Template-Free Double-Stranded Geometric Nucleic Acid Synthesis
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Solution Overview
Problem
Current DNA synthesis technologies are not adequately advanced to meet the increasing demand for long, error-free, and inexpensive DNA sequences, despite advancements in DNA sequencing technology.
Innovation Solution
The development of double-stranded geometric synthesis (gSynth) methods and compositions that utilize partially double-stranded nucleic acid molecules with specific 5' overhangs for efficient ligation, allowing for the production of long, error-free DNA sequences with high accuracy and speed.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional DNA synthesis technology is used, then current synthesis methods can produce DNA sequences, but the synthesis speed and accuracy are insufficient to meet the demand for long, error-free DNA sequences
Solution Approach 1:
The DNA synthesis process is divided into multiple overlapping fragments (e.g., 4-mer sequences) that are synthesized independently and then assembled through ligation. Each fragment contains specific 5' overhangs with unique 4-mer sequences that enable precise alignment and joining, allowing the entire long DNA sequence to be constructed from smaller, manageable segments with high accuracy and speed
Solution Approach 2:
The patent introduces specific 5' overhang sequences (4-mer sequences) as intermediary elements between DNA fragments. These overhangs act as mediators that facilitate accurate alignment and ligation between fragments, ensuring that the assembly process achieves both high speed and high accuracy by providing clear, unambiguous joining points
2Ease of manufacture
If template-free synthesis is used, then the synthesis process becomes simpler and faster, but maintaining high accuracy and purity of long DNA sequences becomes more challenging
Solution Approach 1:
The patent applies local quality by incorporating specific 4-mer sequences with unique 5' overhangs at precise locations within the DNA fragments. These localized unique sequences ensure that each fragment has distinct identification and alignment characteristics, preventing misalignment and ensuring high purity even in template-free assembly of long sequences
3Adaptability or versatility
If long DNA sequences are synthesized, then the utility and application range increase, but the complexity of ensuring error-free assembly increases
Solution Approach 1:
The DNA sequence is segmented into multiple overlapping fragments with standardized 4-mer overhang sequences. This segmentation reduces the complexity of synthesizing and assembling long sequences by breaking them into manageable pieces that follow a consistent, simple ligation protocol, thereby enabling the synthesis of very long sequences without proportionally increasing operational complexity
Solution Approach 2:
The patent creates a universal assembly system where the same 4-mer overhang sequence design and ligation protocol can be applied to assemble any long DNA sequence. The standardized approach provides multi-functionality, allowing the same method to generate various long sequences for different applications (genomics, synthetic biology, therapeutics) without requiring sequence-specific complex procedures
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
Enables the synthesis of nucleic acid sequences of unprecedented lengths with high purity and efficiency, surpassing existing synthesis techniques by ensuring accurate and rapid assembly of long, error-free DNA molecules.
Implementation Method 1
hybridizing the first and the at least second partially double-stranded nucleic acid molecules of the preceding compositions by hybridizing the second 5' overhang of first partially double-stranded nucleic acid molecule and the third 5' overhang of the at least second partially double-stranded nucleic acid molecule
Implementation Method 2
ligating the hybridized first partially double-stranded nucleic acid molecule and the at least second partially double-stranded nucleic acid molecule, thereby producing the target nucleic acid molecule. In some aspects, ligating comprises contacting the hybridized first and at least second partially double-stranded nucleic acid molecules and a ligase
Data Source
Figure 1A
Figure 1B
Figure 1C
AI summary
The present disclosure provides compositions and methods for template-free double stranded geometric enzymatic nucleic acid synthesis of arbitrarily programmed nucleic acid sequences.