Chimeric DNA Polymerase Domain Fusion for Fidelity and Yield
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Solution Overview
Problem
Existing DNA polymerases face challenges in achieving high amplification yield, specificity, continuous synthesis ability, extension rate, thermal stability, and salt resistance, particularly in demanding applications such as long fragment amplification, synthesis, and sequencing, with family A polymerases lacking fidelity and family B polymerases having poor continuous synthesis capability.
Innovation Solution
A chimeric DNA polymerase is created by combining peptide segments from different family B DNA polymerases, specifically 90N, KOD, Pfu, and Pwo, with optimized mutations to enhance properties like high yield, specificity, and salt tolerance, using recombinant cells or microorganisms to express and purify the chimeric enzyme.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If family B DNA polymerase is used to achieve high fidelity through 3'-5' exonuclease proofreading activity, then amplification accuracy is improved, but continuous synthesis ability and amplification yield are reduced
Solution Approach 1:
The patent divides the DNA polymerase into separate functional domains (exonuclease domain from family B for proofreading, polymerase domain from family A for high synthesis ability) and combines them to create a chimeric enzyme that independently performs both functions without mutual interference
Solution Approach 2:
The patent creates a composite chimeric DNA polymerase by fusing amino acid sequences from different polymerase families (e.g., N-terminal domain from Pfu, exonuclease domain from KOD, C-terminal domain from 90N), combining the advantages of each source polymerase into a single functional enzyme
2Productivity
If family A DNA polymerase is used to achieve high continuous synthesis ability and amplification yield, then productivity is improved, but amplification fidelity and proofreading capability are reduced
Solution Approach 1:
The patent merges the high-synthesis-capacity polymerase domain of family A DNA polymerases with the proofreading exonuclease domain of family B DNA polymerases, creating a chimeric enzyme that integrates both functionalities into a single molecular structure
Solution Approach 2:
The patent constructs composite chimeric polymerases by combining amino acid sequences from different families (e.g., Tca polymerase domain with Pfu exonuclease domain), creating a hybrid enzyme with properties superior to either parent polymerase alone
3Ease of operation
If standard DNA polymerase is used for general amplification, then ease of operation is maintained, but amplification specificity and performance under special conditions (high salt, low template) are reduced
Solution Approach 1:
The patent modifies specific amino acid residues in the polymerase structure (e.g., mutations in the finger domain or active site) to alter the enzyme's binding affinity and catalytic properties, enabling enhanced performance under specific conditions like high salt concentrations or with low template amounts while preserving ease of use
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 chimeric DNA polymerase achieves high amplification yield, specificity, and thermal stability, with improved resistance to salt, making it suitable for DNA amplification, synthesis, and sequencing, especially for long fragments, and has a broad application prospect.
Implementation Method 1
DNA polymerase is an enzyme able to synthesize (consequently to replicate), starting from 5′ end, a new DNA strand complementary to a sequence of a template strand... DNA polymerase with its polymerization activity enables additions of free nucleotides to 3′ end of the newly synthesized strand, leading to an extension of the same in the direction from 5′ to 3′ end
Implementation Method 2
some of DNA polymerases are of a 3′-5′ exonuclease activity, which can correct errors occurred during synthesis of the new DNA strand. if there is a mismatched base incorporated during PCR amplification, the DNA polymerases with 3′-5′ exonuclease activity would cut it off
Data Source
AI summary
Provided is a chimeric DNA polymerase, including: a first to seventh peptide segments, which have at least 80% homology with at least part of: the amino acid sequence in the N-terminal domain of a 9°N DNA polymerase; the amino acid sequence in the exonucleolytic domain of a KOD DNA polymerase; the amino acids in the N-terminal domain of the 9°N DNA polymerase; the amino acids in the palm domain of a KOD DNA polymerase; the amino acids in the finger domain of the Pfu DNA polymerase; the amino acids in the palm domain of the KOD DNA polymerase; and the amino acids in the thumb domain of the 9°N DNA polymerase, respectively.