RNA Amplification via Circular Adapter Fusion
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Solution Overview
Problem
Conventional RNA amplification methods suffer from gene loss and amplification bias due to RNA secondary structures and improper adapter ligation, leading to inefficient cDNA conversion and loss of directional information.
Innovation Solution
A method involving the formation of an adapter nucleic acid-RNA fusion construct, followed by self-ligation to create a circular construct, and subsequent amplification using primers complementary to the adapter sequence, which helps retain RNA directionality and minimize gene loss.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional reverse transcription with oligo dT or random primers is used, then RNA can be converted to cDNA, but cDNA conversion efficiency varies and gene loss occurs due to RNA secondary structures
Solution Approach 1:
The patent applies preliminary action by treating RNA with a nuclease to remove the 5' end before adapter ligation. This pre-processing step eliminates the 5' end that would otherwise form secondary structures and interfere with adapter binding, thereby improving cDNA conversion efficiency and reducing gene loss without requiring complex downstream processing
Solution Approach 2:
The patent uses an adapter nucleic acid as an intermediary element that bridges the processed RNA and the amplification primers. The adapter contains both a binding site for the processed RNA and a primer binding site, serving as a mediator that enables efficient and unbiased amplification while maintaining directional information
2Productivity
If adapters are ligated to cDNA ends, then amplification can be performed, but directional information is lost because adapters cannot distinguish between 5' and 3' ends
Solution Approach 1:
The patent introduces asymmetry by creating a non-palindromic adapter sequence with distinct 5' and 3' binding sites. The adapter contains a specific sequence that binds to the processed RNA 5' end and a different sequence that provides primer binding sites, thereby maintaining directional information while enabling amplification
Solution Approach 2:
Instead of using conventional adapters that bind to both ends symmetrically, the patent inverts the approach by using a adapter that binds specifically to the processed 5' end and carries primer binding sites in a configured manner that preserves directionality. This inversion allows the amplification to proceed in the correct directional manner
3Productivity
If PCR with specific sequence primers is used, then amplification can be performed, but amplification bias occurs depending on target sequence sizes and characteristics
Solution Approach 1:
The patent applies universality by designing the adapter nucleic acid to contain multiple functional elements: a binding site for the processed RNA, primer binding sites, and sequences that enable amplification. This multi-functional adapter serves as a universal platform that reduces amplification bias by providing consistent binding and amplification conditions across different target sequences
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 gene loss and amplification bias, allowing for accurate preservation of RNA directionality and enhancing the quality of cDNA libraries for genetic analysis.
Implementation Method 1
forming an adapter nucleic acid-RNA fusion construct by ligating the RNA to an adapter nucleic acid
Implementation Method 2
forming a circular adapter nucleic acid-RNA fusion construct by self-ligating the adapter nucleic acid-RNA fusion construct
Data Source
AI summary
A method of amplifying RNA in a sample and a method of amplifying a pool of RNA is provided. Gene loss and amplification bias may be reduced using the methods. Also, directionality may be preserved in the amplified RNA. The methods may be applied in sequence analysis as well as in general molecular diagnostic areas.