Template Switching DNA Synthesis Eliminates Adaptor Ligation Bias
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Solution Overview
Problem
The use of RNA ligases for attaching oligonucleotide adaptors to RNAs is a time-consuming, expensive, and inefficient step in the cloning and sequencing of cDNAs corresponding to non-polyadenylated RNAs, and it leads to biased representation of cDNAs in constructed libraries due to nucleotide preferences of RNA ligases.
Innovation Solution
Employing non-retroviral reverse transcriptases that facilitate template switching to directly link adaptor sequences to target RNA or DNA sequences without the need for ligation, using methods like template switching from one or more adaptor sequences and non-templated nucleotide residues added to the 3' end of cDNAs, enabling PCR amplification and cloning.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If RNA ligases are used to attach adaptors to RNA templates, then adaptor ligation can be achieved, but the process becomes time-consuming, expensive, and inefficient with biased cDNA representation
Solution Approach 1:
The patent extracts and eliminates the RNA ligase step from the traditional workflow. By using non-retroviral reverse transcriptases with template switching capability, the method removes the need for separate adaptor ligation steps, directly synthesizing cDNA with adaptors incorporated through template switching rather than enzymatic ligation
Solution Approach 2:
The patent replaces the mechanical/enzymatic ligation system (RNA ligases requiring specific nucleotide preferences) with a biochemical template switching system. The reverse transcriptase enzyme naturally switches templates based on sequence complementarity rather than nucleotide preference, eliminating the bias introduced by ligase specificity
2Ease of manufacture
If RNA ligases with distinct nucleotide preferences are used for adaptor ligation, then adaptors can be attached to RNA, but biased representation of cDNAs results in constructed libraries
Solution Approach 1:
The patent changes the fundamental parameter governing adaptor attachment from nucleotide preference (ligase specificity) to sequence complementarity (template switching). By using reverse transcriptases that switch templates based on complementary base pairing rather than nucleotide identity, the method achieves unbiased incorporation of adaptors across all cDNA sequences
Solution Approach 2:
The patent introduces an intermediary mechanism - the template switching process mediated by reverse transcriptase - that bridges the adaptor and cDNA sequences. This intermediary process uses complementary base pairing as the selection criterion rather than nucleotide preference, ensuring equitable representation of all sequences
3Adaptability or versatility
If traditional adaptor ligation methods are used for non-polyadenylated RNA sequencing, then cloning and sequencing can be performed, but the process requires multiple time-consuming ligation steps
Solution Approach 1:
The patent merges the adaptor attachment function with the reverse transcription process itself. Instead of performing adaptor ligation as a separate step before or after reverse transcription, the method combines both functions into a single template switching event, eliminating sequential steps and reducing total processing time
Solution Approach 2:
The patent performs preliminary action by incorporating adaptor sequences into the template switching process before cDNA synthesis is complete. The reverse transcriptase switches to the adaptor-containing template during synthesis, pre-incorporating the adaptor sequence into the growing cDNA strand without requiring subsequent ligation steps
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 reduces the time and cost associated with adaptor ligation, minimizes biases in cDNA libraries, and allows for efficient detection, amplification, and sequencing of RNA and DNA sequences by directly linking adaptors to target sequences, improving the representation of cDNAs.
Implementation Method 1
a reverse transcriptase to extend the 3' end of the DNA primer oligonucleotide to provide a DNA copy polynucleotide that includes a target complementary DNA polynucleotide synthesized using the target polynucleotide as a template
Implementation Method 2
non-templated nucleotide addition to the 3' end of cDNAs
Implementation Method 3
template switching allows a DNA copy to be prepared using a reverse transcriptase that switches from an initial nucleic acid sequence template to the 3' end of a new nucleic acid sequence template
Data Source
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AI summary
A method of preparing a DNA copy of a target polynucleotide using template switching is described. The method includes mixing a double stranded template/primer substrate made up of a DNA primer oligonucleotide associated with a complementary oligonucleotide template strand with a target polynucleotide in a reaction medium and adding a suitable amount of a non- retroviral reverse transcriptase to the reaction medium to extend the DNA primer oligonucleotide from its 3' end to provide a DNA copy polynucleotide. The DNA copy polynucleotide includes a complementary target DNA polynucleotide that is synthesized using the target polynucleotide as a template. Methods of adding nucleotides to the double stranded template/primer substrate are also described. The method can be used to facilitate detection, PCR amplification, cloning, and determination of RNA and DNA sequences.