Controlled Strand-Displacement for Synchronized Paired-End Reads

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Solution Overview

Problem

Existing paired-end sequencing methods struggle to maximize the number of second strands with optimized lengths for sequencing and synchronize their production efficiently, leading to inefficiencies in read alignment and mutation detection.

Innovation Solution

The method involves controlled multiple displacement amplification (MDA) using extension primers and polymerases to generate second strands, synchronized by excisable nucleotides and extension-prevention conditions, ensuring optimal strand production and length synchronization.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If multiple displacement amplification is performed to generate second strands, then the quantity of second strands is increased, but the synchronization of second strand lengths deteriorates

Engineering Contradiction:

Improvenumber of second strandsVSAvoidsynchronization of second strand lengths

Core Design Contradiction:

Quantity of substanceVSManufacturing precision

Solution Approach 1:

The patent applies preliminary action by adding extension-prevention conditions (such as removing dNTPs or adding inhibitors) before the MDA reaction proceeds. This allows the polymerase to bind to templates and primers in advance, and then upon removal of prevention conditions, all extensions are initiated simultaneously, achieving both high yield and length synchronization of second strands.

Inventive Principle:

Principle #10Preliminary action

2Productivity

If strand-displacement polymerase is used for MDA, then the productivity of second strand generation is improved, but the control over extension timing deteriorates

Engineering Contradiction:

Improverate of second strand generationVSAvoidcontrol over extension timing

Core Design Contradiction:

ProductivityVSEase of operation

Solution Approach 1:

The patent applies preliminary anti-action by introducing extension-prevention conditions (such as dNTP depletion or inhibitor addition) that counteract the strand-displacement polymerase activity before the reaction should begin. This allows the high-productivity polymerase to be present and bound, but extension is blocked until prevention conditions are removed, providing both high productivity and precise timing control.

Inventive Principle:

Principle #9Preliminary anti-action

3Productivity

If second strands are produced without synchronization, then the productivity of sequencing is improved, but the accuracy of read alignment deteriorates

Engineering Contradiction:

Improvethroughput of sequencingVSAvoidaccuracy of read alignment

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent applies preliminary action by establishing extension-prevention conditions that allow all polymerase molecules to bind to their respective templates and primers before extension begins. When prevention conditions are removed, all second strands extend simultaneously to synchronized lengths, ensuring accurate read alignment while maintaining high throughput through the efficient MDA process.

Inventive Principle:

Principle #10Preliminary action

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 enhances the yield and efficiency of second strand production, improving read alignment and mutation detection by ensuring synchronized and optimized second strand lengths for sequencing.

Implementation Method 1

performing controlled multiple displacement amplification (MDA) by extending the first read strands or portions thereof with a polymerase having strand-displacement activity to generate a plurality of second strands

Methodology Applied

Scientific EffectStrand-displacement:

Implementation Method 2

extending first read primers hybridized to a plurality of single-stranded DNA concatemers immobilized on an array in the presence of excisable nucleotides to generate first reads of the plurality of single-stranded DNA concatemers, wherein said extending produces first read strands incorporating the exercisable nucleotides

Methodology Applied

Scientific EffectNucleotide excision:

Data Source

PatentUS12448648B2Controlled strand-displacement for paired end sequencing

Publication Date: 2025.10.21 MGI TECH CO LTD

AI summary

This application relates to methods and compositions used for paired-end sequencing. The method comprises providing a DNA array comprising a surface immobilized with DNA concatemers. For each of the plurality of DNA concatemers on the array, the method comprises annealing first read primers to primer binding sites on the DNA concatemer, extending at least some of the first read primers to incorporate dNTPs or dNTP analogs, thereby producing first read strands. Each of the dNTPs or dNTP analogs being incorporated is identified to produce first reads, performing controlled MDA by extending at least some of the first read strands with a polymerase having strand-displacement activity to generate a plurality of partially hybridized second strands. Second read primers are then annealed to the single-stranded branches of the plurality of second strands to generate the second reads.