cDNA Library Construction via Probe-Mediated Inhibition of Reverse Transcriptase
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Solution Overview
Problem
Existing methods for constructing cDNA libraries are inefficient in reducing the content of highly expressed gene clones, often requiring additional steps and leading to nonspecific loss of lowly expressed gene cDNAs, making it difficult to obtain full-length clones of lowly expressed genes.
Innovation Solution
A method involving the use of a double-stranded DNA primer and a probe that inhibits the extension reaction of highly expressed genes during cDNA synthesis, allowing for the selective reduction of highly expressed gene clones by annealing the primer to mRNA with a cap structure and using a probe that binds to the target gene to prevent reverse transcriptase activity, followed by ligating the 3' end to the 5' end of the cDNA heteroduplex for circularization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If conventional cDNA library construction methods are used, then the library contains comprehensive gene representation, but highly expressed genes dominate the clone content, making it difficult to retrieve lowly expressed gene information
Solution Approach 1:
The patent applies preliminary anti-action by introducing a probe that specifically binds to highly expressed gene mRNAs before cDNA synthesis occurs. This probe prevents the reverse transcriptase from extending these abundant transcripts, thereby preemptively counteracting the problem of highly expressed genes dominating the library before it can happen during standard construction
Solution Approach 2:
The patent uses a probe as an intermediary molecule that mediates between the highly expressed gene mRNAs and the reverse transcriptase. The probe binds to the mRNA and physically blocks the enzyme, acting as a mediator that selectively inhibits cDNA synthesis from specific templates without affecting the overall library construction process
2Quantity of substance
If additional steps are added to remove highly expressed gene cDNA after library construction, then the content of highly expressed gene clones is reduced, but the number of construction steps increases and lowly expressed gene cDNAs are nonspecifically lost
Solution Approach 1:
The patent applies preliminary action by performing the selective inhibition of highly expressed gene cDNA synthesis at the very beginning of the library construction process, during the first-strand synthesis step. This eliminates the need for subsequent removal steps, as the problem is addressed proactively before the library is fully constructed
Solution Approach 2:
The patent merges the function of selective inhibition with the standard cDNA synthesis process itself. By incorporating the probe into the first-strand synthesis reaction mixture, the selective reduction of highly expressed genes is achieved as an integrated part of the construction process rather than as a separate subsequent step
3Quantity of substance
If self-association of cDNAs from highly expressed genes is used to remove them, then highly expressed gene clones are reduced, but multiple repeated procedures are required and the process becomes time-consuming
Solution Approach 1:
The patent applies preliminary action by preventing the formation of abundant cDNA templates from highly expressed genes before they can accumulate and require multiple removal cycles. The probe-mediated inhibition occurs during synthesis, eliminating the need for repeated enrichment procedures
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 results in a cDNA library with a significantly reduced content of highly expressed gene clones, allowing for efficient cloning of lowly expressed genes and improved gene information retrieval, particularly for gene diagnosis and pharmaceutical development.
Implementation Method 1
annealing at least one probe that binds to mRNA of the target gene so as to inhibit a reaction with reverse transcriptase
Implementation Method 2
synthesizing 1 st strand cDNA from the double-stranded DNA primer using reverse transcriptase
Implementation Method 3
ligating the 3' end to the 5' end of the cDNA heteroduplex for circularization
Data Source
Figure 1(A)~1(J)
Figure 2
Figure 3
AI summary
A method for efficiently constructing a cDNA library having a reduced content of cDNA clones derived from a highly expressed gene is provided. According to the method for constructing a cDNA library using a double-stranded DNA primer having oligo dT and mRNA as a template, the proportion of cDNA clones derived from a highly expressed gene in a cDNA library was decreased through coexistence with a probe having a property of binding to the mRNA of the highly expressed target gene so as to inhibit a cDNA extension reaction resulting from reverse transcriptase.