PCR Flashcards
What type of method is PCR
In vitro (Cell-free method)
Amplifies large or small amount of DNA?
Small amount of DNA exponentially
Polymerases required?
Taq Polymerase and Pfu polymerase
Taq polymerase properties
- Thermostable, able to withstand heating n cooling
- amplifies DNA at high rate
- Low fidelity due to lack of 3’ to 5’ exonuclease proofreading mechanism
Pfu polymerase
- Thermostable
2. Proofreads DNA due to presence of 3’ to 5’ exonuclease proofreading mechcanism
Building blocks?
Deoxyribonuclueotide Triphosphares
Buffer?
Provides chemical environment for reagents to perform optimally
Forward and reverse primer
DNA primers that attach to specific sites flanking the DNA segment to be amplified
Procedure of PCR?
- Denaturation
- Annealing
- Elongation
- Repeat
- Final elongation
- Samples holding
Explain denaturation steps
Heating of dsDNA strand to 95*C for 1-2 minutes, to break hydrogen bonds between strands to form single template strands
Annealing
Cool down to 55*C for 1-2 minutes to allow annealing of primers. DNA primers bind through hydrogen bonding to specific sites in the template strand flanking the target DNA region to be amplified
Elongation Conditions?
- Temperature increased to 72*C for synthesis of complementary DNA
Polymerase needed in elongation?
Taq polymerase recognises the 3’OH group of the annealed DNA primers, starting synthesis of complementary DNA strand
direction of synthesis?
5’ to 3’ direction
Template strand read from 3’ to 5’ direction
Time taken?
Rule of thumb is 1 minute every 1000 base pairs
