Double-Stranded Splint Adaptors for One-Pot Circular Library Preparation
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Solution Overview
Problem
Existing nucleic acid library preparation methods face inefficiencies in introducing new adaptor sequences and forming covalently closed circular molecules for downstream amplification and sequencing workflows.
Innovation Solution
The use of double-stranded splint adaptors, comprising a first and second splint strand hybridized together, which hybridize to library molecules to form nicks that can be ligated, forming covalently closed circular molecules for amplification and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If existing nucleic acid library preparation methods are used to introduce adaptor sequences and form circular molecules, then the process requires multiple separate steps including adaptor ligation and gap filling, but this results in low efficiency and high complexity of the workflow
Solution Approach 1:
The patent combines multiple separate enzymatic steps (adaptor ligation and gap filling) into a single unified reaction by using a double-stranded splint adaptor structure that allows simultaneous ligation and gap filling in one pot, eliminating the need for separate purification steps between reactions
Solution Approach 2:
The double-stranded splint adaptor serves as an intermediary molecule that bridges the linear library molecule ends and provides the necessary structure for both ligation and gap filling to occur simultaneously, with the splint strands acting as temporary scaffolds that are later removed
2Productivity
If traditional linear library molecules are used for downstream amplification and sequencing, then the workflow is straightforward, but the efficiency of amplification and sequencing is reduced compared to covalently closed circular molecules
Solution Approach 1:
The patent performs preliminary gap filling during the adaptor ligation step itself, ensuring that the circular molecules are properly formed with nicks that can be efficiently ligated in subsequent steps, rather than attempting gap filling after circularization
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
Facilitates efficient introduction of new adaptor sequences and formation of covalently closed circular molecules, enhancing the efficiency of downstream amplification and sequencing processes.
Implementation Method 1
comprising a first and second splint strand hybridized together
Implementation Method 2
which hybridize to library molecules to form library-splint complexes having nicks
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.