PCR & Diagnostics Flashcards
What is the definition of PCR?
Polymerase Chain Reaction is an enzyme-based method used to specifically amplify segments of DNA using a thermal DNA polymerase in a cyclical process.
What is a chain reaction?
A chain reaction is a series of events whereby each one is dependant upon the preceding event to sustain itself in a loop. It is a series of reactions that lead to an exponential increase in the number of events occurring in a sequence. In PCR, there is a doubling of number of molecules produced through each stage of the amplification until the reaction is exhausted.
What is the amplicon?
The segment of DNA that is amplified (within a larger molecule) is the amplicon. It is determined by the sequence at the ends of that section of DNA
What are primers?
Short oligonucleotides that are complementary to sequences at the end of the amplicon and can form a duplex by hybridising/annealing to them. This allows DNA polymerase to recognise these duplexes and form an initiation complex around them.
What factors determine specificity of PCR?
- The uniqueness of the sequences at the end of the amplicon and the complementarity of the primers to these sequences
-Specific only if annealing occurs at the Tm of the primers. By doing this, we are using high stringency conditions where only perfectly matched duplexes will form. This ensures that no mismatch base pairing occurs as only perfectly matched hybrids will form in high stringency conditions.
P.S. Hybridisation of primers = Annealing of primers. Hence melting temperature (Tm) can also be referred to as the annealing temperature.
What is the amplicon dependant on and what is the exponential amplicon dependant on?
The segment amplified (the amplicon) is dependant on the sequence at the ends and the primers complementarity to these sequences
The exponential amplicon is dependant on having two primers, each complementary to one of the two strands (as in diagram) – A reverse primer and a forward primer.
The polymerase is then able to form initiation complexes and make new strands.
What is DNA polymerase? What does it do?
In PCR, the polymerase is a DNA dependent DNA polymerase. It is an enzyme that recognises a specific structure consisting of a partially double stranded DNA molecule and will form an initiation complex with it.
In doing so, the 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 elongating/ non-template strand.
In PCR how is a partially double stranded structure formed?
A partially double stranded structure with a 3’ end and an overhanging 5’end is formed by annealing a primer (short single stranded DNA molecule) to the template strand.
How does the primer anneal to the template?
The double stranded molecule must first be denatured by heating the reaction to a temperature that breaks the hydrogen bonds and thus made into two single stranded molecules. Annealing only takes place after this has occurs – and annealing of the primer under high stringency conditions is achieved using the predicted Tm of the primer-template duplex. The newly formed strand is referred to as the nascent strand.
What is the difference between annealing and renaturation of template. How is the formation of the primer-template favoured over renaturation?
The template has a very low copy number, and the primer has a very high copy number. Annealing and renaturation are competitive processes. The template is at the start of the reaction low in concentration thus the formation of primer-template duplex is driven be favourable kinetic as a result of a huge excess of the primer present in the reaction. Thus, the equilibrium, due to competition, preferentially occurs towards the primer template annealing.
Why and how is RNA converted to DNA before it can be amplified by PCR?
In PCR, DNA polymerase is used. This synthesises a new nucleic acid strand by copying a DNA Molecule. It does not copy an RNA molecule, nor can it make an RNA molecule. Thus, RNA has to be converted into a complementary DNA or cDNA molecule, using reverse transcriptase, before it can be amplified by PCR.
What does the PCR require?
- A template strand with a primer annealed to it (20-30bp) with a 3’ OH group and a 5’ overhanging template strand.
- Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP) to form the elongating strand
- Mg2+ ions as cofactors to DNA polymerase. (If remove mg ions the reaction will be inhibited)
- A roughly neutral pH
What are the three states The PCR reaction transitions between, and hence cycles between what three separate stages?
- Denatured- Where the template becomes single stranded via heat as the hydrogen bonds stabilising the duplex. (Means you need to use an enzyme that is capable of withstanding harsh conditions)
- Annealed- formation of initiating template. The formation of a duplex between the primers and corresponding template strands
- The Native state- where optimal conditions (temp and Ph) for the extension of the initiation complex and enzyme activity occurs.
What is thermostability?
Enzyme being ‘able to retain activity’ upon heating to a temperature that would denature most enzymes
Why must the polymerase be thermostable? (why is Taq polymerase used specifically?)
For PCR to work, the reaction MUST go through multiple rounds of extreme heating and cooling, and most enzymes would not be able to tolerate this. Thus, the polymerase must be thermostable. This is why Taq polymerase is used (it is from a thermophilic bacterium e.g thermus aquaticus)
