PCR

Question 1

In vitro or in vivo?

Answer 1

Vitro

Question 2

What is it used to produce?

Answer 2

Large quantities of specific DNA or RNA from very small quantities
Used to amplify DNA

Question 3

What are the 3 key stages?

Answer 3

Denaturation
Annealing
Elongation

Question 4

What is a primer?

Answer 4

Short sequences of single-stranded DNA that have base sequences complementary to the 3’ end of the template strand of the DNA fragment

Question 5

What is the form of DNA polymerase used called?

Answer 5

TAQ polymerase

Question 6

What do primers identify?

Answer 6

The DNA polymerase where to begin building the new strand

Question 7

Are free nucleotides required?

Answer 7

Yes
Of all 4 types- adenine, cytosine, guanine and cytosine

Question 8

Primers- providing a starting point for DNA polymerase?

Answer 8

Taq DNA polymerase cant start building a new DNA strand from scratch
Can only add nucleotides to an existing strand- primers provided this essential starting point
Once a primer is bound, TAQ polymerase can extend the strand by joining DNA nucleotides together

Question 9

Primers- preventing the original strands from rejoining?

Answer 9

Primers are shorter than the original DNA strands so they anneal (bind) faster
This physically blocks the complementary strands from rejoining

Question 10

Denaturation?

Answer 10

The double-stranded DNA is heated to 95 degrees Celsius, breaking the hydrogen bonds between complementary bases- separating the 2 strands.
Each strand acts as a template strand.

Question 11

Annealing?

Answer 11

The temperature is decreased to 56 degrees Celsius so that the primers can anneal to the ends of the template strands of DNA- forming hydrogen bonds

Question 12

Elongation?

Answer 12

The temperature is increased to 72 degrees Celsius for at least a minute- optimum temperature for TAQ polymerase
TAQ polymerase joins free nucleotides to the primers -> forming new DNA strands by making phosphodiester bonds
Forms new identical double-stranded DNA molecule
Enzyme is thermostable so will not be denatured by the high temperatures used in PCR to separate the DNA strands

Question 13

Is PCR repeated?

Answer 13

Yes- the cycle is repeated multiple times- doubling the DNA each time

Question 14

Calculating DNA fragments in PCR?

Answer 14

2 to the power of n

Question 15

Why might PCR stop or be inefficient?

Answer 15

Primers/nucleotides may run out
Taq polymerase may eventually denature over time
Temperature damages some of the DNA fragments
Once separated, the original DNA strands may re-join, rather than bonding to primers