Nucleic Acid Clonal Amplification by Partial Denaturation

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Existing nucleic acid amplification methods, such as emulsion PCR and solid-phase amplification, are complex, require multiple reagents, and struggle to preserve clonal integrity while scaling to genome-scale sequencing, leading to inefficiencies and high costs.

Innovation Solution

A method involving a single reagent mixture for nucleic acid amplification that preserves clonal integrity by partially denaturing double-stranded nucleic acid molecules using temperature and chemical denaturants, followed by primer hybridization and extension reactions, which can be performed directly on a sequencing instrument without separate automation.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If emulsion PCR is used to amplify multiple clones in individual droplets, then clonal integrity is preserved, but the process complexity increases and scaling to genome scale becomes difficult

Engineering Contradiction:

Improveclonal integrityVSAvoidprocess complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent extracts the amplification reaction from the complex emulsion droplet system and performs it directly on the solid substrate where templates are already immobilized. This eliminates the need for emulsion formation, thermocycling of large volumes, and subsequent bead enrichment steps, while maintaining clonal integrity through spatial confinement of amplicons on the substrate.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent combines template attachment and amplification into a single integrated process on the solid substrate. Templates are immobilized on the substrate and amplified in situ without requiring separation into emulsion droplets, merging the compartmentalization function with the amplification reaction.

Inventive Principle:

Principle #5Merging (Combining)

2Reliability

If a large excess of beads is used in emulsion PCR to decrease polyclonal beads, then clonal integrity improves, but reagent volumes and processing time increase substantially

Engineering Contradiction:

Improveclonal integrityVSAvoidprocessing efficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent performs preliminary attachment of single templates to specific locations on the solid substrate before amplification. This preliminary spatial confinement ensures that subsequent amplification produces only monoclonal amplicons at each location, eliminating the need for excess beads and post-amplification enrichment steps.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent creates multiple copies of the template at the same spatial location on the substrate through amplification. This in-situ copying produces monoclonal populations without requiring physical separation or enrichment, as each location serves as an independent reaction chamber.

Inventive Principle:

Principle #26Copying

3Reliability

If solid-phase amplification methods are used to preserve clonal integrity, then molecular integrity is maintained, but the requirement for multiple reagents and automation increases complexity

Engineering Contradiction:

Improvemolecular integrityVSAvoidautomation requirement

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent uses a universal solid substrate that serves multiple functions: template attachment, amplification reaction platform, and amplicon confinement structure. This multi-functional substrate eliminates the need for separate emulsion formation, thermocycling, and enrichment steps, reducing automation requirements while maintaining molecular integrity.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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

The method allows for rapid, efficient amplification of nucleic acid samples while maintaining clonal integrity, reducing the need for multiple reagents and automation, and is suitable for sample preparation in sequencing applications like cancer detection.

Implementation Method 1

subjecting the double-stranded nucleic acid molecule to conditions sufficient to partially denature the double-stranded nucleic acid molecule

Methodology Applied

Scientific EffectThermal denaturation: Melting

Implementation Method 2

subjecting the double-stranded nucleic acid molecule to conditions sufficient to partially denature the double-stranded nucleic acid molecule using temperature and chemical denaturants

Methodology Applied

Scientific EffectChemical denaturation:

Implementation Method 3

followed by primer hybridization and extension reactions

Methodology Applied

Scientific EffectNucleic acid hybridization:

Data Source

PatentUS12378593B2Nucleic acid clonal amplification and sequencing methods, systems, and kits

Publication Date: 2025.08.05 ULTIMA GENOMICS INC

AI summary

The present disclosure provides methods and systems for processing nucleic acid samples. Methods for processing a nucleic acid sample may comprise providing a double-stranded nucleic acid molecule comprising a partially denaturable region; partially denaturing the partially denaturable region of the double-stranded nucleic acid molecule, thereby generating a region comprising two single strands; and hybridizing a priming sequence to a sequence of one of the single strands. The methods described herein may facilitate amplification without the need for a multitude of complex steps or numerous reagents.