Indexed Splint Oligos for High-Specificity Long DNA Assembly
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Solution Overview
Problem
Current methods for generating DNA libraries are limited by the length of DNA molecules that can be synthesized, with chemical synthesis typically capped at 350 nucleotides, and enzymatic assembly methods suffer from poor hybridization specificity leading to low-quality libraries.
Innovation Solution
A method utilizing indexed splint oligonucleotides for enzymatic ligation of oligonucleotides in solution, where indexed splint molecules with specific index sequences and splint sequences hybridize to terminal ends of assembly components, ensuring precise alignment and ligation without requiring a solid support.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If chemical synthesis is used to generate DNA libraries, then the process is simple and direct, but the maximum length of DNA molecules is limited to 350 nucleotides
Solution Approach 1:
The patent divides long DNA sequences into shorter oligonucleotide fragments that can be chemically synthesized within the 350-nucleotide limit. These fragments are then assembled enzymatically using splint oligos to reconstruct the full-length DNA library, thereby overcoming the synthesis length limitation while maintaining manufacturing simplicity.
Solution Approach 2:
The patent performs preliminary chemical synthesis of oligonucleotide fragments before assembling them into full-length sequences. By pre-synthesizing manageable fragments and preparing splint oligos in advance, the method enables subsequent enzymatic assembly to generate long DNA molecules that would be impossible to synthesize directly.
2Length of moving object
If enzymatic assembly with single-stranded overhangs is used to generate longer DNA constructs, then the DNA length can exceed 350 nucleotides, but hybridization specificity is poor leading to low-quality libraries
Solution Approach 1:
The patent introduces splint oligos as intermediary molecules that facilitate specific hybridization between assembly components. These splint oligos contain sequences complementary to the assembly components and enable precise alignment through hybridization, thereby improving manufacturing precision while allowing enzymatic assembly of long DNA molecules.
Solution Approach 2:
The patent modifies hybridization parameters by using splint oligos with optimized sequences and structures that enhance binding specificity. By changing the hybridization conditions and using indexed splint designs, the method achieves high-specificity assembly of long DNA constructs without the poor hybridization specificity of traditional overhang methods.
3Manufacturing precision
If solid surface DNA arrays are used for enzymatic assembly, then spatial segregation of constructs is achieved, but the method requires solid support and is more complex
Solution Approach 1:
The patent extracts the spatial segregation function from solid surface arrays and implements it through soluble indexed splint oligos in solution. By removing the requirement for solid support while maintaining the ability to segregate and organize constructs spatially through molecular design, the method reduces device complexity while preserving manufacturing precision.
Solution Approach 2:
The patent uses indexed splint oligos as intermediaries that provide spatial organization and segregation of DNA constructs in solution without requiring solid surfaces. These splint molecules mediate the assembly process by bringing specific components together in defined spatial arrangements, achieving the benefits of solid arrays with the simplicity of solution-based methods.
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 assembly of longer DNA constructs with high specificity and accuracy, even in libraries with high sequence homology, allowing for the production of complex and low-homology libraries with minimal dropout constructs and improved read accuracy.
Implementation Method 1
indexed splint molecules with specific index sequences and splint sequences hybridize to terminal ends of assembly components
Data Source
AI summary
The present disclosure relates to a method of assembling long nucleic acids by enzymatically ligating oligonucleotide molecules hybridized to an indexed splint oligonucleotide molecules. Also disclosed are oligonucleotide structures comprising an indexed splint oligonucleotide useful in performing the disclosed method.