PCR

Question 0

List the reagents used in PCR.

Answer 0

Template DNA (~100ng)
dNTPs (200uM)
Primers (forward and reverse - 10pm each)
DNA Polymerase (Taq- 0.5 units)
Standard buffer
MgCl2 (1.5mM) 
Sterile water

Question 1

What is PCR?

Answer 1

Polymerase Chain Reaction is a biochemical technique whereby a single/few copies of DNA is amplified by several orders of magnitude, producing specific DNA product.
-first discovered in Thermus aquaticus in 1976

Question 2

List the 3 steps in a PCR reaction

Answer 2

1) . Denaturation
2) . Annealing
3) . Elongation/Extension

Question 3

Explain the significance of the denaturation step

Answer 3

Opens up template DNA strand, ie produces single stranded DNA template
Activates Taq polymerase
Can be ~10deg lower for smaller templates
92-95degC

Question 4

What are the important things to remember in the annealing step?

Answer 4

Temp: 50-60deg
Usually 5deg lower than the Tm
Tm = 4(G + C) + 2(A+T) degC
Tm= temp at which 50% of primers bind

Question 5

What is the significance of too high/too low Annealing temp?

Answer 5

Too high = primers melt off therefore lower/no yield

Too low = non-specific binding

Question 6

Explain the significance of the Extension step.

Answer 6

Step where Taq poly binds to the primers and start elongating the strands (at 100bp) per sec
72deg
Final extension step for 5min which extends all unfinished/incomplete products

Question 7

How many cycles does a typical PCR reaction have?

Answer 7

25-35 cycles

40-50 cycles for single molecule

Question 8

What are the stages of a PCR reaction?

Answer 8

Exponential - products double with each cycle
Levelling off - Reagents start to run out and polymerase start to lose activity
Plateau - exhaustion of polymerase and reagents

Question 9

How do we visualise our PCR product?

Answer 9

Using gel electrophoresis. Include DNA ladder, and add intercalating agent (SYBR Gold/EtBr) which fluoresces under UV

Question 10

What are the guidelines for designing primers?

Answer 10

Length: 20-40 bp, ideally 17-28bp
50-60%GC content, Tm ~ 50-72deg
GC clamp
Avoid secondary structures (hairpin loops etc)
Avoid complementarity (primer self-annealing)

Question 11

Outline the principles of RFLP.

Answer 11

Restriction Fragment Length Polymorphism
Using restriction enzymes to cut up DNA amplified by PCR, and visualise using gel electropheresis. The different bands produced will suggest different polymorphisms, and the unique patterns are idiosyncratic. Used in DNA fingerprinting, forensics, ancestry studies, dx of diseases

Question 12

Describe the context and significance of VNTRs

Answer 12

Variable Nuclear Tandem Repeats are repeats of nucleotides: