Polymerase Chain Reaction

Question 1

What is it?

Answer 1

In Vitro coloning. Amplification of a section of DNA catalysed by DNA polymerases.

Question 2

What do you need?

Answer 2

Buffer- containing cofactors
Primers - G and C rich, sequence specific
dNTPs
DNA polymerase - eg Taq polymerase, heat resistant
Target DNA

Question 3

Process 1

Answer 3

Initial denaturation - Activate Taq, 94C, 5min

Question 4

Process 2

Answer 4

Denaturation - Melt DNA strand til it breaks open resulting single strand DNA, 94~C, 30sec

Question 5

Process 3

Answer 5

Annealing- Primers anneal to target sequence (5C below Tm, where 50% DNA is single stranded) 62C 30sec

Question 6

Process 4

Answer 6

Extension - Taq catalyses transcription to synthesise new DNA behind the primers 72C 5min

Question 7

Process 5

Answer 7

Amplify- exponential growth - Repeat 2-4 x 30cycles to create millions of copies

Question 8

Process 6

Answer 8

Final Extension- nucleotides fill in 72C 5mins

Question 9

Process 7

Answer 9

Hold at 4C, inactivate Taq, further testing

Question 10

Problems 1

Answer 10

Contamination

• Sterile components
• Use filtered tips
• UV irradiate work area

Question 11

Problems 2

Answer 11

Determination of cycling parameters (Empirical)

• Appropriate buffer
• Sufficient primers/dNTPs
• Temperatures
• Timings

Question 12

Types 1

Answer 12

Real time- Monitors amplification during PCR, quantitative, semi-quantitative, qualitative

Question 13

Types 2

Answer 13

Long and Accurate - Longer amplification times, greater fidelity using a proof reading polymerase

Question 14

Types 3

Answer 14

Hot Start- Avoids non-specific amplification at room temp, antibodies block Taq until required temp

Question 15

Types 4

Answer 15

Touchdown- Cycling programme, gradually reducing temp by 1-2C every two cycles

Question 16

Applications - Research

Answer 16

Human genome project

Diagnostic analysis

Question 17

Applications - Clinical

Answer 17

DNA fingerprinting
Viral and bacterial detection
Viral load monitoring
Genetic diagnosis
Early leukaemia and lymphoma diagnosis