Phi29 DNA Polymerase Mutants Reduce Exonuclease Activity
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Solution Overview
Problem
The high 3'-5' exonuclease activity of phi29 DNA polymerase leads to primer degradation and reduced binding specificity, affecting reaction efficiency in amplification and sequencing, while current methods for determining this activity are costly and pose safety concerns.
Innovation Solution
Development of phi29 DNA polymerase variants with specific amino acid substitutions at positions 61, 96, 58, 94, 119, or 155, and fusion proteins with tags at the N-terminus or C-terminus, which reduce 3'-5' exonuclease activity while maintaining high fidelity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If phi29 DNA polymerase with high 3'-5' exonuclease activity is used, then high fidelity is ensured, but primer degradation occurs and binding specificity is reduced
Solution Approach 1:
The patent extracts or removes the harmful 3'-5' exonuclease activity from the phi29 DNA polymerase through site-directed mutagenesis of critical amino acid residues (D12, D66, Y165, D169) in the Exo-I, Exo-II and Exo-III motifs, while preserving the DNA polymerase activity and fidelity through compensatory mutations
Solution Approach 2:
The patent changes the biochemical parameters of the phi29 DNA polymerase by mutating specific amino acid residues to alter the enzyme's exonuclease activity level, transforming it from high to low activity state while maintaining polymerase function through optimized combination of mutations
2Object-generated harmful factors
If 3'-5' exonuclease activity is reduced through amino acid mutations, then primer degradation is prevented, but fidelity may be compromised
Solution Approach 1:
The patent applies local quality changes by making specific localized mutations at critical amino acid positions (D12N/D12E, D66N/D66E, Y165F/Y165L, D169N/D169E) in the exonuclease motifs to reduce exonuclease activity, while maintaining overall polymerase fidelity through the localized nature of these mutations
Solution Approach 2:
The patent creates mutant variants (copying the wild-type sequence with modifications) that replicate the essential polymerase function while eliminating the harmful exonuclease activity, producing copies with improved properties for amplification and sequencing applications
3Measurement precision
If radioisotope-labelled DNA substrate is used to determine 3'-5' exonuclease activity, then high accuracy is achieved, but cost increases and safety concerns arise
Solution Approach 1:
The patent replaces expensive and hazardous radioisotope labels with non-radioactive fluorescent labels or other safe detection methods, using disposable fluorescently labelled substrates that provide sufficient measurement precision without the safety and cost issues of radioisotopes
Solution Approach 2:
The patent substitutes the radioisotope-based detection system with alternative detection mechanisms such as fluorescent labeling, using optical detection instead of radioactive detection to measure exonuclease activity, thereby eliminating safety concerns and reducing cost while maintaining measurement accuracy
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 phi29 DNA polymerase variants ensure high fidelity and prevent primer digestion, enhancing reaction efficiency and specificity in amplification and sequencing processes without the need for costly radioisotope labeling.
Implementation Method 1
The phi29 DNA polymerase displays its 3′-5′exonuclease activity by continuously cutting the single-stranded DNA substrate longer than 6 nucleotides in the presence of Mg2+ ions as a metal ion activator
Implementation Method 2
the DNA-amplification active site can be initiated by a protein or DNA. Such a phi29 DNA polymerase has effective and persistent DNA-replicating ability thus being useful in isothermal amplification, whole-genome amplification under random primers, rolling circle amplification and the like
Data Source
AI summary
Provided are a phi29 DNA polymerase and an encoding gene and an application thereof. The phi29 DNA polymerase is C1) or C2): C1) is a protein with DNA polymerase activity obtained by substituting at least one of the 58th, 61st, 94th, 96th, 119th, and 155th amino acid residues in the amino acid sequence of a wild type phi29 DNA polymerase as shown in SEQ ID NO: 2 in the sequence listing; and C2) is a fusion protein obtained by linking a label to the N-terminus and/or C-terminus of the protein represented by C1). A 3′-5′exonuclease of the phi29 DNA polymerase has activity lower than that of the wild type phi29 DNA polymerase, and can efficiently and continuously synthesize DNA during amplification and sequencing.