Template Switching Oligonucleotide for mRNA 5' End Tagging

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Solution Overview

Problem

Current methods for adding terminal sequence tags to nucleic acid molecules are inefficient and biased, particularly for amplifying 5' ends of mRNA, and are not universally applicable to unknown sequences or single-stranded nucleic acids.

Innovation Solution

The use of oligonucleotides with a 5' overhanging portion containing a sequence tag and a hybridizing portion that can hybridize to the 3' end of a target nucleic acid molecule, allowing for the addition of a terminal sequence tag that serves as a template for polymerase extension, thereby facilitating amplification and identification of nucleic acid molecules.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If conventional methods (phosphatase treatment, cap removal, T4 RNA ligase) are used to add terminal sequence tags, then sequence tags can be added to mRNA 5' ends, but the process requires two hydrolytic steps causing mRNA loss and has low efficiency

Engineering Contradiction:

Improveterminal sequence tag addition accuracyVSAvoidmRNA loss

Core Design Contradiction:

Manufacturing precisionVSLoss of substance

Solution Approach 1:

The invention extracts and eliminates the harmful hydrolytic steps (phosphatase treatment and cap removal) from the conventional protocol. By using a template switching oligonucleotide that directly extends from the mRNA 5' end during reverse transcription, the method removes the need for these destructive pretreatment steps, thereby preventing mRNA loss while still achieving terminal sequence tag addition.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The template switching oligonucleotide serves as an intermediary that mediates the addition of terminal sequence tags. This oligonucleotide hybridizes to the 3' end of first-strand cDNA and provides a template for second-strand synthesis, thereby introducing the terminal sequence tag without requiring harmful hydrolytic treatments of the original mRNA.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Ease of manufacture

If T4 RNA ligase is used to add arbitrary sequences to mRNA, then terminal priming sites are provided, but the enzyme is less efficient with longer nucleic acid substrates and the process is complex

Engineering Contradiction:

Improveterminal priming site provisionVSAvoidligation efficiency

Core Design Contradiction:

Ease of manufactureVSProductivity

Solution Approach 1:

The invention substitutes the T4 RNA ligase enzymatic system with a template switching mechanism during reverse transcription. Instead of using ligase to join nucleic acid fragments, the method uses the natural template switching capability of reverse transcriptase to incorporate the terminal sequence tag, thereby achieving the same functional result with higher efficiency and simplicity.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Adaptability or versatility

If arbitrary sequences are added to 3' ends of cDNA using T4 RNA ligase, then 5' terminal sequences can be amplified, but the ligation is inefficient requiring extensive exponential amplification

Engineering Contradiction:

Improve5' terminal sequence amplification capabilityVSAvoidligation efficiency

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The invention performs the terminal sequence tag addition as a preliminary action during the first-strand cDNA synthesis step. The template switching oligonucleotide is incorporated into the first strand, and the terminal sequence tag is established before second-strand synthesis begins. This preliminary incorporation eliminates the need for subsequent inefficient ligation steps and extensive amplification.

Inventive Principle:

Principle #10Preliminary action

4Productivity

If conventional amplification methods are used, then nucleic acid sequences can be amplified, but exponential kinetics introduce bias in relative levels of different mRNAs

Engineering Contradiction:

Improvenucleic acid amplificationVSAvoidrelative abundance accuracy

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The invention uses linear amplification via RNA polymerase transcription as a copy mechanism to generate multiple copies of the cDNA template. This linear copying process, as opposed to exponential PCR amplification, maintains the relative abundance relationships of different mRNA species more accurately while still providing sufficient amplification for detection and analysis.

Inventive Principle:

Principle #26Copying

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 enables sensitive and universal amplification of nucleic acid sequences from the 5' ends, reducing sequence bias and improving the efficiency of nucleic acid amplification and identification, particularly for mRNA molecules.

Implementation Method 1

a hybridizing portion that can hybridize to the 3' end of a target nucleic acid molecule

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

allowing for the addition of a terminal sequence tag that serves as a template for polymerase extension

Methodology Applied

Scientific EffectPolymerase extension:

Data Source

PatentUS8304183B2Selective terminal tagging of nucleic acids

Publication Date: 2012.11.06 CELLSCRIPT LLC

AI summary

Methods are provided for adding a terminal sequence tag to nucleic acid molecules for use in RNA or DNA amplification. The tag introduced may be used as a primer binding site for subsequent amplification of the DNA molecule and/or sequencing of the DNA molecule and therefore provides means for identification and cloning of the 5′-end or the complete sequence of mRNAs.