Solid-Phase Nucleic Acid Amplification Without Thermal Cycling
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Solution Overview
Problem
Current sequencing platforms require clonal amplification of template library molecules, which is costly and prone to non-specific amplification artifacts, and existing thermal cycling methods are inefficient and require expensive instrumentation.
Innovation Solution
A novel solid-phase amplification method using controlled thermal and chemical denaturant cycles, where template polynucleotides are annealed to immobilized primers, separated by a chemical denaturant at controlled temperatures, and extended by polymerase to generate complementary strands.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If rapid thermal cycling is used for nucleic acid amplification, then amplification efficiency is improved, but expensive instrumentation is required
Solution Approach 1:
The patent replaces the mechanical thermal cycling system with a chemical denaturation system. Instead of using temperature cycling to denature and anneal nucleic acids, the invention uses chemical denaturants (such as formamide or urea) to achieve strand separation at constant temperature, eliminating the need for expensive thermal cyclers while maintaining amplification efficiency
Solution Approach 2:
The invention changes the physical parameter from temperature cycling to chemical concentration control. By adjusting the concentration and type of chemical denaturant, the patent achieves nucleic acid denaturation and renaturation without requiring rapid temperature changes, thus replacing complex thermal instrumentation with simpler chemical reagent systems
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 that mediate the denaturation process at constant temperature. These chemicals act as intermediaries between the template and primers, controlling the timing and specificity of annealing events while maintaining isothermal conditions, thus preventing non-specific amplification artifacts
Solution Approach 2:
The invention implements periodic addition and removal of chemical denaturants to create cyclic denaturation-annealing conditions at constant temperature. This periodic chemical action mimics the beneficial effects of thermal cycling while maintaining isothermal conditions, improving amplification specificity without requiring complex temperature control instrumentation
3Device complexity
If chemical denaturant is used for strand separation, then thermal cycling equipment is eliminated, but additional chemical reagents are required
Solution Approach 1:
The patent employs a wash step that removes the chemical denaturant after strand separation, allowing the denaturant to be discarded. This approach minimizes the cumulative volume of chemical reagents needed, as the denaturant is used temporarily for denaturation and then removed, rather than being present throughout the entire amplification process
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 improves signal-to-noise ratio in sequencing by reducing non-specific amplification and eliminating the need for expensive thermal cycling equipment, enhancing sequencing quality and efficiency.
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.