Strand-Displacing Polymerase Synthon Assembly
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for assembling double-strand DNA molecules require non-strand displacing polymerases and crowding agents, which are inefficient and costly, and lack the ability to assemble polynucleotides in a single step without gaps or nicks.
Innovation Solution
A composition comprising a 5' exonuclease, a strand-displacing polymerase, a single-strand DNA binding protein, and a non-naturally occurring buffering agent, which excludes crowding agents and non-strand displacing polymerases, enabling efficient assembly of polynucleotides into synthons in a single step by filling gaps left after molecule annealing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If non-strand displacing polymerases and crowding agents are used for DNA assembly, then the assembly can be performed, but the efficiency is low and the cost is high
Solution Approach 1:
The patent changes the fundamental parameter of polymerase selection from non-strand displacing to strand-displacing polymerase. This parameter change eliminates the need for crowding agents and significantly improves assembly efficiency while reducing costs, as the strand-displacing polymerase inherently provides the necessary functionality without requiring additional expensive additives.
2Reliability
If non-strand displacing polymerases are used, then the assembly process can proceed, but gaps and nicks remain after annealing
Solution Approach 1:
The strand-displacing polymerase performs multiple functions simultaneously: it extends the 3' ends of annealed oligonucleotides, displaces any remaining single-stranded regions, and fills gaps without requiring separate processing steps. This multi-functionality achieves both high assembly accuracy and true single-step assembly capability.
3Productivity
If strand-displacing polymerase is used with 5'-3' exonuclease, then assembly efficiency increases, but the combination differs from prior art teachings
Solution Approach 1:
The patent merges the functions of 5'-3' exonuclease and strand-displacing polymerase into a single reaction mixture without crowding agents. The 5'-3' exonuclease prepares the ends by removing 5' flaps, while the strand-displacing polymerase simultaneously extends and displaces strands, creating a streamlined system that improves efficiency despite the enzymatic complexity.
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
This approach increases the efficiency and accuracy of polynucleotide assembly, allowing for the production of functional genes and synthons with high yield and cost-effectiveness, reducing the need for sequencing duplicates and minimizing errors.
Implementation Method 1
a 5' exonuclease
Implementation Method 2
a strand-displacing polymerase
Implementation Method 3
a single strand (ss) DNA binding protein
Data Source
Figure 1A
Figure 1B~1E
Figure 2A
AI summary
This disclosure provides, among other things, a composition comprising a fusion protein, wherein the fusion protein comprises: a strand-displacing DNA polymerase, wherein the strand-displacing DNA polymerase has 3'-5' exonuclease activity; and a heterologous DNA binding domain. A kit and a method for polynucleotide assembly to form a synthon, are also described.