Cren7 Chimeric Protein Enhances Polymerase Processivity
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Solution Overview
Problem
Current nucleic acid modifying enzymes, such as DNA polymerases, face limitations in processivity, salt tolerance, and amplification efficiency, particularly when dealing with low-quality DNA templates from sources like blood, food, and plants.
Innovation Solution
A chimeric protein is created by fusing a nucleic acid modifying enzyme domain with a Cren7 enhancer domain or its variant, which enhances the enzyme's activity, processivity, and salt tolerance, allowing for improved PCR amplification in challenging conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If conventional DNA polymerases are used, then basic PCR amplification can be performed, but processivity is limited resulting in inability to amplify long DNA fragments
Solution Approach 1:
The patent merges a DNA polymerase domain with a Cren7 domain to create a chimeric protein. The Cren7 domain, when fused to the polymerase, significantly enhances processivity and enables amplification of long DNA fragments (up to 20 kb) that conventional polymerases cannot handle effectively.
Solution Approach 2:
The chimeric protein combines two distinct functional domains: the DNA polymerase domain for catalytic activity and the Cren7 domain for enhanced DNA binding and processivity. This composite structure creates a enzyme with superior performance characteristics compared to either domain alone.
2Reliability
If standard PCR conditions are used, then amplification works for high-quality templates, but performance deteriorates with low-quality DNA templates from complex sources
Solution Approach 1:
The chimeric protein alters the functional parameters of the polymerase system by incorporating the Cren7 domain, which changes the enzyme's interaction with DNA substrates. This enables reliable amplification from low-quality templates containing inhibitors or degradation products that would normally prevent successful PCR.
Solution Approach 2:
The invention enables use of degraded or low-quality DNA templates that would otherwise be discarded. By making the most of suboptimal starting materials, the method extracts usable information from samples that would normally be considered unsuitable for amplification.
3Stability of the object's composition
If high salt concentrations are present, then physiological conditions are maintained, but DNA polymerase activity is inhibited
Solution Approach 1:
The fusion of Cren7 domain to the polymerase domain creates a chimeric protein that combines the catalytic function with enhanced environmental tolerance. The Cren7 domain confers salt resistance while the polymerase domain maintains catalytic activity, allowing function in high salt conditions.
4Productivity
If PCR amplification is attempted from low-quality DNA templates, then amplification may fail, but extending reaction time increases risk of non-specific products
Solution Approach 1:
The chimeric protein fundamentally changes the kinetic parameters of the PCR reaction by enhancing processivity. This allows efficient amplification from low-quality templates in shorter times, eliminating the trade-off between amplification efficiency and specificity that plagues conventional methods.
Data Source
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AI summary
There is provided a chimeric protein comprising a nucleic acid modifying enzyme domain having nucleic acid modifying activity joined with an Cren7 enhancer domain or variant thereof, in which the Cren7 enhancer domain or variant thereof enhances the activity of the nucleic acid modifying enzyme domain compared with a corresponding protein lacking the Cren7 enhancer domain or variant thereof. There is also provided an isolated nucleic acid encoding the chimeric protein of the invention and methods utilising the protein.