PCR and its role in diagnostics Flashcards
What is the PCR reaction?
An enzyme based chain reeaction used to amplify specific segments of DNA using thermostable DNA polymerase in a cyclical process
What is a chain reaction?
A series of events that rely on the events preceding to sustain itself, typically a series of reactions that lead to an exponential increase in the number of events occuring in a sequence
What determines the segment amplified, and what are the requirmnets for amplification?
The sequence at the ends of the segment, which if known can have specific primers produced for it and must requre a reverse and forward primer for the exponential growth
Define Stringency and what it means when there is high stringency conditions?
Stringency is the quality of being very strict, High stringency conditions ensure that the specific annealing taking place is the one desired, usually acheived through havign annealing occur at the melting temperature of the DNA strand
What is te melting temperature Tm?
The melting temperature is the temperature at which 50% of the DNA in a sample has denatured charactherised by hyperchromcity at 260nm
What is hyperchromicity and how does it help us to observe the melting point of a DNA strand?
It is the increased absorbrance of light at 260nm, single stranded DNA absorbs light more than double stranded so the increase in absorption can signal when the deanturation has occured and highlights the temperature that caused that
What is DNA dependent DNA polymerase’s function?
To detect a partially double stranded molecule from the 3 prime end and extending the molecule by combining the DNA nucleotides in reference to the template
Annealing and renaturation are both competitive processes, how do we acheive favourable kinetics for annealing?
Through the use of high concnetrations of primers, this drives the process of annealing rather than waiting for renaturation to occur
You cannot copy or makr RNA in PCR, so what must be done in order for the amplification of a sequence from the use of RNA?
Meausre the amount of transcript of RNA out of the while population of mRNA, which must then be converted into cDNA, realistically you never use PCR to amplify RNA
What does the DNA dependant DNA polymerase need?
- A template strand with a primer (20-30bp long) annealed to it
- Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
- Mg2+ ions needed for the normal functioning of DNA polymerase
- Roughly neutral pH
Why is Taq polymerase used in PCR?
Due to its thermostability, it is able to retain its function upon extreme heating which is essential for PCR due to its cyclical nature which occurs in three stages (denaturing, annealing, native state for optimal extension and pH for enzyme activity)
PCR doubles the amount of molecules each cycle giving it a log2 scale, but what causes the sigmoidal curve?
- The acidifciation of the solution due to the addition of dATP
- The reduction in the quantitiy of the reactants
- Effects DNA polymerase and “poisons” the reaction
What can PCR be used for medically, what are the 3 examples of this?
- Used for the identification, quantifcation and confirmation of the DNA sequence of interest
- e.g. Prescence abscence calling TB
- e.g. differentiating between closely related diseases such as swinew flu and human influenza
- e.g. to determine the viral load (how much there is) such as the HIV viral load
PCR usually isnt quantitative, what is th effect of using a low copy number versus a high copy number?
- The end point is the same
- The larger the copy number at the start the more of a shift taken to the right by the graph and the lower the copy number at the start the more of a shift taken to the left
What are the modifications amde to PCR to make it “real time” (quantitative)?
The use of flourescent detection where we observe in real time the growth and observe the accumulation, making it quantitative
