5. PCR Amplification

Question 1

Optimal PCR annealing temperature?

Answer 1

0-5C BELOW the Tm of primers (therefore primer pairs should have similar Tm)

Question 2

What is PCR processivity?

Answer 2

Nucleotide addition rate

High processivity = high rate of extension

Question 3

Another name for template strand

Answer 3

Antisense strand

Question 4

Major source of DNA contamination when it comes to PCR amplification?

Answer 4

Previously-amplified DNA

Question 5

What defines the length of an amplicon?

Answer 5

5’ end of each primer (forward/reverse)

Primers sequences are IN each amplicon

Question 6

Another name for sense strand

Answer 6

Coding strand

Question 7

Taq extension temeprature

Answer 7

72C

Question 8

To which end of a primer does Taq start adding nucleotides?

Answer 8

3’ end of primer

Because 5’-3’ activity

Question 9

After primer design, what are the 2 primary determinants of amplification specificity and sensitivity

Answer 9

• Annealing temperature

- Mg2+ concentration

Question 10

Primer that anneals to coding strand

Answer 10

Reverse primer

Question 11

High PCR fidelity = ________ dNTP mis-incorporation

Answer 11

low

Question 12

3 things that determine the number of cycles a PCR assay should have

Answer 12

1. Number of starting copies of target
2. Efficiency of amplification reaction (presence of inhibitors, competition for reagents..)
3. Amount of DNA required for detection or further analysis

Question 13

Denaturation temperature range

Answer 13

83 - 97C

Question 14

Why should the annealing temperature be lower than that of DNA polymerase

Answer 14

So that primers can anneal before Taq starts working

Question 15

Primer that anneals to antisense strand

Answer 15

Forward primer

Question 16

What is PCR fidelity?

Answer 16

Nucleotide incorporation specificity