PCR: Its use in molecular biology and clinical diagnostics

Question 1

Define Polymerase chain reaction

Answer 1

An enzyme-based method to specifically amplify segments of DNA using a thermal DNA polymerase in a cyclical process.

Question 2

Define chain reaction

Answer 2

A series of events each one of which is dependent upon the preceding event to sustain itself.

Question 3

What type of reaction is PCR?

Answer 3

It is an exponential reaction which results in a doubling the number of molecules present within the reaction mixture at every cycle.

Question 4

What is PCR used for?

Answer 4

It is a method to specifically amplify segments of DNA.

Question 5

What is the specificity dependent on?

Answer 5

It is dependent on the complementarity of the primers themselves.

Question 6

How are specific target molecules amplified?

Answer 6

They are dependent on the properties and behaviour of the primers used and the sequences of the primers in the context of PCR.

Question 7

At what temperature is the annealing stage performed?

Answer 7

It is performed at or near to the Tm of the primer.

Question 8

Why is annealing performed at or near the Tm of the primer?

Answer 8

It will prevent mismatch-based pairing therefore have a high specificity towards the reaction.

Question 9

Why is the sequence that we are annealing important?

Answer 9

It is important so that we can make the primers unique and adjacent to the position where we want to amplify the product.

Question 10

Why is it important to pick a sequence that is unique?

Answer 10

Primers that are complementary to sequences that are frequently found in the population of molecules that we want to amplify, there would be a lot of specificity irrespective of the temperature that we perform the annealing at.

Question 11

How many primers are needed for an exponential reaction?

Answer 11

two primers

Question 12

Why are two primers needed for an exponential reaction?

Answer 12

One primer that is complementary to each of the two strands and these primers would be orientated in opposite directions to each other such that the polymerase would produce a template which would move towards the opposing primer.

Question 13

What DNA polymerase is used to elongate the template molecule?

Answer 13

DNA dependent DNA polymerase

Question 14

What is the structure of the primer/template dimer?

Answer 14

Consists of a partially double-stranded DNA with a 3’ end forming an initiation complex with it.

Question 15

How is a partially double stranded structure formed?

Answer 15

It is formed by annealing a short-single stranded DNA molecule (primer) to a denatured and thus a single stranded DNA molecule.

Question 16

Why does annealing occur?

Answer 16

It results from the formation of base-pairing, stabilised by hydrogen-bonding between the complementary bases.

Question 17

What is annealing an alternative for?

Answer 17

Hybridisation

Question 18

What is annealing?

Answer 18

It is distinctive from renaturation as we are introducing a foreign molecule to the reaction mixture and competing for the formatino of duplex against the renaturation of the template strand itself. It is performed only after the template is denatured by heat.

Question 19

Why is annealing of the primer in preference to than renaturation?

Answer 19

There is a vast excess of the primer (molar excess) present in the reaction drives the kinetics.

Question 20

Summarise the competitive process

Answer 20

• Denaturing the template
• Hybridising/annealing the primer to one/both template strands in a competitive process driven by the high molar excess of the primer.

Question 21

Why is the enzyme DNA dependent DNA polymerase used in PCR?

Answer 21

• It synthesises a new nucleic acid strand by coping a DNA molecule.
• It cannot copy RNA nor make RNA

Question 22

What needs to happen to RNA before it can be measured by PCR?

Answer 22

The RNA needs to be converted to cDNA (complementary DNA) by reverse transcription before it can be amplified by PCR.

Question 23

What does the DNA dependent DNA polymerase require?

Answer 23

• A template strand with a primer
• Deoxynucleotide triphosphates
• Mg2+ ions
• A roughly neutral pH

Question 24

What is used to stop/terminate a polymerase reaction?

Answer 24

Mg2+ is a cofactor and if removed will inhibit the DNA polymerase which will stop/terminate a polymerase reaction.

Question 25

When trinucleotide phosphate is converted into a mononucleotide phosphate, what is released?

Answer 25

Pyrophosphate and hydrogen ions

Question 26

What are the three different states that the reaction transitions through?

Answer 26

• Denatured
• Annealed
• Native state at the optimal extension temperature and pH for enzyme activity

Question 27

For PCR to work, what needs to happen to the reaction?

Answer 27

It must go through multiple rounds of extreme heating and cooling.

Question 28

What does the reaction need to work?

Answer 28

A thermostable polymerase.

Question 29

What type of polymerase needs to be used to withstand the extreme conditions?

Answer 29

A polymerase which originates from a Thermus aquaticus is used e.g. Taq polymerase

Question 30

Define thermostability

Answer 30

Able to retain activity upon repeated heating to temperatures that would “destroy” most enzymes.

Question 31

Explain the steps of PCR

Answer 31

1. Start with a reaction mixture containing all the reaction components.
2. Denature the reaction mixture, separate the strands, heating the temperature at which the bonds break which is around 95 degrees.
3. The reaction mixture is cooled, and the primer is annealed to the template strand. The temperature needs to be close to the Tm of the duplex which would be formed between the primer and the template.
4. 72 degrees optimum temp for elongation with the polymerase
   - initiation complex formed
5. cycle repeated multiple times between denature, annealed and elongate.

Question 32

Why is PCR useful?

Answer 32

It provides a large number of molecules from a small amount of starting material.