PCR Role In Diagnosis Flashcards
Define the Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction is an enzyme based method to specifically amplify segments of DNA using a Thermal DNA polymerase in a cyclical process.
Define what is meant by a chain reaction.
Series of reactions that lead to an exponential increase in the number of events occurring in a sequence.
Under what conditions is PCR specific
Only under High Stringency conditions
- Only if annealing is undertaken at the melting temperature Tm of the primers
- Preventing miss matched base pairing
How do we use PCR to amplify a specific segment of DNA?
We can do this by choosing primers complementary to the unique sequence at the ends of the desired DNA sample.
How does DNA polymerase function?
An enzyme that recognises a specific structure consisting of a partially double stranded DNA forming an initiation complex with it.
In what direction does DNA polymerase work?
From 3’ to 5’.
How is a Primer/ Template duplex formed?
- The double stranded DNA molecule is denatured and thus made into single stranded molecule using HEAT
- Newly formed strand is sometimes referred to as the nascent strand
- Then annealing of the primer molecule occurs
What is annealing?
An alternative way of describing hybridisation.
Describe the competitiveness of annealing the primer and Renaturation?
Annealing of the primer occurs in preference to Renaturation - due to a favour in kinetics as a result of the vast excess of the primer present in the reaction.
What enzyme is used during PCR?
DNA dependent DNA polymerase.
What is the function of DNA polymerase?
It synthesises a new nucleic acid strand by copying a DNA molecule.
CANNOT copy RNA nor make RNA - RNA must be converted to DNA by reverse transcription before it can be amplified by PCR.
What are the required conditions for a Polymerase Chain Reaction occur?
- A template with opposing primers annealed to respective stands
- Deoxy nucleotide triphosphate
- Mg2+ ions
- A roughly neutral pH
What is the function of Mg 2+ in PCR?
Functions as a cofactor for activity of DNA polymerases by enabling incorporation of dNTPs during polymerisation.
What type of process is a Polymerase Chain Reaction?
Cyclical Process with an exponential accumulation of product.
What 3 states does PCR rely on?
- Denatured (template becomes single stranded)
- Annealed (formation of initiating template)
- Native state at the optimal extension temperature and pH for enzyme activity
What MUST occur for PCR to occur?
- MUST go though multiple rounds of extreme heating and cooling
- Polymerase MUST be thermostable (polymerase from a thermophilic bacterium is often used)
What is meant by the term Thermostability?
“Able to retain activity” upon repeated heating to temperature that would “destroy” most enzymes.
Describe the exact 3 temperature changes that occur during PCR.
- Denature (95 degrees Celsius)
- Anneal at the Tm of the primers (55 degrees Celsius)
- Extend from the 3’ end of the primer (72 degrees Celsius)
How are the characteristic kinetics for PCR determined?
- Depletion of reactants
- Acidification of the reaction
How is PCR used in Diagnostics?
Routine diagnostic tool used for identification, conformation and quantification of specific DNA sequence.
What methods are used to quantify PCR for the use of diagnostics?
- Real-time PCR
- Quantitative PCR
These techniques utilise fluorescent detection of the amplification.
What is SNP (single nucleotide polymorphism)?
Single nucleotide genetic variants
What are the two approaches to SNP detection?
- High resolution melting (HRM)
- Probe based version of qPCR (Alleilic descrimination)
What is High resolution Melting (HRM)?
Tm of the amplified product is used to determine which sequence variant is present.
What is probe based version of qPCR process?
Where specific binding of the probe to the amplified region containing the SNP is detected.
When is PCR of genetic markers used?
- Parentage or Kinship: immigration or inheritance
- identification: military casualties, missing persons or environmental disasters
- Matching biological materials from two sources: placing an individual at a crime scene.
- Authentificatio of biological material: cell lines, purity of foods