Polymerase Chain Reaction (PCR) cont.
Choice of Polymerases for PCR
One of the important advances which allowed development of PCR was the availability of thermostable polymerases.
This allowed initially added enzyme to survive temperature cycles approaching 100 °C.
Thermostable DNA polymerases and their sources
DNA Polymerase | Natural or recombinant | Source |
Taq | Natural | Thermus aquaticus |
Amplitaq? | Recombinant | T. aquaticus |
Amplitaq (Stoffel fragment)? | Recombinant | T. aquaticus |
Hot Tub? | Natural | Thermus flavis |
Pyrostase? | Natural | T. flavis |
Vent? | Recombinant | Thermococcus litoralis |
Deep Vent? | Recombinant | Pyrococcus GB-D |
Tth | Recombinant | Thermus thermophilus |
Pfu | Natural | Pyrococcus furiosus |
ULTma? | Recombinant | Thermotoga maritima |
Properties of DNA polymerases used in PCR
Taq/Amplitaq? | Stoffel fragment | Vent? | Deep Vent? | Pfu | Tth | ULTma? | |
95 °C half-life | 40 min | 80 min | 400 min | 1380 min | >120 min | 20 min | >50 min |
5'3' exo | + | + | |||||
3'5' exo | + | + | + | + | |||
Extension rate (nt/sec) | 75 | >50 | >80 | ? | 60 | >33 | ? |
RT activity | Weak | Weak | ? | ? | ? | Yes | ? |
Resulting ends | 3' A | 3' A | >95% blunt | >95% blunt | blunt | 3' A | blunt |
Strand displacement | + | + | |||||
M.W. (kDa) | 94 | 61 | ? | ? | 92 | 94 | 70 |