PCR

Question 1

What does PCR stand for?

Answer 1

Polymerase Chain Reaction

Question 2

What do methods of molecular analysis make use of?

Answer 2

DNA replication processes

Question 3

What are the 2 widely used methods of using DNA replication for molecular analysis?

Answer 3

polymerase chain reaction (PCR)

dideoxynucleotide DNA sequencing

Question 4

Describe the PCR

Answer 4

an automated version of DNA replication that segments millions of copies of a short target DNA segment

Question 5

T or F: PCR reactions are carried out in large volumes

Answer 5

false! they are carried out in small volumes (20-50 uL)

Question 6

What amount of volumes are PCR reactions normally done in?

Answer 6

20-50 uL

Question 7

What are the 5 components of PCR?

Answer 7

double-stranded DNA template containing target sequence for amplification

supply of the 4 DNA nucleotides

a heat-stable DNA polymerase

2 different single-stranded DNA primers

a buffer solution

Question 8

What is the most commonly used DNA polymerase?

Answer 8

Taq

Question 9

What is Taq isolated from?

Answer 9

bacteria that live in hot springs

Question 10

What does Taq polymerase do?

Answer 10

adds nucleotides

Question 11

What does Taq polymerase require to function?

Answer 11

2 PCR primers

Question 12

How many PCR primers does Taq polymerase require?

Answer 12

2

Question 13

What are PCR primers and what is their function?

Answer 13

DNA sequences (2) that provide a starting point for Taq polymerase to add nucleotides

Question 14

What boundaries do the PCR primers define on the replication products?

Answer 14

5’ and 3’

Question 15

How many steps are involved in PCR?

Answer 15

3

Question 16

What is the result of PCR?

Answer 16

exponential amplification of large numbers of the target DNA

Question 17

What are the 3 steps of PCR?

Answer 17

1. denaturation
2. primer annealing
3. primer extension

Question 18

Describe denaturation in PCR

Answer 18

the reaction mixture is heated to ~95C to denature the DNA into single strands

Question 19

What is the purpose of the denaturation step of PCR?

Answer 19

to denature the DNA into single strands

Question 20

Describe the primer annealing step in PCR

Answer 20

reaction temperature reduced to 45-68C to allow primers to hybridize to their complementary sequences in the target DNA

Question 21

What is the purpose of primer annealing?

Answer 21

to hybridize the primers to their complementary sequences in the target DNA

Question 22

Describe the primer extension step of PCR

Answer 22

reaction temperature raised to 72C to allow Taq polymerase to synthesize DNA by extending primers

Question 23

What is the purpose of the primer extension step?

Answer 23

to allow Taq polymerase to synthesize DNA by extending primers