PCR

Question 1

What does PCR stand for?

Answer 1

Polymerase chain reaction

Question 2

What is PCR?

Answer 2

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

Question 3

What is a chain reaction?

Answer 3

-A series of events each one of which is dependant upon the preceding event to sustain itself

Question 4

What does a chain reaction lead to?

Answer 4

-An exponential increase in the number of events occurring in a sequence

Question 5

What is the amplicon?

Answer 5

-Segment within the DNA that is being amplified

Question 6

What is the amplicon determined?

Answer 6

-Determined by the sequence at the ends of that section of DNA.

Question 7

How do primers bind to the amplicon?

Answer 7

-These primers are complementary to sequences at the ends of the amplicon and are able form a duplex by hybridising to them.

Question 8

What happens when the DNA polymerase bind to the duplex?

Answer 8

-DNA polymerase recognises these duplexes and forms an initiation complex around them

Question 9

What factors determine specificity of PCR?

Answer 9

• Uniqueness of the sequences at the ends of the amplicon
• Hybridising the primers at the Tm or melting temperature of the duplex, we are using high stringency conditions under which only perfectly matched duplex will form, preventing mis-match base pairing occurring

Question 10

What is exponential amplification dependent upon?

Answer 10

-Two primers each complementary to one of the two strands

Question 11

What is the polymerase used in PCR?

Answer 11

-DNA dependent polymerase

Question 12

What is the purpose of DNA polymerase?

Answer 12

• Recognises a specific structure consisting of a partially double stranded DNA forming an initiation complex with it
• Polymerase will extend the strand that has a free 3’ end using the 5’ overhang as a template as it adds nucleotides to the 3’ carbon of the of the elongating or non-template strand

Question 13

In PCR, how is a partially double stranded structure formed?

Answer 13

• Formed by annealing (hybridising) a primer to the template strand
• Double stranded template has first to be denatured separating the two strands and thus made into two single stranded molecules
• Heating the reaction to a temperature that breaks the hydrogen bonds stabilising the duplex
• Newly formed strand is sometimes referred to as the nascent strand

Question 14

What type of reaction is annealing and renaturation?

Answer 14

-Competitive process

Question 15

What are the concentrations of template and primers?

Answer 15

• Template is low conc

- Primer is high conc

Question 16

What is the benefit of having these concentrations?

Answer 16

• Template is at the start of the reaction in a low concentration
• Formation of primer-template duplex is driven by favourable kinetics provided by a huge molar excess of the primer
• Equilibrium due to competition between renaturation of the double stranded template and annealing of the primer to the template preferentially occurs towards the primer template annealing

Question 17

Can polymerase copy or make RNA?

Answer 17

• No

- Must first be converted to a complementary DNA or cDNA molecule by using reverse transcription

Question 18

What does PCR require?

Answer 18

• A template strand with a primer (20-30 bases long)
• Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
• Mg2+ ions
• A roughly neutral pH

Question 19

What is the purpose of the deoxy nucleotide triphosphate?

Answer 19

• To form the elongating strand
• We hydrolyse the triphosphate adding a mono-phostphate to the strand
• Releasing pyrophosphate and hydrogen ions.
• This leads to an acidification of the reaction and depletion of reactants

Question 20

Purpose of Mg2+ ions?

Answer 20

• Essential co factor

- If we remove or chelate Magnesium

Question 21

Why is a neutral ph required?

Answer 21

-The reaction requires buffering since the reaction itself is producing hydrogen ions

Question 22

What is the PCR reaction dependent upon?

Answer 22

-Based on transition between three states reliant upon hybridisation of primers and formation of a partial duplex

Question 23

What are the 3 states of PCR?

Answer 23

• Denatured - where the template is single stranded where the template has been heated and the hydrogen bonds stabilising the duplex broken, this also means we need to use an enzyme that is capable of withstanding the harsh conditions used
• Annealed - the formation of a duplex between the primers and corresponding template strands
• The Native state – this is where optimal conditions for the extension of the initiation complex and enzyme activity occurs

Question 24

Why must Polymerase be thermostable?

Answer 24

-reaction MUST go through multiple rounds of extreme heating and cooling

Question 25

What does thermostability mean?

Answer 25

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

Question 26

From which organism is the polymerase used?

Answer 26

-Polymerase from a thermophilic bacterium

Question 27

Describe the PCR process

Answer 27

• Firstly we need to assemble the reaction components : template , enzyme cofactors, buffer and other reactants
• First stage is denaturation where we heat the reaction to temperatures in excess of 95 degrees
• Cooling to the tm of the primer –template duplex followed by adjusting these conditions to the optimal conditions for the enzyme to elongate the first round product which the can act as template in subsequent rounds

30 cycles of PCR will amplify a single molecule 1 billion times so an incredibly powerful technique

Question 28

What is the kinetics of the reaction dependent upon?

Answer 28

• Depletion of reactants

- Acidification of the reaction as a result of producing those hydrogen ions during elongation