Double-Stranded Splint Adaptors for Library Circularization
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Solution Overview
Problem
Existing nucleic acid library preparation methods face challenges in efficiently forming covalently closed circular molecules for downstream amplification and sequencing workflows, particularly due to the lack of effective methods for circularizing linear library molecules.
Innovation Solution
The use of double-stranded splint adaptors that hybridize with library molecules to form nicks, which are then ligated to create covalently closed circular molecules, facilitating downstream amplification and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional library preparation methods are used, then the process is simpler, but the efficiency of forming covalently closed circular molecules is low
Solution Approach 1:
The patent introduces double-stranded splint adaptors as intermediary molecules that facilitate the circularization of linear library molecules. These splint adaptors hybridize to the library molecules and provide a scaffold for ligation, enabling efficient formation of covalently closed circular molecules without requiring complex multi-step protocols
Solution Approach 2:
The method performs preliminary hybridization of the double-stranded splint adaptors to the library molecules before ligation. This preliminary action positions the adaptors correctly and creates the necessary structure for efficient circularization, improving overall process efficiency
2Reliability
If existing circularization methods are used, then the protocol is established, but the reliability of forming covalently closed circular molecules is insufficient
Solution Approach 1:
The patent changes key parameters of the circularization process by using double-stranded splint adaptors with specific hybridization properties and ligation conditions. These parameter changes include the use of temperature-controlled hybridization and optimized ligation buffers, which significantly improve the reliability of forming covalently closed circular molecules
Solution Approach 2:
The double-stranded splint adaptors serve as reliable intermediary structures that ensure proper alignment and stabilization during the circularization process, increasing the reliability of forming covalently closed circular molecules
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 enhances the efficiency of forming covalently closed circular molecules, improving the reliability and effectiveness of nucleic acid library preparation for sequencing workflows.
Implementation Method 1
The double-stranded splint adaptors can hybridize to portions of library molecules to form library-splint complexes having nicks
Implementation Method 2
the nicks can be ligated to form covalently closed circular molecules
Data Source
AI summary
The present disclosure provides compositions comprising nucleic acid double-stranded splint adaptors, including kits, and methods that employ the double-stranded splint adaptors. The double-stranded splint adaptors (200) can be used in a one-pot, multi-enzyme reaction to introduce one or more new adaptor sequences into a library molecule. The double-stranded splint adaptor (200) comprises a first splint strand (long splint strand (300)) and a second splint strand (short splint strand (400)), where the first and second splint strands are hybridized together to form the double-stranded splint adaptor (200) having a double-stranded region and two flanking single-stranded regions. The second splint strand (400) carries the new adaptor sequence(s) to be introduced, such as for example a universal binding sequence and/or an index sequence.