DNA Polymerase Variant for Modified Nucleotide Sequencing
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Solution Overview
Problem
Current DNA polymerases struggle to effectively polymerize artificially modified nucleotides or their analogs, limiting DNA replication efficiency in sequencing methods like sequencing-by-synthesis, particularly due to poor recognition and binding mechanisms.
Innovation Solution
A DNA polymerase variant with specific amino acid mutations at positions 408, 409, and 485, along with additional mutations at other sites, is developed to enhance polymerization efficiency and reduce adsorption on biochips, allowing for improved DNA sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type DNA polymerase is used, then DNA replication can proceed, but polymerization efficiency with artificially modified nucleotides is poor
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the DNA polymerase active site (positions 408, 409, and 485) to alter the enzyme's substrate binding properties. These parameter changes in the protein structure enable the polymerase to effectively incorporate artificially modified nucleotides while maintaining DNA replication functionality.
2Reliability
If DNA polymerase strongly adsorbs to biochip surface, then stable binding occurs, but elution and subsequent sequencing cycles are interfered with
Solution Approach 1:
The patent applies parameter changes by introducing amino acid mutations that modify the surface properties of the DNA polymerase, altering its adsorption characteristics to the biochip surface. This enables the enzyme to maintain sufficient binding stability during the reaction while allowing for easy elution afterward, facilitating multiple sequencing cycles without interference.
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 variant exhibits enhanced polymerization activity and easy elution from biochips, facilitating efficient DNA sequencing with artificially modified nucleotides without interference in subsequent cycles.
Implementation Method 1
DNA polymerase catalyzes the rapid and accurate replication of DNA in chromosomes of a living organism
Implementation Method 2
the polymerases exhibit different recognition mechanisms for a nucleotide and its analogues because they have significant different motifs
Data Source
AI summary
Disclosed in the present disclosure is a recombinant DNA polymerase. The recombinant DNA polymerase is any one selected from: A) a protein, having amino acid modifications at positions 408, 409 and 485, and at least one of amino acid modification(s) at positions 53, 59, 199, 243, 526, 558, 613, 641, 671, 673, 674, 692 and 709 compared to the amino acid sequence of a wild-type KOD DNA polymerase; B) a protein derived from the protein in A), formed by deleting amino acids 1 to 29 from a C-terminus of the protein in A) and keeping the remaining amino acids unchanged; and C) a protein derived from the protein in A) or B), formed by connecting a tag to the N-terminus or C-terminus of the amino acid sequence of the protein in A) or B), wherein the protein in A), B) and C) has DNA polymerase activity.