Modified DNA Polymerases for Single Molecule Sequencing Accuracy
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Solution Overview
Problem
Current DNA polymerases used in single molecule sequencing face challenges such as high branching fractions, reduced stability of closed polymerase-DNA complexes, and inefficient kinetic properties, leading to inaccuracies and reduced processivity in sequencing processes.
Innovation Solution
Development of modified recombinant DNA polymerases with specific mutations, such as L253, E375, A484, and K512 mutations, that reduce branching fractions, enhance closed complex stability, and alter catalytic cycle rates, improving processivity and accuracy in single molecule sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If wild-type DNA polymerases are used in single molecule sequencing, then the sequencing process can proceed, but high branching fractions occur leading to inaccuracies
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations (L253, E375, A484, K512) in the DNA polymerase enzyme to alter its kinetic properties. These mutations change the enzyme's catalytic cycle rates and closed complex stability, thereby reducing the branching fraction and improving sequencing accuracy without changing the fundamental polymerase structure or function
2Stability of the object's composition
If standard DNA polymerases are used, then the sequencing reaction can proceed, but reduced stability of closed polymerase-DNA complexes occurs
Solution Approach 1:
The patent uses parameter changes by mutating specific residues (particularly in the L253, E375, A484, and K512 positions) to optimize the stability of the closed polymerase-DNA complex. These mutations enhance the residence time of the polymerase on the template, improving processivity and ensuring complete sequencing reactions
3Productivity
If conventional polymerases are used, then the catalytic cycle proceeds at natural rates, but inefficient kinetic properties reduce sequencing performance
Solution Approach 1:
The patent applies parameter changes by introducing mutations that specifically alter the kinetic parameters of the catalytic cycle. The L253, E375, A484, and K512 mutations are designed to optimize the timing and efficiency of nucleotide incorporation steps, reducing idle time in the catalytic cycle and improving overall sequencing throughput
Data Source
AI summary
Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions and/or heterologous or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include reduced reaction rates at one or more steps of the polymerase kinetic cycle, increased closed polymerase/DNA complex stability, enhanced metal ion coordination, reduced exonuclease activity, decreased branching fractions, and the like. Polymerases that exhibit branching fractions that are less than the branching fractions of the polymerases from which they were derived, or branching fractions that are less than about 25% for a phosphate-labeled nucleotide analog, are also provided. 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.