PCR and its role in diagnostics

Question 1

What type of method is PCR?

Answer 1

An enzyme based method to specifically amplify segments of DNA using a thermal DNA polymerase in a cyclical process

Question 2

What is the definition of a chain reaction?

Answer 2

Series of events in which each one of which is dependent upon the preceding event to sustain itself

Question 3

Where does specificity stem from in PCR?

Answer 3

Specificity stems from complementarity of primers

Primers must be in opposite directions

• Is specific only if annealing is undertaken at the melting temperature/Tm of the primers
• High stringency conditions need to be carried out to prevent mis-matched base pairing and allows for high specificity for amplification of the product
• The segment amplified in PCR is determined by the sequence at the ends and exponential amplification requires two primers corresponding to these sequences

Question 4

When is PCR method only specific and what does this prevent?

Answer 4

Specific only if annealing is undertaken at melting temperature Tm of the primers Prevents mismatched base pairing

Question 5

What polymerase is used in PCR?

Answer 5

DNA dependent DNA polymerase is used

Question 6

What does DNA polymerase recognise?

Answer 6

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

Question 7

What does annealing result from and when is it performed?

Answer 7

Results from the formation of base pairing, stabilised by hydrogen bonding Performed only after template is denatured by heat

Question 8

What type of process is annealing and renaturation?

Answer 8

Annealing and renaturation are a competitive process

Question 9

Between annealing and renaturation, what occurs in preference and due to what reason?

Answer 9

Annealing of primer occur in preference to renaturation due to the vast excess of the primer

Question 10

What does DNA polymerase require to function in PCR?

Answer 10

• -Template strand with primer
• -Deoxynucleotide triphosphates - (dATP, dGTP, dCTP, dTTP) - KNOWWWW!!!!!!
• -Mg2+ (removing/kelating can stop polymerase reaction)
• -Roughly neutral pH = acidification pauses the reaction

Question 11

What are the 3 states that PCR relies on and what are they reliant upon?

Answer 11

PCR based on 3 states reliant upon hybridisation of primers and formation of partial duplex: -Denatured -Annealed -Native state at the optimal extension tempearture and pH for enzyme activity

Question 12

What must be thermostable and due to what reason?

Answer 12

Polymerase must be thermostable as in order for PCR to work, the reaction must go through multiple rounds of extreme heating and cooling

Question 13

What is the definition of thermostability?

Answer 13

The ability to retain activity upon repeated heating to temperatures that would destroy most enzymes

Question 14

From what bacterium is a polymerase is used due to thermostability?

Answer 14

A polymerase from a thermophilic bacterium is used The bacterium used is called thermos aquaticus

Question 15

Steps involved in PCR

Answer 15

1. Template, primers (in opposite direction), enzyme and reactants mixed
2. Denaturation at 95 degrees celcius - H bonds in base pairs are broken and strands separated by heating
3. Anneal at the Tm of the primers (in excess so that hybridisation is favoured as opposed to renaturation)
4. Temperature changed to 72 degrees Initiation complex formed and polymerase recognises partial duplex elongating from 3’ end.

Process is repeated many times, every cycle results in a doubling of product = exponential accumulation

Question 16

What is PCR used for?

Answer 16

Used in diagnostics -Identification, confirmation and quantification of specific DNA sequence e.g presence of TB -detection in sputum determining the treatment response Differentiate between closely related organisms ‘swine flu vs human influenza’ both H1N1 subtypes

Question 17

What is QPCR used for?

Answer 17

• Used for quantifying the amount of target DNA molecule
• Involve techniques which utilise fluorescent detection of the amplification
• The more product you accumulate the more fluorescence you gain

Question 18

What is the technique utilised in QPCR?

Answer 18

Involve techniques which utilise fluorescent detection of the amplification

Question 19

What are 2 methods to detect SNPs during PCR?

Answer 19

-HRM Tm of amplified product is used to determine which sequence is present

• • Amplicon with SNP will have a different melting curve
• Probe based version of qPCR: Where specific binding of the probe to the amplified region containing SNP is detected
• Uses a probe which identifies the position of the SNP and you differentiate between the binding of two probes

Question 20

Identify two applications of SNP detection using PCR?

Answer 20

1) Antibiotic resistance testing: Can discriminate between two organisms which have resistance to a particular antibiotic providing the resistance is dependant on the site of mutation within a gene

2)Identification of genetic markers: Markers of disease, cancer, treatment response (HCV)

Question 21

Explain this graph in PCR

Answer 21

• The reaction has characteristic kinetics determined by depletion of reactants and the acidification of the reaction
• Initially exponential to log base 2 (doubles every cycle)
• Acidification: As reaction progresses there is acidification due to dNTPs elongating strand resulting in accumulation of H+ ions and pyrophosphates overcoming the buffering capacity and moving away from the optimum pH
• Enzyme activity of dependant DNA polymerase will also be affected by the pH
• Depletion of reactants: Reduction in concentration of the primers and dNTPs causes the reaction to not continue infinitely due to reducing kinetic

Question 22

How are STRs detected using PCR in forensics

Answer 22

• Multiple sets of labelled Primers are designed such that the products span different STRs o The product of the amplification will depend of the number of repeats present in that loci • The more STRs investigated the more unique the pattern of sizes produced providing a “DNA fingerprint” of STRs around the genome