B103 DNA Polymerase Variants for Isothermal Amplification
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Solution Overview
Problem
There is a need for DNA polymerases with improved properties such as increased efficiency, specificity, yield, reduced sequence bias, and ability to function well at elevated temperatures and high salt conditions for applications like rolling circle amplification and whole genome amplification.
Innovation Solution
Development of DNA polymerase variants with specific amino acid substitutions, such as lysine replacing alanine at position 147 and glycine replacing alanine at position 318, which exhibit increased thermostability, reaction rate, and reduced bias in DNA amplification, allowing for efficient amplification at temperatures up to 42°C and improved yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type Phi29 DNA polymerase is used for DNA amplification, then the polymerase maintains stability and activity at lower temperatures, but the reaction rate and yield are limited, and sequence bias occurs
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid residues at specific positions (147, 221, 318, 339, 359, 372, 503, 511, 544, 550) in the Phi29 DNA polymerase sequence. These substitutions alter the enzyme's biochemical parameters to achieve improved reaction rate, yield, and reduced sequence bias while maintaining thermostability and specificity
2Productivity
If the temperature is increased to improve reaction rate, then the amplification efficiency increases, but the polymerase loses stability and activity
Solution Approach 1:
The patent modifies the polymerase's amino acid composition to change its thermal stability parameters. Specific substitutions at positions such as 147, 221, and 318 enable the enzyme to maintain structural stability and catalytic activity at elevated temperatures (37-42°C), thereby decoupling the trade-off between temperature-induced rate enhancement and enzyme denaturation
3Ease of operation
If standard Phi29 DNA polymerase is used, then the amplification works well at low temperatures, but non-specific amplification and background artifacts increase
Solution Approach 1:
The patent introduces amino acid substitutions that modify the polymerase's specificity parameters. Changes at positions including 318, 359, and 544 enhance the enzyme's discrimination between specific and non-specific templates, reducing background artifacts and non-specific amplification while preserving the ability to function at low temperatures
Data Source
AI summary
Variants of the bacteriophage B103 DNA polymerase are described herein. The variant has improved properties, that include when compared to wild-type Phi29 DNA polymerase, at least one of the following: increased thermostability, improved reaction rate for DNA amplification, reduced background and a reduction of bias. Methods of using the DNA polymerase variant are also described herein.