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Question
Statement 1: Thermostable Taq polymerase has an effective 3’-5’ proofreading activity.
Statement 2: Thermostable Pfu polymerase does not have any proofreading activities.
Statement 1: Thermostable Taq polymerase has an effective 3’-5’ proofreading activity.
Statement 2: Thermostable Pfu polymerase does not have any proofreading activities.
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Solution
The correct option is D Both the statements are false
Polymerase chain reaction is a technique used for selective amplification of a fragment of DNA in vitro so as to make many copies of a particular DNA sequence. This process of duplication of DNA is achieved in three steps-
Denaturation at 94−98°C.
Annealing at 40−60°C.
Extension at 72°C.
This means that a thermostable DNA polymerase enzyme has to be employed in the process. Taq DNA Polymerase and Pfu DNA polymerase are two such enzymes.
Taq DNA ploymerase was obtained from a thermophilic bacterium Thermus aquaticus, living in hot springs. This enzyme reads the template in 3'- 5' direction and synthesizes the new strand against the template in 5'- 3' direction. It does not have proofreading activity, resulting in relatively low replication fidelity.
Pfu DNA polymerase is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus, found in geothermally heated marine sediments. Pfu DNA polymerase has superior thermostability and proofreading properties compared to Taq DNA polymerase. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3' to 5' exonuclease proofreading activity, meaning that as the DNA is assembled from the 5' end to 3' end, the exonuclease activity immediately removes nucleotides misincorporated at the 3' end of the growing DNA strand. Consequently, Pfu DNA polymerase generated PCR fragments will have fewer errors than Taq generated PCR inserts.
Polymerase chain reaction is a technique used for selective amplification of a fragment of DNA in vitro so as to make many copies of a particular DNA sequence. This process of duplication of DNA is achieved in three steps-
Denaturation at 94−98°C.
Annealing at 40−60°C.
Extension at 72°C.
This means that a thermostable DNA polymerase enzyme has to be employed in the process. Taq DNA Polymerase and Pfu DNA polymerase are two such enzymes.
Taq DNA ploymerase was obtained from a thermophilic bacterium Thermus aquaticus, living in hot springs. This enzyme reads the template in 3'- 5' direction and synthesizes the new strand against the template in 5'- 3' direction. It does not have proofreading activity, resulting in relatively low replication fidelity.
Pfu DNA polymerase is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus, found in geothermally heated marine sediments. Pfu DNA polymerase has superior thermostability and proofreading properties compared to Taq DNA polymerase. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3' to 5' exonuclease proofreading activity, meaning that as the DNA is assembled from the 5' end to 3' end, the exonuclease activity immediately removes nucleotides misincorporated at the 3' end of the growing DNA strand. Consequently, Pfu DNA polymerase generated PCR fragments will have fewer errors than Taq generated PCR inserts.
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