Fill-In Adaptor Ligation for Selective DNA Fragment Amplification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for attaching oligonucleotide adaptors to DNA fragments, particularly protein-bound DNA, suffer from low efficiency and contamination issues due to uneventful adaptor forms like dimers, which interfere with sequencing processes and consume reagents.
Innovation Solution
Design of fill-in adaptors with a single-stranded amplification sequence and a nicking site, allowing discrimination between free adaptors and those attached to DNA fragments through selective amplification, using a ribonucleotide to create a fork structure that only becomes functional upon ligation, and a strand-displacing polymerase to elongate the complementary strand.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If adaptor ligation is performed with excessive adaptor molecules to compensate for low efficiency, then ligation efficiency is improved, but adaptor contamination increases
Solution Approach 1:
The patent introduces a ribonucleotide at a specific position within the adaptor sequence, creating a structural parameter change that enables RNase H to specifically recognize and process adaptor-DNA hybrids. This parameter change allows differentiation between ligated adaptors (containing DNA- RNA hybrids) and free adaptors (lacking the hybrid structure), enabling selective amplification of desired products while eliminating contamination without requiring excess adaptor molecules
Solution Approach 2:
The patent employs RNase H as an intermediary enzyme that specifically degrades RNA in DNA-RNA hybrids. This intermediary selectively processes adaptor molecules that have successfully ligated to DNA fragments (creating hybrids) versus free adaptors, enabling discrimination and removal of unwanted adaptor forms through enzymatic mediation rather than relying on concentration-based approaches
2Ease of manufacture
If routine beads or column-based size selection methods are used to remove excessive free adaptor, then clean-up is performed, but small size differences prevent efficient removal
Solution Approach 1:
The patent changes the chemical composition parameter of the adaptor by incorporating a ribonucleotide, which creates a DNA-RNA hybrid structure upon ligation to DNA fragments. This structural parameter enables specific recognition by RNase H, providing a molecular fingerprint that distinguishes ligated adaptors from free adaptors based on composition rather than size, allowing precise removal of contamination through enzymatic specificity
Solution Approach 2:
The patent replaces the mechanical size-based separation system (beads or columns) with a biochemical recognition system using RNase H enzyme. Instead of relying on physical size differences that are too subtle for routine methods, the invention uses enzymatic recognition of the DNA-RNA hybrid structure, substituting mechanical separation with biochemical specificity to achieve precise discrimination and removal of free adaptor contamination
3Productivity
If adaptor dimers or concatemers are formed at high adaptor concentration, then ligation reaction proceeds, but these uneventful adaptor forms are difficult to remove
Solution Approach 1:
The patent modifies the adaptor structure by adding a ribonucleotide, creating a parameter change that enables RNase H to recognize and process only those adaptor molecules involved in DNA-RNA hybrid formation. This structural modification ensures that adaptor dimers and concatemers (which lack the hybrid structure) are differentially processed or removed, enabling selective elimination of these harmful forms while preserving desired ligation products
Solution Approach 2:
The patent uses RNase H as an intermediary enzyme that specifically targets RNA-containing structures. This intermediary selectively processes adaptor molecules based on their structural state: adaptor-DNA hybrids (desired product) versus adaptor dimers/concatemers (contamination). The enzyme mediates discrimination and removal of harmful forms through its specificity for the hybrid structure, enabling clean-up without affecting the desired ligation products
4Reliability
If fill-in adaptors with ribonucleotide are used, then selective amplification of ligated adaptors is enabled, but adaptor design complexity increases
Solution Approach 1:
The patent introduces a ribonucleotide at a specific position within the adaptor sequence, creating a minimal but critical parameter change. This single nucleotide modification enables RNase H to recognize the DNA-RNA hybrid structure formed upon ligation to DNA fragments, providing a molecular signature for selective amplification. The simplicity of this single parameter change (adding one ribonucleotide) achieves high reliability in discriminating ligated adaptors from free adaptors without requiring complex multi-component designs
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
Enhances ligation efficiency, reduces adaptor contamination, and allows more aggressive size selection, resulting in higher library diversity and improved mapping of transcription factor footprints.
Implementation Method 1
using a ribonucleotide to create a fork structure that only becomes functional upon ligation
Implementation Method 2
a strand-displacing polymerase to elongate the complementary strand
Implementation Method 3
a nicking site, allowing discrimination between free adaptors and those attached to DNA fragments through selective amplification
Data Source
AI summary
The present invention relates to the field of ligation of oligonucleotides to DNA fragments. The ligation methods of the invention may be used for attaching oligonucleotides comprising for example adaptors, primer binding sites, promoters, tags, barcodes or any combination of the aforementioned to DNA fragments.