PCR

Question 1

What are the advantages and disadvantages of nested and semi nested PCR?

Answer 1

Advantage: Both nested and semi nested PCR increase the sensitivity and specificity of the PCR reaction.

Disadvantage: if the PCR reaction vessel is opened to do a second round of PCR there is a risk of contamination.

Question 2

Describe nested pcr?

Answer 2

In nested pcr there are two rounds of amplification with two primer pairs.
On the first round of amplification, the first primer pair amplifies an amplicon.
In the second round of amplification, the second pair of primer hybridizes just inside of the first primer pair and amplifies an smaller region of the target.

Question 3

Describe semi-nested PCR?

Answer 3

Like nested PCR, there are two rounds of amplification but there are only 3 primers. Amplification round 1 and 2 share a primer. But primer 3 used in the second round of amplification hybridizes inside the sequence of the primer used only in round 1.

Question 4

What are the advantages of multiplex pcr?

Answer 4

Internal controls indicates inhibitors false negative
Improve efficiency
Indicates template quantity or quality

Question 5

What are the disadvantages of multiplex PCR?

Answer 5

Multiplex pcr is difficult to optimize and may have decreased sensitivity.

Preferential amplification of one target over another.

Question 6

List applications of nested PCR

Answer 6

Long target PCR

Insufficient template

Question 7

List application of multiplex PCR

Answer 7

Infectious diseases

Internal controls

Question 8

Describe touch down PCR

Answer 8

Touch down PCR utilizes changes in annealing temperatures.
Typically starting 5-10 C above the Tm of the primers. The annealing temperature is subsequently decreased by ~2 C until it is 2-5 C below the Tm.

Question 9

What are the advantages and disadvantages of touchdown PCR?

Answer 9

Advantage- enhance specificity and efficiency

Disadvantage - non specific amplification