Modified Polymerase Dissociation and Accuracy
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Solution Overview
Problem
Existing polymerases used in nucleic acid synthesis and sequencing have limitations such as high dissociation rates from templates, incorporation of incorrect nucleotides, and reduced processivity, which affect the accuracy and length of sequencing reactions.
Innovation Solution
Development of modified polymerases with specific amino acid mutations that enhance their dissociation time constant, processivity, and accuracy, allowing for improved nucleic acid sequencing and library generation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If existing polymerases are used in nucleic acid synthesis, then the basic polymerization function is achieved, but high dissociation rates from templates reduce processivity and read length
Solution Approach 1:
The patent applies parameter changes by modifying amino acid residues at specific positions (e.g., positions 77, 261, 264, 272, 281, 480, 528, 572, 577, 579) in the polymerase protein structure. These amino acid substitutions alter the biochemical parameters of the polymerase-template interaction, increasing the dissociation time constant and thereby improving processivity and read length in sequencing applications
2Reliability
If existing polymerases are used for nucleic acid synthesis, then amplification occurs, but incorporation of incorrect nucleotides reduces sequencing accuracy
Solution Approach 1:
The patent modifies specific amino acid residues in the polymerase active site and nucleotide binding regions to change the kinetic parameters of nucleotide selection. These modifications enhance the discrimination between correct and incorrect nucleotides, reducing misincorporation rates and improving sequencing accuracy while maintaining acceptable amplification efficiency
3Length of stationary object
If existing polymerases are used in sequencing reactions, then nucleic acid synthesis occurs, but reduced processivity limits read length
Solution Approach 1:
By changing amino acid residues at positions that contact the DNA template and influence polymerase stability (e.g., positions 77, 261, 264, 272, 281, 480, 528, 572, 577, 579), the patent increases the duration the polymerase remains bound to the template, thereby extending the read length achievable in single-molecule sequencing reactions
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 demonstrate increased accuracy, longer read lengths, and enhanced processivity, leading to improved sequencing performance and the ability to amplify larger numbers of nucleic acid templates in a single reaction.
Implementation Method 1
a modified polymerase or a biologically active fragment thereof, where the modified polymerase or the biologically active fragment thereof includes one or more amino acid modifications relative to a reference polymerase
Data Source
AI summary
The present disclosure provides compositions, methods, kits, systems and apparatus that are useful for nucleic acid polymerization. In particular, modified polymerases and biologically active fragment thereof are provided that allow for nucleic acid amplification. In one aspect, the disclosure relates to modified polymerases useful for nucleic acid sequencing, genotyping, copy number variation analysis, paired-end sequencing and other forms of genetic analysis. In some aspects, the disclosure relates to modified polymerases useful for the generation of nucleic acid libraries or nucleic acid templates for use in various downstream processes. In some aspects, the disclosure relates to the identification of homologous amino acid mutations that can be transferred across classes or families of polymerases to provide novel polymerases with altered catalytic properties. In some aspects, the disclosure provides modified polymerases having enhanced catalytic properties as compared to a reference polymerase.