Fergus - Introduction to PCR

Question 1

What is DNA cloning?

Answer 1

The isolation, purification and amplification of a specific DNA sequence

Question 2

What is Taq polymerase?

Answer 2

A DNA polymerase that exists in an organism called thermos aquaticus

Question 3

What are the two main steps of DNA cloning using a PCR based approach?

Answer 3

Design complimentary oligonucleotide primers

Amplify clone using Taq Polymerase

Question 4

What is the first thing you must do when designing a PCR?

Answer 4

Decide on the sequence you want to amplify

Question 5

What do you do after you have decided on the sequence you want to amplify?

Answer 5

Decide on suitable primers for this sequence

Question 6

What do you do after deciding on your primers?

Answer 6

Denature the DNA

Question 7

How and why do you denature the DNA?

Answer 7

By heating the reaction up to about 50-65 degrees Celsius

This breaks the hydrogen bonds between the two strands of DNA

Results in single strands of DNA

We can now anneal our primers to the single strands using the correct optimal annealing temperature

Question 8

What do we do after annealing our primers?

Answer 8

We need to add our Taq polymerase

Question 9

How do we add our Taq polymerase?

Answer 9

Increase heat up to 72 degrees (optimum temperature for Taq)

Question 10

What does Taq do?

Answer 10

Taq extends the primers we have added (cloning our DNA)

Question 11

What do we do after Taq has extended to give us clones?

Answer 11

We repeat this PCR to give us multiple clones

Question 12

After 20 cycle repeats how many copies of the target gene will we have?

Answer 12

A million

Question 13

What is PCR?
(5)

Answer 13

Polymerase chain reaction

Method of amplifying DNA sequences in vitro

Exponential DNA replication process

Based on thermostable DNA polymerase

Requires some knowledge of sequence to be amplified

Question 14

Who invented PCR?

Answer 14

Kary Mullis

Question 15

List the three steps in a PCR cycle

Answer 15

Denaturation
Annealing
Elongation

(These are repeated again and again)

Question 16

What is the gene that causes cystic fibrosis?

Answer 16

CFTR

Question 17

What is CFTR?

Answer 17

A Cl- ion channel

Question 18

What mutation is the most common cause of cystic fibrosis?

Answer 18

A single deletion of phenylalanine

Question 19

How does a single deletion of phenylalanine cause cystic fibrosis?

Answer 19

The mutant protein does not fold or insert into the membrane correctly

Question 20

What temperature is needed to denature DNA?

Answer 20

50-65 Degrees Celsius

Question 21

What temperature is needed to anneal Taq polymerase?

Answer 21

72 degrees Celsius