Adaptor Hybridization for Nucleic Acid Ligation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The formation of adapter-dimers during the ligation of adaptors to target nucleic acids interferes with subsequent processing steps, skewing amplification and making quantitation of nucleic acid libraries difficult, particularly in sequencing applications.
Innovation Solution
A method involving the hybridization of adaptors to form a hybridized second adaptor with a single-stranded 3′-end, which is then coupled to the target nucleic acid, utilizing pseudo-intramolecular interactions and specific enzymes like ligases to minimize dimer formation and enhance ligation efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional ligation methods are used to attach adaptors to target nucleic acids, then ligation efficiency can be improved by using enhanced ligation buffer and longer incubation time, but adaptor-dimers are formed which skew amplification and make quantitation difficult
Solution Approach 1:
The adaptor is divided into two separate adaptors: a first adaptor that attaches to the target nucleic acid, and a second adaptor that hybridizes to the first adaptor and then attaches to the target. This segmentation prevents the formation of adaptor-dimers by ensuring that the two adaptor components are added in a controlled sequence rather than simultaneously, thereby resolving the contradiction between maintaining high ligation efficiency and preventing harmful dimer formation.
Solution Approach 2:
The first adaptor is attached to the target nucleic acid in a preliminary step before the second adaptor is introduced. This preliminary action establishes a foundation that guides the subsequent hybridization and attachment of the second adaptor, ensuring that ligation occurs efficiently on target molecules while preventing premature dimer formation between free adaptor molecules.
2Device complexity
If adaptors are ligated to both ends of target nucleic acids in a single reaction, then library preparation can be simplified, but adaptor-dimers form and distort sequence representation
Solution Approach 1:
The single ligation reaction is segmented into two sequential steps: first adaptor attachment followed by second adaptor hybridization and attachment. This segmentation maintains relative simplicity in the overall workflow while dramatically improving sequence representation accuracy by preventing adaptor-dimer formation that would otherwise distort the library.
Solution Approach 2:
The first adaptor serves as an intermediary between the target nucleic acid and the second adaptor. It mediates the interaction by first binding to the target and then providing a hybridization site for the second adaptor, ensuring that both adaptors are attached to genuine target molecules rather than forming dimers with each other.
3Loss of time
If standard ligation protocols are used, then the process is simple and quick, but adaptor-dimers skew amplification results
Solution Approach 1:
The ligation process is segmented into two rapid sequential steps rather than one extended step. The first adaptor attaches quickly to the target, then the second adaptor hybridizes and attaches in a second quick step. This segmented approach maintains overall process speed while eliminating the time during which adaptor-dimers would form and compromise amplification accuracy.
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 significantly reduces or prevents the formation of adaptor-dimers, reducing bias and amplification issues in nucleic acid libraries, accelerating ligation kinetics, and improving the efficiency of library preparation for sequencing.
Implementation Method 1
A portion of a second adaptor is hybridized to a portion of the coupled first adaptor to form a hybridized second adaptor
Implementation Method 2
The hybridized second adaptor is coupled to a second end of the target nucleic acid to form an adaptor-flanked product
Data Source
AI summary
Methods of coupling adaptors to a target nucleic acid include coupling a first adaptor to a first end of the target nucleic acid to form a coupled first adaptor. A portion of a second adaptor is hybridized to a portion of the coupled first adaptor to form a hybridized second adaptor having a single-stranded 3′-end. The hybridized second adaptor is coupled to a second end of the target nucleic acid to form an adaptor-flanked product having at least a part of the first adaptor coupled to the first end of the target nucleic acid and at least a part of the second adaptor coupled to the second end of the target nucleic acid. These methods can minimize the formation of adaptor-dimers that may be problematic in subsequent complementary nucleic acid strand synthesis, amplification, and sequencing.