Single-Stranded DNA Synthesis Using Uracil Template Segmentation
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Solution Overview
Problem
Conventional methods for synthesizing single-stranded DNA struggle to produce sequences longer than 80 bases with acceptable purity, limiting their application in techniques requiring longer DNA strands or increased molecular weight/ionic charge.
Innovation Solution
A method involving the use of a template molecule with uracil bases to anneal and then digest, allowing the assembly of longer target DNA molecules through ligation or polymerization, followed by enzymatic separation using uracil-N-glycosylase, enabling the production of longer, purer DNA sequences with increased molecular weight or ionic charge.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If conventional nucleotide polymerization methods are used to synthesize longer DNA sequences, then the length of the DNA strand increases, but the purity of the resulting product decreases due to increased errors in the desired sequence
Solution Approach 1:
The patent divides the long target DNA sequence into multiple shorter segments (e.g., 50-80 base pairs each), which are synthesized separately using conventional methods. These segments are then ligated together to form the complete long DNA molecule. This segmentation allows each segment to be synthesized with high purity using standard techniques, while the overall length is achieved through assembly of multiple segments.
Solution Approach 2:
The patent uses a template DNA molecule as an intermediary carrier. The template contains the complete desired sequence and serves as a guide during the ligation and polymerization processes. By annealing short synthesized segments to complementary regions on the template, the correct sequence is ensured, and the template is eventually removed by uracil-N-glycosylase digestion. This intermediary approach maintains sequence accuracy while enabling long DNA synthesis.
2Adaptability or versatility
If the DNA sequence length is increased beyond 80 bases, then more complex applications become possible, but the conventional synthesis methods can no longer produce acceptable purity
Solution Approach 1:
The patent applies segmentation by dividing long DNA sequences into multiple manageable segments that can be synthesized by conventional methods. Each segment is typically 50-80 base pairs long, and multiple segments are ligated together to achieve the desired long sequence length for applications such as multi-loci gene sequencing, while maintaining acceptable purity through controlled synthesis and ligation processes.
Solution Approach 2:
The template DNA molecule serves as an intermediary that guides the assembly of long DNA sequences. The template contains the complete desired sequence and facilitates the annealing of short synthesized segments to the correct positions. After ligation, the template is removed by uracil-N-glycosylase, leaving the purified long DNA product. This intermediary mechanism enables high-purity synthesis of long sequences for diverse applications.
3Reliability
If uracil-N-glycosylase digestion is used to remove the template molecule, then the template is selectively removed, but additional purification steps may be required
Solution Approach 1:
The patent extracts the template DNA molecule from the reaction mixture by selective digestion with uracil-N-glycosylase. The template contains uracil bases (replacing thymine), which are recognized and cleaved by the enzyme, removing the template while leaving the target DNA intact. This extraction method provides reliable template removal and can be combined with standard purification techniques such as gel electrophoresis or column chromatography to achieve high purity.
Solution Approach 2:
The patent utilizes parameter changes in the enzymatic digestion process. Uracil-N-glycosylase specifically targets uracil-containing DNA under controlled conditions (temperature, pH, enzyme concentration), allowing selective removal of the template. By optimizing these parameters, efficient template removal is achieved while minimizing impact on the target DNA. Additional purification steps can be adjusted based on these parameter optimizations to balance reliability with process 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 allows for the reliable synthesis of single-stranded DNA molecules exceeding conventional length limits while maintaining purity, enabling applications such as multi-loci gene sequencing and improved separation techniques.
Implementation Method 1
annealing the at least one part of the target molecule to the template molecule
Implementation Method 2
digesting the template molecule with a digestion enzyme specific to molecules containing uracil bases, e.g. uracil-N-glucocylase
Data Source
AI summary
A method of producing single-stranded DNA. In one form, the method involves providing a uracil-containing oligonucleotide template molecule having a sequence that is complementary to a part of a target single-stranded DNA molecule of length greater than the template molecule; providing one or more parts of the target molecule including a base sequence complementary to a part of the template molecule; annealing the part(s) of the target molecule to the template molecule and forming the complete target molecule by ligating together at least two adjacent parts of the target molecule while annealed to the template molecule, and/or extending at least one part of the target molecule to form a sequence complementary to a remainder of the template molecule by nucleotide polymerization, and then separating the template molecule from the target molecule. In another form, an intermediate molecule is annealed to the template and then enzymatically cut, and one part is then extended by DNA polymerization using monomers of increased molecular weight or ionic charge compared to the monomers used to form the intermediate molecule.