Fusion Polymerase Enhances DNA Assembly Processivity
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Solution Overview
Problem
Current methods for increasing the processivity of DNA polymerases often face counterproductive effects due to the proofreading activity, and existing assembly techniques require non-strand displacing polymerases and crowding agents, which are inefficient and costly.
Innovation Solution
The use of a fusion protein combining a DNA polymerase with a sequence-specific DNA binding domain and a strand-displacing polymerase, along with a 5′-3′ exonuclease and a single-strand DNA binding protein, eliminates the need for crowding agents and non-strand displacing polymerases, enhancing processivity and assembly efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of moving object
If the processivity of a DNA polymerase is increased, then the ability to copy and amplify longer templates is improved, but the proofreading activity counteracts the positive effects
Solution Approach 1:
The patent divides the DNA polymerase into separate functional components: a high-processivity polymerase domain and a separate proofreading domain. This segmentation allows each domain to perform its specialized function optimally without interference, resolving the contradiction between processivity and fidelity
Solution Approach 2:
The patent creates a fusion protein that combines the high-processivity polymerase domain with the proofreading domain in a single molecular complex. This merging allows coordinated action where the polymerase domain synthesizes DNA rapidly while the proofreading domain simultaneously corrects errors, achieving both high processivity and high fidelity
2Duration of action of moving object
If a sequence-specific DNA binding domain is added to anchor the polymerase to the template, then processivity is improved, but the polymerase is prevented from moving to target sequences
Solution Approach 1:
The patent introduces a sequence-specific DNA binding domain that provides localized anchoring at specific target sequences. This creates a system where the polymerase can selectively bind to and process specific sequences with high processivity while maintaining the ability to move between different target sequences through the specific binding activity
Solution Approach 2:
The sequence-specific DNA binding domain acts as an intermediary between the polymerase and the DNA template. It mediates the interaction by providing specific sequence recognition and anchoring, allowing the polymerase to efficiently process target sequences without losing mobility to other targets
3Ease of manufacture
If non-strand displacing polymerases and crowding agents are used for DNA assembly, then assembly can be performed, but the process is inefficient and costly
Solution Approach 1:
The patent extracts and eliminates the requirement for crowding agents from the DNA assembly system. By using a strand-displacing polymerase with enhanced processivity, the invention achieves efficient DNA assembly without needing crowding agents, thereby improving productivity and reducing cost
Solution Approach 2:
The patent changes the key parameter of polymerase processivity to a level that enables efficient strand displacement and DNA assembly. This parameter change allows the reaction to proceed efficiently without requiring crowding agents, resolving the contradiction between ease of manufacture and productivity
Data Source
AI summary
This disclosure provides, among other things, a composition comprising: comprising a fusion protein comprising: (a) a DNA polymerase; and (b) a heterologous sequence-specific DNA binding domain. A method for copying a DNA template, as well as a kit for performing the same, are also described.