PCR Flashcards
What does PCR stand for?
Polymerase chain reaction
What is PCR?
-An enzyme based method to specifically amplify segments of DNA using a Thermal DNA polymerase in a cyclical process.
What is a chain reaction?
-A series of events each one of which is dependant upon the preceding event to sustain itself
What does a chain reaction lead to?
-An exponential increase in the number of events occurring in a sequence
What is the amplicon?
-Segment within the DNA that is being amplified
What is the amplicon determined?
-Determined by the sequence at the ends of that section of DNA.
How do primers bind to the amplicon?
-These primers are complementary to sequences at the ends of the amplicon and are able form a duplex by hybridising to them.
What happens when the DNA polymerase bind to the duplex?
-DNA polymerase recognises these duplexes and forms an initiation complex around them
What factors determine specificity of PCR?
- 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
What is exponential amplification dependent upon?
-Two primers each complementary to one of the two strands
What is the polymerase used in PCR?
-DNA dependent polymerase
What is the purpose of DNA polymerase?
- 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
In PCR, how is a partially double stranded structure formed?
- 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
What type of reaction is annealing and renaturation?
-Competitive process
What are the concentrations of template and primers?
- Template is low conc
- Primer is high conc
What is the benefit of having these concentrations?
- 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
Can polymerase copy or make RNA?
- No
- Must first be converted to a complementary DNA or cDNA molecule by using reverse transcription
What does PCR require?
- A template strand with a primer (20-30 bases long)
- Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
- Mg2+ ions
- A roughly neutral pH
What is the purpose of the deoxy nucleotide triphosphate?
- 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
Purpose of Mg2+ ions?
- Essential co factor
- If we remove or chelate Magnesium
Why is a neutral ph required?
-The reaction requires buffering since the reaction itself is producing hydrogen ions
What is the PCR reaction dependent upon?
-Based on transition between three states reliant upon hybridisation of primers and formation of a partial duplex
What are the 3 states of PCR?
- 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
Why must Polymerase be thermostable?
-reaction MUST go through multiple rounds of extreme heating and cooling
What does thermostability mean?
-Able to retain activity” upon repeated heating to temperatures that would “destroy” most enzymes
From which organism is the polymerase used?
-Polymerase from a thermophilic bacterium
Describe the PCR process
- 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
What is the kinetics of the reaction dependent upon?
- Depletion of reactants
- Acidification of the reaction as a result of producing those hydrogen ions during elongation
