Modified DNA Polymerases Enhancing Thermostability and Strand Displacement
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Solution Overview
Problem
Current DNA polymerases, such as Bst DNA polymerase, are limited by their thermostability and strand displacement activity, making them unsuitable for methods requiring high temperatures like PCR or PCDR, and lacking efficiency in amplifying long DNA sequences.
Innovation Solution
Development of modified DNA polymerases within the Type-A family with specific amino acid substitutions at positions 738 and 743, enhancing thermostability and strand displacement activity, allowing for efficient DNA sequencing and amplification in LAMP, PCDR, and PCR processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Bst DNA polymerase is used for isothermal amplification, then strand displacement activity is improved, but thermostability deteriorates (cannot be used at temperatures above 68-70°C)
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (738F and 743F) in the DNA polymerase sequence to modify its thermal stability parameters while preserving strand displacement activity. This allows the enzyme to function at higher temperatures than the original Bst polymerase.
Solution Approach 2:
The patent creates a composite enzyme structure by combining features of different DNA polymerases (Bacillus stearothermophilus and Thermus aquaticus) into a chimeric enzyme that exhibits both strong strand displacement activity and enhanced thermostability, allowing use in both isothermal and thermal cycling methods.
2Temperature
If Taq DNA polymerase is used for PCR, then thermostability is improved, but strand displacement activity deteriorates
Solution Approach 1:
The patent creates a composite enzyme structure by combining features of different DNA polymerases (Bacillus stearothermophilus and Thermus aquaticus) into a chimeric enzyme that exhibits both strong strand displacement activity and enhanced thermostability, allowing use in both isothermal and thermal cycling methods.
3Measurement precision
If initial heat denaturation is applied to increase sensitivity, then detection sensitivity is improved, but enzyme inactivation worsens (for Bst polymerase)
Solution Approach 1:
The patent applies parameter changes by modifying the thermal stability parameters of the DNA polymerase through amino acid substitutions, enabling the enzyme to withstand initial heat denaturation temperatures (92-95°C) that were previously damaging, thereby allowing sensitivity enhancement without enzyme inactivation.
Data Source
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AI summary
The technology provided herein relates to novel variants of DNA-Polymerases exhibiting high termo- stability as well as a strong strand displacement activity; to nucleic acid molecules encoding said DNA- Polymerases, vectors, host cells containing the nucleic acids and methods for preparation and producing such enzymes; compositions comprising at least one of the DNA-Polymerases; and methods for using such enzymes in DNA sequencing and/or DNA amplification processes.