PCR Role In Diagnosis

Question 1

Define the Polymerase Chain Reaction (PCR)

Answer 1

Polymerase Chain Reaction is an enzyme based method to specifically amplify segments of DNA using a Thermal DNA polymerase in a cyclical process.

Question 2

Define what is meant by a chain reaction.

Answer 2

Series of reactions that lead to an exponential increase in the number of events occurring in a sequence.

Question 3

Under what conditions is PCR specific

Answer 3

Only under High Stringency conditions
- Only if annealing is undertaken at the melting temperature Tm of the primers
- Preventing miss matched base pairing

Question 4

How do we use PCR to amplify a specific segment of DNA?

Answer 4

We can do this by choosing primers complementary to the unique sequence at the ends of the desired DNA sample.

Question 5

How does DNA polymerase function?

Answer 5

An enzyme that recognises a specific structure consisting of a partially double stranded DNA forming an initiation complex with it.

Question 6

In what direction does DNA polymerase work?

Answer 6

From 3’ to 5’.

Question 7

How is a Primer/ Template duplex formed?

Answer 7

• 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

Question 8

What is annealing?

Answer 8

An alternative way of describing hybridisation.

Question 9

Describe the competitiveness of annealing the primer and Renaturation?

Answer 9

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.

Question 10

What enzyme is used during PCR?

Answer 10

DNA dependent DNA polymerase.

Question 11

What is the function of DNA polymerase?

Answer 11

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.

Question 12

What are the required conditions for a Polymerase Chain Reaction occur?

Answer 12

• A template with opposing primers annealed to respective stands
• Deoxy nucleotide triphosphate
• Mg2+ ions
• A roughly neutral pH

Question 13

What is the function of Mg 2+ in PCR?

Answer 13

Functions as a cofactor for activity of DNA polymerases by enabling incorporation of dNTPs during polymerisation.

Question 14

What type of process is a Polymerase Chain Reaction?

Answer 14

Cyclical Process with an exponential accumulation of product.

Question 15

What 3 states does PCR rely on?

Answer 15

• Denatured (template becomes single stranded)
• Annealed (formation of initiating template)
• Native state at the optimal extension temperature and pH for enzyme activity

Question 16

What MUST occur for PCR to occur?

Answer 16

• MUST go though multiple rounds of extreme heating and cooling
• Polymerase MUST be thermostable (polymerase from a thermophilic bacterium is often used)

Question 17

What is meant by the term Thermostability?

Answer 17

“Able to retain activity” upon repeated heating to temperature that would “destroy” most enzymes.

Question 18

Describe the exact 3 temperature changes that occur during PCR.

Answer 18

• 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)

Question 19

How are the characteristic kinetics for PCR determined?

Answer 19

• Depletion of reactants
• Acidification of the reaction

Question 20

How is PCR used in Diagnostics?

Answer 20

Routine diagnostic tool used for identification, conformation and quantification of specific DNA sequence.

Question 21

What methods are used to quantify PCR for the use of diagnostics?

Answer 21

• Real-time PCR
• Quantitative PCR

These techniques utilise fluorescent detection of the amplification.

Question 22

What is SNP (single nucleotide polymorphism)?

Answer 22

Single nucleotide genetic variants

Question 23

What are the two approaches to SNP detection?

Answer 23

• High resolution melting (HRM)
• Probe based version of qPCR (Alleilic descrimination)

Question 24

What is High resolution Melting (HRM)?

Answer 24

Tm of the amplified product is used to determine which sequence variant is present.

Question 25

What is probe based version of qPCR process?

Answer 25

Where specific binding of the probe to the amplified region containing the SNP is detected.

Question 26

When is PCR of genetic markers used?

Answer 26

• 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