Nucleic Acid Amplification With Small-Range Temperature Oscillation
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Solution Overview
Problem
Conventional PCR methods require expensive and complex thermal cycling instrumentation, while isothermal amplification techniques are slow and unreliable, making them unsuitable for low-cost, point-of-care nucleic acid diagnostics.
Innovation Solution
A method for nucleic acid amplification using oscillating temperature cycles with a variation of no more than 20°C, employing a reaction mixture with primers, DNA polymerase, and destabilizing agents, allowing for rapid and robust amplification without precise temperature control.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional PCR thermal cycling is used, then nucleic acid amplification is achieved, but expensive and complex thermal cycling instrumentation is required
Solution Approach 1:
The patent changes the temperature cycling parameters from conventional large幅度 cycling (e.g., 95°C to 55°C) to small-range oscillating cycling (e.g., 65°C to 75°C). This parameter change allows the use of simpler heating mechanisms without compromising amplification efficiency, as the destabilizing agents enable strand separation at lower temperature variations.
Solution Approach 2:
The patent introduces destabilizing agents (e.g., DMSO, formamide, betaine) as intermediaries that facilitate DNA strand separation at lower temperatures. These agents mediate the interaction between temperature and DNA stability, allowing amplification to proceed with reduced temperature cycling requirements and simpler instrumentation.
2Device complexity
If isothermal amplification techniques are used, then instrumentation complexity is reduced, but amplification speed decreases and reliability is compromised
Solution Approach 1:
The patent employs periodic temperature oscillations (small-range cycling) instead of strictly isothermal conditions. This periodic action maintains the simplicity of instrumentation while restoring amplification speed and reliability by periodically destabilizing DNA strands to enable primer binding and extension cycles.
Solution Approach 2:
The patent transitions from static isothermal conditions to dynamic oscillating temperature conditions. This dynamic approach allows the system to adapt between stabilization and destabilization phases, achieving both rapid amplification and robustness while requiring minimal instrumentation complexity.
3Reliability
If conventional PCR with large temperature cycling is used, then amplification is achieved, but precise temperature control is required
Solution Approach 1:
The patent changes the temperature parameters from large-range cycling requiring precise control to small-range oscillating cycling that is inherently more tolerant of temperature variations. The destabilizing agents further reduce the precision requirements by chemically facilitating strand separation.
Solution Approach 2:
The patent employs chemical destabilizing agents that can be added to the reaction mixture to temporarily reduce DNA stability during specific phases of the cycling process. These chemical helpers compensate for imprecise temperature control, making the system more robust and less dependent on sophisticated temperature regulation.
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
Enables efficient nucleic acid amplification in a cost-effective and portable manner, tolerant to temperature fluctuations, reducing the need for sophisticated instrumentation and improving sample preparation processes.
Implementation Method 1
A temperature of the reaction is oscillated between an upper temperature and a lower temperature wherein the change in temperature is no greater than about 20° C. during a plurality of temperature cycles
Implementation Method 2
A (generally thermostable) DNA polymerase is used to extend hybridized primers in the 5′→3′ direction by adding deoxynucleoside-triphosphates (dNTPs) in order to 'copy' the target sequence
Implementation Method 3
A PCR reaction employs two oligonucleotide primers that hybridize to opposite strands of the DNA double helix either upstream (5′) or downstream (3′) of the target sequence to be amplified
Implementation Method 4
By cycling the temperature of the reaction mixture (typically 95° C. Celsius), the two strands of DNA can be separated at high temperature allowing them to serve as templates for further primer binding and polymerization
Data Source
AI summary
One embodiment of the present invention provides for a method for amplifying a template of nucleic acid target sequence contained in a sample. The method includes contacting the sample with an amplification reaction mixture containing a primer complementary to the template of nucleic acid target sequence. A temperature of the reaction is oscillated between an upper temperature and a lower temperature wherein the change in temperature is no greater than about 20° C. during a plurality of temperature cycles. The template of nucleic acid target sequence is amplified.