Recombinant Bst Polymerase Mutations for Accurate Nucleotide Incorporation
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Solution Overview
Problem
Existing polymerases exhibit limitations such as high systematic error rates, low accuracy, and inefficient nucleotide incorporation, which hinder the performance of nucleic acid sequencing and amplification processes.
Innovation Solution
Development of recombinant Bst DNA polymerases with specific amino acid mutations, such as E30K, G209Q, and H528T, that enhance processivity, accuracy, and reduce systematic errors, particularly in sequencing by synthesis reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type polymerase is used for nucleic acid synthesis, then the polymerase maintains natural catalytic activity, but systematic error rates are high and accuracy is low
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (E30K, G209Q, H528T) into the polymerase enzyme to alter its catalytic properties. These parameter changes in the enzyme's structure result in improved accuracy and reduced systematic error rates while maintaining nucleotide incorporation efficiency.
Solution Approach 2:
The patent replaces the wild-type polymerase mechanism with a mutant polymerase that has modified catalytic properties. The mutant enzyme substitutes the natural catalytic mechanism with an improved version that reduces erroneous nucleotide incorporations while maintaining faithful base pairing.
2Duration of action of moving object
If polymerase catalytic properties are enhanced via amino acid substitution, then processivity and read length increase, but enzyme structure complexity increases
Solution Approach 1:
The patent applies local quality by introducing specific amino acid substitutions at particular positions (E30K, G209Q, H528T) within the polymerase enzyme structure. These localized changes at specific sites improve overall processivity and read length without requiring global restructuring of the entire enzyme, thereby limiting the increase in structural complexity.
3Productivity
If polymerase is modified to increase nucleotide incorporation rate, then sequencing throughput increases, but accuracy of nucleotide incorporation may decrease
Solution Approach 1:
The patent simultaneously optimizes multiple parameters through specific amino acid substitutions. The E30K, G209Q, and H528T mutations work together to enhance nucleotide incorporation rate while maintaining high accuracy, resolving the trade-off between productivity and measurement precision through coordinated parameter changes.
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 recombinant Bst DNA polymerases demonstrate improved accuracy, reduced error rates, and increased signal-to-noise ratios, leading to enhanced nucleic acid sequencing and amplification efficiency.
Implementation Method 1
polymerases, which can catalyze the polymerization of biomolecules (e.g., nucleotides or amino acids) into biopolymers (e.g., nucleic acids or peptides)
Implementation Method 2
The ability of enzymes to catalyze biological reactions is fundamental to life
Implementation Method 3
Many nucleic acid sequencing methods monitor nucleotide incorporations during in vitro template-dependent nucleic acid synthesis catalyzed by a polymerase
Implementation Method 4
such modification can be performed to favorably alter a polymerase's rate of nucleotide incorporation, affinity of binding to template, processivity
Data Source
AI summary
The present disclosure provides compositions, methods, kits, systems and apparatus that are useful for nucleic acid polymerization. In particular, recombinant polymerases and biologically active fragments thereof are provided that allow for nucleic acid amplification. In some aspects, the disclosure provides recombinant polymerases that yield lower systematic error rates and/or improved accuracy, when used in sequencing by synthesis reactions as compared to a control polymerase. In one aspect, the disclosure relates to recombinant polymerases useful for nucleic acid sequencing, genotyping, copy number variation analysis, paired-end sequencing and other forms of genetic analysis. In another aspect, the recombinant polymerases are useful for the amplification of nucleic acid templates during PCR, emPCR, isothermal amplification, recombinase polymerase amplification, rolling circle amplification, strand displacement amplification and proximity ligation amplification. In some aspects, the disclosure relates to recombinant polymerases useful for the generation of nucleic acid libraries and/or nucleic acid templates.