Template Switching Oligonucleotide for Low-Input DNA Sequencing

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Solution Overview

Problem

Current DNA sequencing protocols are inefficient in detecting novel DNA fusion events, particularly from small amounts of starting material, due to time-consuming ligation steps that require large DNA quantities.

Innovation Solution

The use of a template switching mechanism to add adaptors to DNA sequences, allowing for efficient detection of fusion events by incorporating a template switching oligonucleotide (TSO) that switches templates and adds adaptor sequences to both strands of DNA, enabling PCR with target-specific primers and an adaptor-specific primer.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If traditional ligation-based protocols are used, then adaptor sequences can be added to DNA, but the process is time-consuming and requires large amounts of DNA

Engineering Contradiction:

Improveamount of starting materialVSAvoidtime for ligation steps

Core Design Contradiction:

Quantity of substanceVSLoss of time

Solution Approach 1:

The patent replaces the mechanical/chemical ligation process with a template switching mechanism that uses polymerase enzyme to incorporate adaptors. The TSO (template switching oligonucleotide) binds to the template DNA and directs polymerase to add adaptor sequences, eliminating the need for separate ligation steps and reducing both time and material requirements

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The template switching oligonucleotide (TSO) acts as an intermediary that facilitates adaptor addition. The TSO binds to the template DNA and serves as a bridge for the polymerase to transition from copying the template to adding the adaptor sequence, enabling efficient and low-material consumption adaptor ligation

Inventive Principle:

Principle #24Intermediary (Mediator)

2Ease of manufacture

If traditional ligation protocols are used, then adaptors can be added to DNA strands, but the workflow is complex and material-intensive

Engineering Contradiction:

Improveworkflow simplicityVSAvoidDNA quantity required

Core Design Contradiction:

Ease of manufactureVSQuantity of substance

Solution Approach 1:

The patent merges multiple functions into a single template switching reaction: the TSO simultaneously serves as a binding site for the polymerase, a template for adaptor synthesis, and a marker for strand identification. This consolidation eliminates multiple separate steps and reduces the total DNA material needed compared to traditional multi-step ligation protocols

Inventive Principle:

Principle #5Merging (Combining)

3Productivity

If conventional methods are used for detecting DNA fusion events, then fusion detection is possible, but efficiency is low especially with limited starting material

Engineering Contradiction:

Improvedetection efficiencyVSAvoidstarting material amount

Core Design Contradiction:

ProductivityVSQuantity of substance

Solution Approach 1:

The template switching mechanism creates a copy of the template DNA with adaptors added, allowing the original limited starting material to be amplified through subsequent PCR steps. This copying approach enables efficient detection of fusion events from minimal input material by generating sufficient copies for 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 method significantly reduces the amount of starting material needed, simplifies the workflow, and enhances the ability to detect novel fusion events, improving upon the inefficiencies of traditional ligation-based methods.

Implementation Method 1

the second adaptor sequence comprises a hybridization site for a template switching oligonucleotide (TSO)

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

a polymerase to extend the primer and add the adaptor sequence to the 3' end of the template

Methodology Applied

Scientific EffectDNA synthesis:

Data Source

PatentUS20220380823A1System and method for nucleic acid library preparation via template switching mechanism

Publication Date: 2022.12.01 KAPA BIOSYSTEMS INC

AI summary

The disclosure provides a composition comprising a double-stranded deoxyribonucleic acid (dsDNA) sequence comprising from 5′ to 3′, a sequence comprising a first adaptor sequence, a template sequence, and a second adaptor sequence, wherein the second adaptor sequence comprises a hybridization site for a template switching oligonucleotide (TSO). The disclosure provides methods for making the compositions of the disclosure using a template switching mechanism to add non-templated basepairs to the ends of a DNA molecule, hybridize a TSO to the non-templated basepairs, and then extend the sequence complementary to the TSO to add an adaptor.