Circular Nucleic Acid Assembly Vectors with Annealable Linkers
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Solution Overview
Problem
Current methods for assembling DNA segments are time-consuming, laborious, and often introduce extraneous nucleic acid sequences, requiring careful selection of restriction sites and primer optimization, limiting the efficiency and accuracy of polynucleotide assembly.
Innovation Solution
The use of circular nucleic acid assembly vectors with annealable linkers and primer binding segments allows for the rapid and ordered assembly of polynucleotides through splicing by overlap extension (SOE) and PCR, eliminating the need for extensive primer optimization and minimizing intermediate product manipulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If traditional restriction enzyme and ligation methods are used for assembling DNA segments, then the assembly can be performed with standard enzymatic tools, but the process becomes time-consuming and laborious requiring multiple sub-cloning steps and screening
Solution Approach 1:
The patent divides the polynucleotide assembly process into modular components with standardized ends. DNA segments are designed with specific terminal structures that enable direct ligation without restriction enzymes, allowing parallel assembly of multiple segments simultaneously rather than sequential sub-cloning steps
Solution Approach 2:
The patent introduces standardized terminal structures as intermediaries between DNA segments. These standardized ends act as universal connectors that facilitate direct ligation without requiring restriction enzyme digestion and ligase treatment, eliminating multiple intermediate purification and screening steps
2Manufacturing precision
If restriction enzyme sites are selected for sticky-end ligation, then fragments can be joined with directional control, but the selection is limited by the compositions of the pieces being joined and requires careful consideration to avoid interrupting sequences of interest
Solution Approach 1:
The patent creates universal terminal structures that can be used across different DNA segments regardless of their internal sequences. These standardized ends provide consistent ligation interfaces that work with any fragment composition, eliminating the need to search for compatible restriction sites and avoiding interference with sequences of interest
3Ease of manufacture
If traditional ligation methods are used, then DNA fragments can be joined together, but extraneous nucleic acid sequences are introduced to the resulting clone that can interfere with the structure and function of desired gene products
Solution Approach 1:
The patent extracts and removes the problematic extraneous sequences (restriction sites, ligase recognition sequences, and other vector backbone elements) from the final assembled polynucleotide. The design allows ligation to occur without incorporating these interfering sequences into the functional regions of the gene product
4Adaptability or versatility
If the number of nucleic acid molecules to be ligated is increased, then more complex assemblies can be created, but the intrinsic limitation on the number of molecules that can be ligated together in a single reaction restricts efficiency
Solution Approach 1:
The patent merges multiple DNA segments with standardized terminals into a single ligation reaction mixture. The standardized ends enable all segments to be ligated simultaneously in one reaction without requiring sequential sub-cloning, dramatically increasing the number of fragments that can be assembled in parallel and improving overall productivity
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 enables efficient assembly of multiple polynucleotides into assembled polynucleotides with high accuracy and speed, reducing the need for labor-intensive steps and minimizing extraneous sequences, while allowing for the assembly of various types of polynucleotides, including synthetic genes and expression vectors.
Implementation Method 1
annealable linker sequences that flank a DNA segment within an assembly vector... Upon mixing and denaturation of these amplicons, strands having complementary sequences at their 3' ends overlap and act as primers for each other
Implementation Method 2
Extension of this overlap by DNA polymerase produces a nucleic acid molecule in which the original sequences are 'spliced' together
Implementation Method 3
Upon mixing and denaturation of these amplicons... By repeating the separation and synthesis steps in an automated system
Data Source
Figure 1A~1B
Figure 2
Figure 3
AI summary
The present invention provides compositions and methods for rapid assembly of one or more assembled polynucleotides from a plurality of component polynucleotides. The methods of the invention utilize circular nucleic acid vectors that comprise a DNA segment D flanked by an annealable linker sequence, annealable linker sequence pairs LA and LB, or annealable linker sequence / primer binding segment pairs LA and PB or PA and LB. Restriction endonuclease digestion of a plurality of vectors containing the DNA segments to be assembled generates a plurality of DNA fragments comprising the elements PA-D-LB, LA-D-LB, and LA-D-PB or D-LB, LA-D-LB, and LA-D. The sequences of annealable linker sequences LA and LB provide complementary termini to the DNA fragments, which are utilized in host cell mediated homologous recombination or together with promer binding segments PA and PB in a polymerase cycling assembly reaction for the ordered assembly of the various DNA segments into one or more assembled polynucleotides.