TOPO-adaptor DNA Library Preparation via Topoisomerase I

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Solution Overview

Problem

Current methods for preparing DNA libraries for Next Generation Sequencing (NGS) are time-consuming, prone to significant sample loss, and result in low coverage of genetic material, with issues such as adaptor dimer formation affecting the efficiency of sequencing.

Innovation Solution

The use of activated topoisomerase adaptors (TOPO-adaptors) for preparing DNA libraries, which reduces adaptor dimer formation and expedites the library preparation process by covalently linking adaptors to DNA duplexes, improving yield and reducing preparation time.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If ligase-only dependent linkage of standard oligonucleotide adaptors is used, then the library preparation process can be completed, but the process is time-consuming and results in low yield of library DNA product

Engineering Contradiction:

Improveyield of library DNA productVSAvoidpreparation time

Core Design Contradiction:

ProductivityVSLoss of time

Solution Approach 1:

The patent uses topoisomerase I as an intermediary enzyme to facilitate adaptor-DNA linkage. The topoisomerase I-adaptor complex acts as a mediator that enables covalent attachment of adaptors to DNA fragments through its endonuclease and ligase activities, replacing the traditional ligase-only approach and significantly improving reaction efficiency and yield

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent changes the enzymatic parameters by using topoisomerase I instead of traditional DNA ligase. This enzyme substitution alters the reaction mechanism, enabling faster adaptor-DNA linkage with higher yield. The topoisomerase I enzyme operates under different biochemical parameters that favor rapid covalent bond formation between adaptors and DNA fragments

Inventive Principle:

Principle #35Parameter changes

2Productivity

If standard oligonucleotide adaptors are used with ligase, then library preparation can proceed, but adaptor dimers are formed which reduce sequencing efficiency

Engineering Contradiction:

Improvesequencing efficiencyVSAvoidadaptor dimer formation

Core Design Contradiction:

ProductivityVSObject-generated harmful factors

Solution Approach 1:

Topoisomerase I acts as an intermediary that prevents adaptor dimer formation by specifically facilitating the attachment of adaptors to DNA fragments through a controlled enzymatic mechanism. The enzyme's specificity ensures that adaptors are linked only to DNA targets rather than to each other, eliminating the harmful dimer side product

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent converts the potential harm of adaptor self-ligation into a benefit by using topoisomerase I's unique mechanism. The enzyme's ability to create transient breaks and rejoin DNA in a controlled manner transforms what would be a harmful side reaction (adaptor dimer formation) into a beneficial specific ligation event between adaptor and DNA fragment

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

3Quantity of substance

If traditional library preparation methods are used, then sequencing can be performed, but the coverage of genetic material is low

Engineering Contradiction:

Improvecoverage of genetic materialVSAvoidlibrary preparation time

Core Design Contradiction:

Quantity of substanceVSLoss of time

Solution Approach 1:

The topoisomerase I-mediated ligation enables continuous and efficient adaptor-DNA linkage without the time losses associated with traditional ligase methods. The enzymatic reaction proceeds continuously with high efficiency, maintaining optimal conditions throughout the reaction to maximize library yield and genetic material coverage in reduced time

Inventive Principle:

Principle #20Continuity of useful action

Solution Approach 2:

The patent replaces the mechanical mixing and incubation process of traditional ligase-based library preparation with an enzymatic system driven by topoisomerase I. This substitution creates a more efficient biochemical reaction system that continuously produces high-quality library product with better coverage and faster kinetics

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

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 significantly enhances the yield and efficiency of DNA library preparation, reducing the time required for library preparation and minimizing adaptor dimer formation, leading to improved sequencing outcomes.

Implementation Method 1

a) attaching a topoisomerase (TOPO) enzyme to a 3' end of a adaptor DNA

Methodology Applied

Scientific EffectEnzyme-mediated bonding: Enzyme

Implementation Method 2

b) covalently linking the 5' end of a target DNA to the TOPO-DNA complex to form a TOPO-adaptor-target DNA complex

Methodology Applied

Scientific EffectPhosphodiester bonding: Chemical Bonding

Data Source

PatentUS11274333B2Compositions and methods for preparing sequencing libraries

Publication Date: 2022.03.15 MOLECULAR CLONING LABORATORIES MCLAB LLC

AI summary

Compositions comprising activated topoisomerase adaptors (TOPO-adaptors) and methods of using the activated TOPO-adaptors are provided for preparing a library of target DNA duplexes derived from sample polynucleotides (e.g., DNA, RNA) for the streamlined preparation of a large number of samples. Such libraries may be used for Next Generation Sequencing (NGS).