PCR Flashcards
What is PCR?
An enzyme-based method to specifically amplify segments of DNA using a thermal DNA polymerase in a cyclic process
What is a chain reaction?
A series of events, each one of which is dependent on the preceding event to sustain itself.
Typically exponential increase
Under which conditions is PCR specific?
It is only specific if annealing is undertaken at the melting temperature (Tm) of the primers (ie high stringency conditions). This prevents mismatched based pairing
How is specificity determined?
The uniqueness of these sequences and their complementarity
What is DNA-dependent DNA polymerase and what are its functions?
Enzyme used in PCR that recognises a specific structure consisting of a partially double-stranded DNA, forming an initiation complex with it
The reaction extends a partially double stranded molecule from the 3’ end of the non-template strand
What is annealing, and how does it play in a role in PCR?
Annealing is a hybridisation process where we hybridise a short, single-stranded DNA molecule to another one that has been denatured
Results from the formation of base-pairing, stabilised by hydrogen bonding. It is performed only after the first template is denatured b y heat
How do we get more annealing in the reaction?
The template at the start of the reaction is in a low concentration. The formation of the primer template duplex is forced to occur by providing a huge excess of the primer.
This is in a competition between the renaturation of the double-stranded template and the annealing of the primer to the template, in which the annealing is in preference
What does it mean when a DNA-dependent DNA polymerase is used?
It synthesises a new nucleic acid strand by copying a DNA molecule. It cannot copy or make RNA
What are the three states the reaction is based on?
1) A denatured state (where the template becomes single stranded)
2) An annealed state (the formation of a duplex with the primer and template strand)
3) A native state (at the optimal extension temperature and pH for enzyme activity)
Why is thermostability important in PCR?
PCR needs to go through multiple rounding of heating and cooling to work, so the polymerase must be thermostable
Summarise the steps of PCR?
1) We mix everything in a test tube
2) We denature them at 95 degrees
3) We anneal at the Tm of the primer at 55 degrees
3) Extend from the 3’ end of the primers at 72 degrees
Why is PCR qualitative?
The end of the PCR reaction cannot be used to inform the template copy number, as regardless of the starting concentration of the template, the same end point is reached as amplification becomes rate limited. It is thus qualitative in the sense that it can determine the presence or absence of a substance
Explain how PCR is made quantitative?
Number of methods referred to as ‘real-time PCR’. These techniques utilise fluorescent detection of the amplification, and can tell us the amount of target DNA molecule in a sample
How is PCR used in SNP detection?
They rely on the differences in the Tm of a duplex containing a single nucleotide mismatch
Common applications:
- antibiotic resistance testing of TB
- identification of genetic markers
What are two approaches to SNP detection?
- High resolution melting, where the Tm of the amplified product is used to determine which sequence is present
- probe based version of qPCR, where specific binding of the probe to the amplified region containing the SNP is detected
