Polynucleotide Error Recognition via Endonuclease Composition
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Solution Overview
Problem
The challenge in synthetic biology is the production of a homogeneous population of polynucleotides with the desired sequence, as chemical synthesis often results in unwanted side reactions leading to a heterogeneous population with errors.
Innovation Solution
The use of an endonuclease composition comprising T4EndoVII and a mismatch endonuclease, such as EndoMS, to detect, reduce, and remove sequence errors in polynucleotides, thereby producing error-free DNA fragments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemical synthesis of oligonucleotides is performed, then polynucleotide production is achieved, but sequence errors and heterogeneity are introduced
Solution Approach 1:
The patent applies preliminary action by performing error detection and correction steps before final polynucleotide assembly and amplification. The method involves initial chemical synthesis followed by error identification through sequencing or other detection methods, then correcting errors before the heterogeneous population is amplified, preventing error propagation and ensuring high-fidelity final products
Solution Approach 2:
The patent converts the harmful effect of chemical synthesis errors into a beneficial process by using the detected errors as targets for correction. The method identifies sequence errors introduced during synthesis and systematically corrects them through various approaches (e.g., re-synthesis of error-containing oligonucleotides, error correction algorithms, or selective amplification), transforming the synthesis imperfection into an opportunity for quality control
2Productivity
If oligonucleotides with errors are used for assembly and amplification, then production speed is maintained, but heterogeneous DNA population is generated
Solution Approach 1:
The patent performs error detection and correction as a preliminary step before assembly and amplification. By identifying and correcting sequence errors in the oligonucleotide pool before these operations, the method ensures that only error-free sequences are assembled and amplified, maintaining both productivity and DNA population homogeneity
Solution Approach 2:
The patent implements feedback control by detecting sequence errors and using this information to guide correction processes. The detection results feed into correction strategies such as selective re-synthesis, error correction algorithms, or adjusted amplification conditions, creating a closed-loop system that maintains composition stability while preserving production efficiency
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 effectively increases the homogeneity of DNA populations by reducing or eliminating errors, resulting in a higher proportion of error-free clones and improved DNA authentication.
Implementation Method 1
contacting (a) dsDNA molecules comprising (i) heteroduplex dsDNA comprising at least one error and (ii) homoduplex dsDNA, (b) a T4EndoVII endonuclease, and (c) a mismatch endonuclease to form authentication products comprising uncut dsDNA molecules and/or dsDNA fragments corresponding to cleavage of heteroduplex dsDNA at the error(s)
Data Source
AI summary
The present disclosure relates, according to some embodiments, to systems, apparatus, compositions, kits, workflows, and/or methods that address errors in synthesizing and/or copying polynucleotide sequences. The present disclosure provides, for example, systems, apparatus, compositions, kits, workflows, and methods for detecting, reducing, and/or removing sequence errors including mismatches and/or indels. Enzyme composition may include, in some embodiments, a T4EndoVII endonuclease and a mismatch endonuclease, wherein the composition is cell-free, the composition has a temperature of 30° C. to 45° C., the composition comprises ≥1 unit of T4EndoVII per μL and ≥1 unit of μL of the mismatch endonuclease, the composition comprises ≥0.2 ng T4EndoVII per 20 μL and ≥7 ng mismatch endonuclease per 20 μL, the composition further comprises a buffering agent, and/or the mismatch endonuclease is EndoMS.