CMB2000/L02 PCR Amplifying DNA Flashcards
Give 5 pros of PCR.
Sensitive
Specific
Cheap
Rapid
Robust
Give the reactants and products of the DNA synthesis reaction.
3’ OH + 5’ 3P -> Phosphate backbone + 2P + H2O
Give the 6 reactants required for PCR.
dsDNA template
2 Primers
Polymerase
dNTPs
Magnesium
Buffer
Describe the 2 primers required for DNA synthesis. (3)
Small ssDNA molecule
6-30 bases
Chemically synthesised
What is the role of polymerase?
Copies template
Extends from 3’ end of primer
Give the 4 dNTPs.
dATP
dCTP
dGTP
dTTP
Why is magnesium required for PCR?
Co-factor for DNA polymerase enzyme
Describe the role of Taq polymerase.
Copies DNA accurately (1 mistake per 1 billion nucleotides)
Give the 3 regions of Taq polymerase.
Synthesis
Proof reading
Primer removal
Why can’t primers be too long or too short?
Long - hybridise too slowly
Short - won’t be specific
What GC content should primers contain and why?
40-60% as they have stronger H bonding and aid stability
Should also start and end with 1-2 G/C pairs
What should the melting temperature of primers be?
50-60C
What is the relationship between the Tmax of 2 primers used in a PCR?
Should be within 5C of each other
Define co-factor.
A non-protein component of the reaction that’s needed to enable activity of catalysis
Metallic or a compound
Which enzyme does magnesium act as a cofactor for?
DNA polymerase
What is the optimum pH for PCR?
8-9.5
Describe the ideal buffer for PCR.
Tris HCL
KCl promotes annealing
May be replaced by ammonium sulphate which destabilises base pairing bonds
Give the 3 stages of PCR.
Denaturation
Annealing
Elongation
Describe the first cycle of PCR.
Boil dsDNA
Anneal primer to ssDNA
Extend with polymerase and dNTPs
Boil, anneal different primer, complementary to new DNA and other strand of original DNA
Extend with polymerase
How many copies of DNA would be present after 30 cycles of PCR?
2^30
>1 billion
What is the role of Nancy520?
Dye intercalates between base pairs
How can PCR products be detected?
Run products of agarose gel
Use intercalating dye to stain DNA to determine size and yield
What PCR products can be identified from agarose gel electrophoresis? (4)
Molecular weight markers
PCR products
Primers
Template
