Bio 2c03: Polymerase chain reaction week 12-13

Question 1

What does PCR use?

Answer 1

It uses dna polymerase which is an in vitro replication of specific dna sequences

Question 2

What does pcr generate?

Answer 2

Can generate tens of billions of copies of a particular dna fragment from a dna extract

Question 3

What is TAQ?

Answer 3

A dna polymerase which serves as a standard reagent for pcr reaction

Question 4

At what temperature is TAQ stable? why is this useful?

Answer 4

95 degrees, extreme temperatures are needed to make it go from double stranded to single strand

Question 5

What is the processivity of Taq polymerase?

Answer 5

50-60 nucleotides (nt) per second at 72 degrees celsius.

Question 6

What are the two primers used for a polymerase chain reaction? how long are they?

Answer 6

forward and reverse primers- 18-30 nucleotides in length

Question 7

What do primers do?

Answer 7

Primers are no longer than 50 nucleotides and determine the beginning and end of the region to be amplified; the polymerase synthesizes the complementary sequences from each primer

Question 8

What do the primers depend on?

Answer 8

• primer length
• melting point
• specificity
• complementary primer sequences

Question 9

What is the processitivity for the extension step for taq

Answer 9

1000 nt per minute

Question 10

What is the fidelity of taq?

Answer 10

Low fidelity because it is not accurate, leads to mutations, suitable for 500 base pairs or less.

Question 11

When should high fidelity dna polymers be used?

Answer 11

should be used for experiments involving mutation detection and dna sequencing

Question 12

What are the 3 main processes involved in polymerase chain reaction?

Answer 12

denaturing/melting, annealing, elongation/extension step

Question 13

What is the denaturing/melting step

Answer 13

Tax polymerase used, temperature increased to 94-96 degrees for 10-20 mins, causing 2 complementary strands

Question 14

What is the annealing (hybridization) step?

Answer 14

the temperature is lowered 2-5 degrees from the melting/denature temperature, and hence the primers attach to single dna strands

Question 15

What happens in the extension step for a polymerase chain reaction?

Answer 15

The dna polymerase fills the missing strands; the temperature involved will be dependant on the processivity of taq polymerase

Question 16

What is the temperature involved in the extension step for taq polymerase and how long does it take to start?

Answer 16

72 degrees, takes 5-15 minutes

Question 17

How is the application of dna polymerase done?

Answer 17

on a logarithmic scale

Question 18

What is the amplified product of the pcr process called?

Answer 18

an amplicon

Question 19

How many different types of polymerase chain reactions are there? name 2 important ones

Answer 19

roughly 31 so far, two important ones are multiplex pcr and nested-semi nested pcr

Question 20

What is multiplex pcr?

Answer 20

used to amplify several targeted regions in a single reaction

Question 21

Why is the design for primers for each region in a multiplex pcr different?

Answer 21

It uses different primers so they can stick to different parts of the dna that needs to be amplified

Question 22

What are the drawbacks of multiplex pcr?

Answer 22

• the melting temperature of the primers has to be close enough to anneal at the same annealing temperature
• the base pair lengths should be different to form distinct bands because varying sizes of different genes are targeted

Question 23

What is nested-semi nested pcr

Answer 23

it involves amplifying a larger sequencing and then amplifying a smaller part of that large sequence

Question 24

How many primers are used for a single locus point for nested-semi nested pcr?

Answer 24

Two primers are used for a single locus point