Week 5 - Manipulation of DNA, Polymerase

Question 1

What is polymerization?

Answer 1

It is the process of adding nucleotides via phosphodiester bonds to 3’OH with the use of polymerase enzymes

Question 2

What is proof reading in polymerases?

Answer 2

It is the function to remove mis-paired nucleotides, it is a exonuclease activity the helps increase accuracy

Question 3

DNA polymerase 1

Answer 3

Nick translation activity:
5’ to 3’ exonuclease removes existing nucleotides downstream of nick, before gap is filled and existing downstream nucleotides are replaced by DNA polymerase activity.

Question 4

What is the Klenow fragment

Answer 4

It is a modified DNA polymerase 1, it lacks the 5’ to 3’ exonuclease activity and cant remove downstream nucleotides from the nick. it just fills in the gap.

Its uses:
making dsDNA from ss templates
filling in 3’ ends of DNA fragments to make 5’ overhangs
digestion away 3’ overhangs

Question 5

Reverse transcriptase

Answer 5

Isolated from Retroviruses, template dependent DNA polymerases that use RNA as a template.

Synthesizes DNA using RNA, can be used to make cDNA from ssRNA

Question 6

Uses for PCR

Answer 6

Cloning
cDNA synthesis
DNA sequencing
Diagnostics:
-Genotyping
-Recombinant construct checking
-Gene expression studies

Question 7

What does PCR require

Answer 7

Template DNA
2 DNA primers
DNA polymerase
dNTPS (all 4 DNA nucleotides)
buffer containing Mg2+
Thermal cycler

Question 8

PCR primers

Answer 8

ss 15 to 20 bp in length
forward primer to 5’ end of the sequence while the reverse primers to the 3’ end of the sequence

Question 9

DNA polymerases used for PCR

Answer 9

Taq DNA polymerase:
- from thermus aquaticus
- thermophilic bacterium of hot springs
- optimal growth at 65-70 degrees
- error prone 1 error per 1000 bases

Pful DNA polymerase
- derived from Pyrococcus furiosus
- hyperthermophilic archaeon of marine sediments
- optimum growth at 100 degrees
- high fidelity, 1 error in > 1 million bases

Question 10

In a PCR programme, what variables can change?

Answer 10

The temperature used to cause denaturing is dependent on the melting point of the primers used, the time for DNA polymerisation step also changes as different polymerases have differing amount of nt.s-1