Recombinant Polymerases for Large Analog Incorporation
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Solution Overview
Problem
Existing DNA polymerases struggle with incorporating large nucleotide analogs efficiently, leading to challenges in single-molecule sequencing and nucleic acid amplification, particularly in terms of readlength, stability, and accuracy.
Innovation Solution
Development of recombinant DNA polymerases with specific mutations, such as amino acid substitutions, to enhance the incorporation of large nucleotide analogs, improving properties like readlength, thermostability, and accuracy, and incorporating them into sequencing systems like zero-mode waveguides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing DNA polymerases are used for incorporating large nucleotide analogs, then the basic replication function is maintained, but the readlength and accuracy are limited
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations (e.g., K138Q, L142K, Y224K, E239G, V250I, L253A, A256S, E375Y, A437G, A484E, S487A, E508R, D510K, K512Y, E515Q, D570E, D570T, D570M, T571V) into the polymerase sequence. These mutations alter the enzymatic parameters to enhance its ability to incorporate large nucleotide analogs, thereby increasing readlength while maintaining accuracy in single-molecule sequencing applications
2Temperature
If existing DNA polymerases are used for nucleic acid amplification, then the amplification function is achieved, but thermostability is insufficient
Solution Approach 1:
The patent modifies the polymerase through specific amino acid substitutions that enhance thermostability. These mutations (such as those at positions K138, L142, Y224, E239, V250, L253, A256, E375, A437, A484, S487, E508, D510, K512, L513, E515, D570, T571) stabilize the polymerase structure at elevated temperatures, enabling it to maintain amplification efficiency during high-temperature cycling in nucleic acid amplification reactions
3Productivity
If large nucleotide analogs are incorporated by wild-type polymerases, then the basic synthesis occurs, but the incorporation efficiency is reduced
Solution Approach 1:
The patent introduces specific mutations into the polymerase sequence that optimize its interaction with large nucleotide analogs. These parameter changes (amino acid substitutions at multiple positions including K138Q, L142K, Y224K, E239G, V250I, L253A, A256S, E375Y, A437G, A484E, S487A, E508R, D510K, K512Y, E515Q, D570E, D570T, D570M, T571V) enhance the polymerase's ability to efficiently incorporate large nucleotide analogs while maintaining high accuracy, thereby resolving the contradiction between productivity and measurement precision
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The modified polymerases exhibit enhanced performance with large nucleotide analogs, increasing readlength, stability, and accuracy, facilitating efficient single-molecule sequencing and nucleic acid amplification.
Implementation Method 1
a polymerase modified to optimize usage of the large analog, e.g., in single-molecule sequencing or another application
Data Source
AI summary
Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing or nucleic acid amplification. Such properties include enhanced performance with large nucleotide analogs, increased stability, increased readlength, and improved detection of modified bases, and can also include resistance to photodamage, enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased accuracy, altered speed, increased cosolvent resistance, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.