Adaptor-Invaded DNA Cleavage for Circular Library Prep
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Solution Overview
Problem
Current methods for molecular biology, such as sequencing library preparation, face challenges in efficiently adding adaptors to DNA fragments for manipulation and sequencing, particularly in producing circular DNA molecules for amplification and sequencing.
Innovation Solution
A method involving hybridization of genomic DNA to an adaptor with a double-stranded and single-stranded region, followed by flap endonuclease cleavage, ligation, and intramolecular ligation to form a circular DNA molecule, which can be enzymatically processed for amplification and sequencing using PCR primers binding to the adaptor's sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional adaptor ligation methods are used, then adaptors can be added to DNA fragments, but the process is inefficient and complex
Solution Approach 1:
The adaptor structure enables self-service through the flap endonuclease mechanism. The 5' overhang of the adaptor invades the double-stranded DNA, displaces a flap, and the flap endonuclease automatically cleaves the displaced flap to create a ligatable end. This self-cleaving mechanism eliminates the need for separate complex enzymatic steps, thereby improving productivity while reducing process complexity.
Solution Approach 2:
The adaptor is segmented into distinct functional regions: a 5' overhang region for DNA invasion and flap displacement, a double-stranded region for ligation, and a 3' end for circularization. This segmentation allows each region to perform its specific function efficiently, improving the overall adaptor addition process while maintaining manageable complexity through modular design.
2Adaptability or versatility
If adaptor is added to DNA fragment for sequencing library preparation, then manipulation capability is provided, but additional steps are required for circularization and amplification
Solution Approach 1:
The method merges multiple functions into a single integrated process: adaptor hybridization, flap displacement, endonuclease-mediated cleavage, and ligation all occur in one reaction体系. The intramolecular circularization step further combines fragmentation and circularization functions. This merging improves productivity by eliminating sequential steps while maintaining the adaptability needed for sequencing library preparation.
Solution Approach 2:
The flap endonuclease acts as an intermediary that facilitates the conversion of linear adaptor-DNA hybrids into ligatable circular structures. This intermediary enzyme mediates the complex transformation required for efficient sequencing library preparation, enabling both manipulation capability and high productivity through its specific catalytic activity.
3Manufacturing precision
If linear adaptor-DNA hybrids are produced, then adaptor is attached to genomic DNA, but further processing steps are needed for amplification
Solution Approach 1:
The adaptor is designed with pre-positioned 5' overhangs that are primed for immediate flap displacement and cleavage upon hybridization to double-stranded DNA. This preliminary configuration of the adaptor structure allows the reaction to proceed directly to ligatable products without requiring additional activation steps, thereby maintaining manufacturing precision while reducing total processing time through streamlined intramolecular 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
This method enables efficient production of adaptor-ligated DNA for sequencing and amplification, facilitating whole genome or targeted sequencing applications by generating circular DNA molecules that can be processed using standard PCR techniques.
Implementation Method 1
cleaving the substrate using the flap endonuclease to produce a product comprising the adaptor and a flap endonuclease-generated fragment of the genomic DNA
Implementation Method 2
ligating the recessed end of the double stranded region to the fragment to produce an adaptor-ligated DNA
Implementation Method 3
intramolecularly ligating the ends of the adaptor-ligated DNA to produce a circular DNA molecule
Implementation Method 4
amplifying the circular DNA molecule using PCR with primers that bind to those sites
Data Source
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AI summary
This disclosure provides method for adding an adaptor to a genomic sequence by invasive cleavage, as well as a kit for performing the method. In some embodiments, the method comprises: a) hybridizing genomic DNA to an adaptor comprising a double stranded region and a single stranded region comprising a 5' overhang to produce a substrate for a flap endonuclease; b) cleaving the substrate using the flap endonuclease; c) ligating the recessed end of the double stranded region to the fragment to produce an adaptor-ligated DNA; d) intramolecularly ligating the ends of the adaptor-ligated DNA to produce a circular DNA molecule; and e) enzymatically processing the circular DNA molecule using an oligonucleotide that hybridizes to the adaptor and an enzyme. A kit for performing the method is also provided.