Hairpin Oligonucleotides Prevent Adapter Dimer Formation
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Solution Overview
Problem
The formation of adapter dimers during the ligation of adapters to target nucleic acid sequences reduces the efficiency of amplification and sequencing reactions, as these dimers do not contain target nucleic acids and consume amplification components, thereby limiting the amplification of adapter-target-adapter nucleic acids and producing useless information in sequencing techniques.
Innovation Solution
The method involves contacting target nucleic acid sequences with 5′ and 3′ adapters in the presence of hairpin oligonucleotides to reduce adapter dimer formation, where the hairpin oligonucleotides bind to the adapters, thereby forming 5′-adapter-target-3′-adapter sequences, and subsequent ligation with second adapter oligonucleotides to form library sequences, which can be amplified and sequenced.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If adapters are ligated to target nucleic acid sequences, then amplification and sequencing can be performed, but adapter dimers are formed which consume amplification components and reduce reaction efficiency
Solution Approach 1:
The patent applies preliminary action by performing a pre-ligation step where first adapter oligonucleotides are ligated to target nucleic acid sequences before the main ligation step. This preliminary ligation creates intermediate products that are then used as templates in the subsequent ligation with second adapter oligonucleotides, thereby preventing adapter dimer formation by establishing proper adapter-target complexes first
Solution Approach 2:
The patent segments the adapter ligation process into two distinct steps: first ligation (pre-ligation) and second ligation (main ligation). The first ligation attaches first adapters to targets, and the second ligation attaches second adapters to the first ligation products. This segmentation allows each step to be optimized independently and prevents premature dimer formation by maintaining proper molecular complexes throughout the process
2Quantity of substance
If standard adapter ligation is performed, then libraries can be prepared, but adapter dimers consume amplification components and limit amplification of adapter-target-adapter sequences
Solution Approach 1:
The pre-ligation step creates stable adapter-target complexes before the main ligation reaction, ensuring that amplification components are reserved for productive adapter-target-adapter sequences rather than being wasted on adapter dimers. This preliminary establishment of proper complexes maximizes the efficiency of subsequent amplification
Solution Approach 2:
The first ligation products serve as intermediaries between the target nucleic acids and the second adapters. These intermediate complexes act as mediators that guide the second ligation step to produce only useful adapter-target-adapter sequences, preventing direct adapter-dimer interactions that would waste amplification resources
3Loss of information
If adapters are ligated to target sequences, then amplification can proceed, but adapter dimers produce useless sequencing information
Solution Approach 1:
By performing preliminary ligation to establish correct adapter-target associations before final adapter attachment, the method ensures that only sequences containing actual target nucleic acids proceed to amplification and sequencing. This preliminary step filters out potential dimer formations that would generate useless sequencing data
Solution Approach 2:
The patent converts the potential harm of adapter dimer formation into a benefit by using the first ligation step to selectively create productive complexes. The conditions and design of the first ligation are optimized to favor adapter-target binding over adapter-dimer binding, thereby turning what would be a harmful side reaction into a beneficial selection step
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 adapter dimer formation, allowing for the preparation of libraries with low levels of dimers, enhancing the efficiency of amplification and sequencing reactions by ensuring that a higher percentage of adapters form adapter-target-adapter sequences, thereby improving the yield of useful nucleic acid information.
Implementation Method 1
contacting a sample comprising target nucleic acid sequences with 5′ and 3′ adapters in the presence of one or more hairpin oligonucleotides under conditions to form 5′-adapter-target-3′-adapter sequences
Implementation Method 2
contacting first adapter oligonucleotides with a sample comprising target nucleic acid sequences under conditions to form first ligation products
Data Source
AI summary
Provided herein is a method of reducing adapter dimer formation comprising contacting a sample comprising target nucleic acid sequences with 5′ and 3′ adapters in the presence of one or more hairpin oligonucleotides. Also provided is a method of preparing a library of nucleic acid sequences comprising contacting first adapter oligonucleotides with a sample comprising target nucleic acid sequences under conditions to form first ligation products, contacting the sample with one or more hairpin oligonucleotides that binds to the first adapter oligonucleotides, and contacting the sample with second adapter oligonucleotides under conditions to bind to the first ligation products and form second ligation products, wherein the second ligation products form the library of nucleic acid sequences.