - Vary temperature

Question 3

What are the 3 stages of the PCR process and their temperatures

Answer 3

Separation of DNA strand - 95°
Annealing of the primers - 55°
Synthesis of DNA - 72°

Question 4

What happens in separation of DNA strand stage?

Answer 4

DNA fragments, primers and DNA polymerase and placed in thermocycler at 95°
2 strands of DNA fragments to separate as H bonds are broken

Question 5

What happens in the annealing stage?

Answer 5

Thermocycler cooled to 55°, causing primers to anneal to complementary bases at ends of DNA fragment. This provides starting point for DNA polymerase and prevents strands rejoining.

Question 6

What happens in the synthesis of DNA stage?

Answer 6

Temp increased to 72°, optimal temp for DNA polymerase to add complementary nucleotides along DNA strands. Starts at primer and finishes at the ends of the chain

Question 7

Advantages of in vitro

Answer 7

Extremely rapid
Does not require living cells

Question 8

Advantages of in vivo

Answer 8

Useful when introducing gene to another organism
Almost no risk of contamination
Very accurate
Cuts out specific genes
Transformed bacteria can produce large quantities of gene products