PCR

Question 1

PCR

Answer 1

In vitro technique that allows the amplification of a DNA fragment from DNA template.
Research: detection of specific genes.
Medicine: Identification of pathogens.
Biotech: Recombinant systems.
Other: Forensics, paternity test.

Question 2

Polymerase Chain Reaction

Answer 2

PCR is based on the ability of the DNA polymerase to synthesize a new strand of DNA complementary to the template strand.

Question 3

PCR sequence

Answer 3

Denaturation
Cycling: denaturation, annealing, elongation
Extension
Amplification > Electrophoresis

Question 4

PCR equipment

Answer 4

Gloves/ice/pipettes/tips
Microtubes and PCR tubes
PCR instrument
Electrophoresis

Question 5

PCR Reaction mix

Answer 5

DNA template
DNA polymerase
DNA polymerase reaction buffer
Mg2+
dNTPS (dATP, dCTP, dGTP, dTTP)
2x Primers, forward and reverse
Water.

Question 6

DNA polymerase

Answer 6

Thermus aquaticus:
Thermophilic bacterium, Taq polymerase.
Pyrococcus furiosus:
Pfu polymerase
Proof reading activity (3’ to 5’ exonuclease), higher accuracy.
Processivity (Taq DNApol)
Fidelity (Pfu DNApol)

Question 7

Oligonucleotide Primers

Answer 7

18-30 nucleotides long
Melting temperature of the two primers should be between 60 and 65 and within 2deg of each other. Tm 2 x (A+T) + 4 x (C+G)
Each primer should contain between 40 and 60% C and G
The 3’ end of a primer ending in C or G to promote binding.
Balanced distribution of GC-rich and AT-rich domains avoids localised differences in Tm.

Question 8

Dimers

Answer 8

Intra-primer homology, more than 3 bases that complement opposite each other within the primer, can create hairpins.
Inter-primer homolgy (forward and reverse primers have complementary sequences.
Dimers formed instead of annealing to desired DNA sequences.
Primers always in ‘5 to 3’