Clonal Nucleic Acid Amplification by Controlled Partial Denaturation
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Solution Overview
Problem
Existing nucleic acid amplification methods, such as emulsion PCR and solid-phase amplification, face challenges in preserving clonal integrity, are complex, require multiple reagents, and are inefficient or difficult to scale for genome-scale sequencing, leading to significant limitations in amplifying nucleic acid molecules without loss of molecular integrity.
Innovation Solution
A method involving a single reagent mixture for nucleic acid amplification that preserves clonal integrity by using a controlled denaturation process with temperature-dependent melting points to separate and extend primer molecules, allowing for efficient amplification directly on a sequencing instrument without separate automation, using a support to immobilize adapters and primers for rapid amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If emulsion PCR is used to amplify multiple clones in individual droplets, then clonal integrity is preserved, but the process becomes complex and time-consuming with multiple steps including emulsion generation, thermocycling, disruption, and enrichment
Solution Approach 1:
The patent extracts the amplification process from the complex emulsion droplet system and transfers it to a solid surface, eliminating the need for emulsion generation, disruption, and enrichment steps while maintaining clonal integrity through surface-bound amplification
Solution Approach 2:
The patent replaces the mechanical emulsion droplet system with a solid-phase surface binding system, substituting the complex fluid mechanics of emulsion handling with simpler surface chemistry-based amplification
2Reliability
If emulsion PCR is used to amplify large numbers of nucleic acid molecules, then clonal integrity is preserved, but substantial volumes of reagents and a large number of beads are required
Solution Approach 1:
The patent extracts the amplification process from the bulk emulsion phase and concentrates it on a solid surface, dramatically reducing reagent volumes from milliliters to microliters or nanoliters while maintaining clonal integrity
Solution Approach 2:
The patent changes the physical state from liquid emulsion droplets to solid surface-bound molecules, enabling reduced reagent volumes and improved scalability for genome-scale sequencing
3Reliability
If solid-phase amplification methods are used to preserve clonal integrity, then molecular integrity is maintained, but the methods are inefficient or difficult to scale for genome-scale sequencing
Solution Approach 1:
The patent creates a universal solid-phase amplification platform that can handle both small and large scale amplification needs, enabling scalability from individual clones to genome-scale sequencing through the same basic mechanism
Solution Approach 2:
The patent optimizes parameters such as surface density, primer concentration, and thermal cycling conditions to achieve high amplification efficiency on solid surfaces, making the method scalable for genome-scale applications
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 enables rapid, efficient, and cost-effective amplification of nucleic acid samples while preserving clonal integrity, suitable for sample preparation in sequencing applications like cancer detection, with potential for high-throughput and reduced reagent usage.
Implementation Method 1
subjecting the double-stranded nucleic acid molecule to conditions sufficient to partially denature the double-stranded nucleic acid molecule, thereby separating the first sequence of the first strand from the third sequence of the second strand
Implementation Method 2
wherein the first sequence hybridized to the third sequence has a first melting point and the second sequence hybridized to the fourth sequence has a second melting point higher than the first melting point
Implementation Method 3
bringing a primer molecule having sequence complementarity with the third sequence of the second strand in contact with the second strand under conditions sufficient to permit the primer molecule to hybridize to the third sequence of the second strand
Implementation Method 4
subjecting the second strand comprising the primer molecule hybridized to the third sequence of the second strand to a primer extension reaction under conditions sufficient to generate a third strand hybridized to at least a portion of the second strand
Data Source
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.