DNA-tagged Methanol-Responsive Polymer for ssDNA Production
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Solution Overview
Problem
Current methods for producing high-purity, long single-stranded DNA (ssDNA) are expensive and involve cumbersome protocols, limiting their accessibility and scalability for applications in diagnostics and therapeutics.
Innovation Solution
A method using a DNA-tagged methanol-responsive polymer (MeRPy) in PCR, which allows for the selective recovery of ssDNA through denaturing precipitation and site-specific cleavage, enabling the production of high-purity ssDNA molecules ranging from 89 to 3315 nucleotides in length.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If standard PCR methods are used to produce single-stranded DNA, then DNA amplification is achieved, but the protocol becomes cumbersome and purification becomes expensive
Solution Approach 1:
A methanol-responsive polymer (MeRPy) is introduced as an intermediary component attached to the forward primer during PCR. This polymer acts as a selective tag that enables simple methanol precipitation to separate the desired single-stranded DNA from reaction components, transforming a complex purification process into a straightforward procedure.
Solution Approach 2:
The invention changes the chemical parameters of the PCR system by incorporating a polymer-tagged primer. This modification allows the use of methanol precipitation conditions that selectively precipitate the polymer-DNA complex while leaving other components in solution, enabling easy separation and purification of the single-stranded DNA product.
2Manufacturing precision
If commercial methods are used to obtain high-purity long ssDNA, then purity and yield are improved, but cost increases significantly
Solution Approach 1:
The invention uses a disposable methanol-responsive polymer tag that is inexpensive and single-use. The polymer is attached to the primer, performs its purification function through simple precipitation, and is then discarded, replacing expensive commercial purification reagents and procedures with a low-cost alternative.
Solution Approach 2:
By changing the solubility parameters through methanol addition, the invention achieves high-purity ssDNA separation based on differential precipitation. The polymer-tagged DNA precipitates selectively at specific methanol concentrations, allowing purification without expensive commercial kits while maintaining high purity and yield.
3Measurement precision
If selective recovery of ssDNA strands is achieved through denaturing precipitation, then strand separation is improved, but the procedure becomes more complex
Solution Approach 1:
The methanol-responsive polymer serves as an intermediary that provides a simple physical basis for strand separation. By attaching the polymer to one strand and using methanol precipitation, the invention achieves selective recovery of individual strands through a single straightforward step rather than multiple complex operations.
Solution Approach 2:
The invention exploits phase transition principles through methanol-induced precipitation. Adding methanol changes the solvent phase properties, causing the polymer-tagged DNA strand to precipitate selectively while the other strand remains in solution, achieving simple and efficient strand separation.
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 method provides a rapid, scalable, and user-friendly approach to producing high-purity ssDNA, suitable for applications such as CRISPR/Cas9 homology-directed repair, DNA origami folding, and fluorescent in situ hybridization, with yields exceeding 50% and maintaining the integrity of the ssDNA.
Implementation Method 1
selective recovery of both strands from the amplicon, via denaturing precipitation
Implementation Method 2
site-specific creation and cleavage of an abasic site (AB-site)
Data Source
AI summary
Provided herein, in some embodiments, are methods and compositions for the production of long single-stranded DNA.