A Single Amino Acid Change to Taq DNA Polymerase Enables Faster PCR, Reverse Transcription and Strand-Displacement

Area of Science:

• Molecular Biology
• Enzymology
• Biochemistry

Background:

• PCR (Polymerase Chain Reaction) is a cornerstone of molecular biology.
• Thermus aquaticus (Taq) DNA polymerase is widely used in PCR.
• Optimizing PCR protocols, including reducing extension times, is crucial for efficiency.

Purpose of the Study:

• To characterize a gain-of-function mutation in Taq DNA polymerase.
• To investigate the novel enzymatic activities of the mutant Taq D732N.
• To explore the potential of Taq D732N in one-step nucleic acid amplification assays.

Main Methods:

• Site-directed mutagenesis was used to create the Taq D732N mutant.
• PCR amplification assays were performed with varying extension times.
• Enzymatic activities, including strand displacement and reverse transcriptase activity, were assessed.
• Reverse Transcription PCR (RT-PCR) and Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) assays were conducted using the mutant enzyme.

Main Results:

• The Taq D732N mutation significantly reduced PCR extension times by 2-3 fold.
• The mutant enzyme demonstrated unexpected strand-displacement and reverse transcriptase activities.
• Taq D732N could catalyze both RT-PCR and RT-LAMP assays without additional enzymes.
• The mutated residue (D732) is located on the enzyme surface, away from the active site.

Conclusions:

• The Taq D732N mutation represents a significant advancement in PCR technology, enabling faster amplification.
• The discovery of dual DNA polymerase and reverse transcriptase activity in Taq D732N opens new possibilities for molecular diagnostics.
• This single-point mutation enhances enzyme function and broadens its application in nucleic acid amplification techniques.