LASSO Probe Assembly via DNA Recombinase
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Solution Overview
Problem
Previous methods for generating mature LASSO probes result in significant amounts of discordant probes and DNA artifacts, leading to reduced capture efficiency and purity, particularly limiting their use in complex eukaryotic genomes like the human genome.
Innovation Solution
The method involves DNA recombinase mediated assembly that omits the self-circularization step and initial fusion PCR, using a double-stranded pre-LASSO probe and a linear pLASSO vector to generate mature ssDNA LASSO probes with specific ligation and extension arms, ensuring high specificity and purity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If self-circularization step and initial fusion PCR are used in LASSO probe assembly, then the assembly process can be completed, but discordant probes and DNA artifacts are generated leading to reduced capture efficiency and purity
Solution Approach 1:
The patent extracts and removes the problematic self-circularization step and initial fusion PCR from the assembly process. By replacing these steps with a streamlined recombinase-mediated assembly approach, the method eliminates the generation of discordant probes and DNA artifacts while maintaining assembly completion, thereby resolving the contradiction between ease of manufacture and manufacturing precision.
Solution Approach 2:
The patent introduces a recombinase-mediated assembly mechanism as an intermediary process to replace the traditional self-circularization and fusion PCR steps. This intermediary approach enables precise and specific assembly of LASSO probes without generating harmful byproducts, thus improving probe purity while maintaining manufacturing feasibility.
2Productivity
If self-circularization step is used in LASSO probe assembly, then probe formation can occur, but intermolecular ligation of different LASSO probe precursors generates discordant probes
Solution Approach 1:
The patent removes the self-circularization step that causes intermolecular ligation of different LASSO probe precursors. By extracting this problematic step and replacing it with a more controlled assembly method, the patent maintains productive probe formation while eliminating the generation of discordant probes that reduce capture efficiency.
Solution Approach 2:
The patent implements a controlled assembly mechanism with built-in specificity checks through recombinase-mediated recombination. This feedback mechanism ensures that only correct intramolecular ligation events proceed, preventing the formation of discordant probes from intermolecular ligation while maintaining overall productivity.
3Productivity
If two consecutive PCR steps are used in LASSO assembly, then probe amplification can be achieved, but DNA polymerase errors and probe-probe fusion products are introduced
Solution Approach 1:
The patent extracts and eliminates the two consecutive PCR steps from the assembly process. By removing these amplification steps, the patent prevents the introduction of DNA polymerase errors and probe-probe fusion products, thereby maintaining sequence accuracy while still achieving the necessary probe production through alternative means.
Solution Approach 2:
The patent introduces a recombinase-mediated assembly process as an intermediary that replaces the need for consecutive PCR amplification. This intermediary mechanism achieves probe production with high sequence accuracy by avoiding the error-prone PCR amplification steps, thus resolving the contradiction between productivity and manufacturing precision.
4Ease of manufacture
If traditional LASSO assembly method is used, then probe generation can proceed, but significant reduction of capture efficiency occurs due to discordant probes
Solution Approach 1:
The patent extracts and removes the self-circularization step that is responsible for generating discordant probes. By eliminating this step while maintaining an feasible assembly process through recombinase-mediated assembly, the patent preserves manufacturing ease while significantly improving capture efficiency by preventing discordant probe formation.
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 results in a pure population of mature LASSO probes with significantly improved capture efficiency, enabling effective targeting and sequencing of kilobase-sized DNA fragments in complex genomes.
Implementation Method 1
DNA recombinase mediated assembly
Implementation Method 2
annealing to target sequences that flank a desired DNA fragment
Data Source
AI summary
Methods of generating mature ssDNA LASSO probes using DNA recombinase mediated assembly are provided. Also provided are mature ssDNA LASSO probes made by the methods, methods of their use, and kits including such.