Blocking Oligonucleotide Reduces Adapter Dimer in RNA Ligation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for characterizing RNAs face challenges with adapter dimer formation during ligation reactions, leading to significant background noise and reduced yield in cloning and sequencing processes, as excess adapters can form dimers that interfere with subsequent reactions.
Innovation Solution
A method involving a first ligation between a 3′ adapter and a single strand polynucleotide, followed by the addition of a blocking oligonucleotide to prevent dimer formation with a 5′ adapter, allowing for efficient ligation without adapter dimer contamination, and enabling the use of adapters in excess without reducing yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If an excess of adapters is used in ligation reactions, then the ligation efficiency is improved, but adapter dimer formation increases leading to background noise and reduced yield
Solution Approach 1:
A blocking oligonucleotide is introduced as an intermediary substance that binds to the 3' adapter, preventing it from forming dimers with the 5' adapter. This mediator allows the use of excess adapters for high ligation efficiency while eliminating the harmful dimer formation through competitive binding.
Solution Approach 2:
The blocking oligonucleotide is added after the first ligation step to preemptively prevent adapter dimer formation before the second ligation step. This preliminary anti-action stops the harmful effect (dimer formation) from occurring in the first place, allowing excess adapters to be used without penalty.
2Object-generated harmful factors
If gel purification is used to remove excess 3' adapter, then background noise is reduced, but adapter yield and productivity decrease
Solution Approach 1:
The blocking oligonucleotide serves as a selective intermediary that binds to excess 3' adapters in solution, preventing them from participating in harmful dimer formation. This allows the desired ligated products to be purified efficiently while maintaining high adapter yield, as the blocking oligo specifically targets only the free 3' adapters without affecting the ligated complexes.
3Object-generated harmful factors
If a spanning oligonucleotide is used to block cDNA synthesis of adapter dimer, then dimer background is reduced, but the yield of ligated target RNA decreases due to adapter consumption
Solution Approach 1:
The blocking oligonucleotide acts as a selective intermediary that binds to 3' adapters to prevent dimer formation, unlike the spanning oligonucleotide that consumes adapters. This mediator approach reduces dimer background without depleting adapters needed for target RNA ligation, thereby maintaining high yield of ligated target RNA.
Solution Approach 2:
The blocking oligonucleotide provides local quality control by specifically binding to free 3' adapters in the solution phase, preventing them from forming dimers. This localized action at the 3' adapter binding site prevents harmful effects without interfering with the 5' adapter ligation to target RNA, maintaining high productivity.
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 effectively reduces adapter dimer formation, enhancing the yield of ligated target RNA and minimizing background noise, thereby improving the efficiency of cloning, sequencing, and amplification processes.
Implementation Method 1
adding a blocking oligonucleotide for hybridizing to the 3' adapter so that the 3' adapter is no longer capable of being ligated to a 5' adapter
Implementation Method 2
performing a first ligation between a 3' end adapter and a single strand polynucleotide to form a first ligation product
Data Source
AI summary
Methods are provided for ligating a 3′ adapter and a 5′ adapter to a target polynucleotide so as to avoid adapter dimer formation. Embodiments of the methods include adding a blocking oligonucleotide after the first ligation in which a 3′ adapter is ligated to the target polynucleotide so that the blocking oligonucleotide is capable of hybridizing to excess 3′ adapter and the ligated 3′ adapter. Subsequently, a 5′ adapter is ligated to the target polynucleotide thus avoiding adapter dimer formation.