Solid-Phase Nucleic Acid Amplification Without Rapid Thermal Cycling
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Solution Overview
Problem
Current sequencing platforms require clonal amplification of initial template library molecules to create clusters, which is costly and prone to non-specific amplification artifacts, and existing thermal cycling methods are expensive and inefficient.
Innovation Solution
A novel solid-phase amplification method using chemical and thermal denaturant cycles, where template polynucleotides are annealed to immobilized primers at controlled temperatures, followed by chemical denaturation and primer extension, to generate complementary polynucleotides without the need for rapid thermal cycling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If rapid thermal cycling is used for nucleic acid amplification, then amplification efficiency is improved, but instrumentation cost and complexity increase
Solution Approach 1:
The patent replaces the mechanical thermal cycling system with a chemical denaturation system. Instead of using expensive thermal cyclers to denature DNA through temperature changes, the invention uses chemical denaturants (such as formamide or urea) to achieve DNA strand separation at constant temperature, thereby eliminating the need for complex thermal cycling instrumentation while maintaining amplification efficiency
Solution Approach 2:
The invention changes the physical parameter used for DNA denaturation from temperature (thermal cycling) to chemical composition (denaturant concentration). By using chemical denaturants at constant temperature, the system achieves the same DNA separation effect without requiring rapid temperature changes, thus reducing instrumentation complexity while preserving amplification productivity
2Device complexity
If isothermal nucleic acid amplification is used, then instrumentation cost is reduced, but non-specific amplification artifacts increase
Solution Approach 1:
The patent introduces chemical denaturants as intermediary substances to mediate the DNA denaturation process at constant temperature. These denaturants specifically disrupt hydrogen bonding between DNA strands without causing non-specific amplification, thereby maintaining amplification specificity while enabling isothermal conditions that reduce instrumentation cost
Solution Approach 2:
The invention implements periodic addition and removal of chemical denaturants in a controlled manner. By cycling the denaturant presence rather than maintaining constant isothermal conditions, the system achieves specific amplification while keeping instrumentation simple, as the periodic chemical action replaces the need for complex thermal control
3Measurement precision
If clonal amplification to create clusters is performed, then signal-to-noise ratio is improved, but amplification cost and time increase
Solution Approach 1:
The patent performs preliminary attachment of template molecules to the solid support surface before amplification begins. By pre-positioning templates on the surface and using surface-immobilized primers, the system creates localized amplification sites that immediately generate detectable signals without requiring extensive clonal expansion time, thus improving signal-to-noise ratio while reducing amplification time
Solution Approach 2:
The invention segments the amplification process into surface-immobilized localized reactions rather than bulk solution amplification. Each template molecule on the surface undergoes independent amplification, creating discrete signal sources that improve measurement precision. This segmented approach reduces the time required compared to traditional clonal amplification methods
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 reduces the cost and complexity of amplification, enhances signal-to-noise ratio, and improves sequencing accuracy by minimizing non-specific amplification artifacts.
Implementation Method 1
contacting the complement template polynucleotide and the template polynucleotide with a chemical denaturant at a second temperature thereby separating the complement template polynucleotide from the template polynucleotide
Implementation Method 2
wherein the second temperature is greater than the first temperature by 12° C. to 18° C.
Implementation Method 3
extending the first primer with a polymerase to generate a complement template polynucleotide
Implementation Method 4
annealing a template polynucleotide to a first immobilized primer on a solid support at a first temperature, wherein the first temperature is about 25° C. to about 45° C.
Data Source
AI summary
Disclosed herein, inter alia, are novel methods pertaining to nucleic acid amplification and detection. Devices, compositions, and kits for use in such methods are also provided.