Isothermal Linear Amplification for Uniform Nucleic Acid Libraries
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Solution Overview
Problem
Current nucleic acid sequencing methods, particularly next-generation sequencing technologies, face challenges due to amplification biases and errors inherent in PCR, which compromise the accuracy and clinical utility of sequencing data, especially in multiplex reactions.
Innovation Solution
A method employing isothermal linear amplification with primers that prevent exponential amplification biases, using techniques such as 3′ product hairpin priming, enzymatic removal of primers, or quadruplex formation to generate dsDNA fragments, combined with cycled linear amplification to increase yield while minimizing errors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If PCR amplification is used to generate large quantities of target nucleic acid amplicons, then amplification yield is improved, but amplification bias increases
Solution Approach 1:
The patent changes the fundamental parameter of amplification from exponential (PCR) to linear (LAMP), transforming the mathematical model of nucleic acid amplification. This parameter change fundamentally alters the behavior of the system, eliminating the exponential growth that causes bias while maintaining sufficient yield through linear accumulation over multiple cycles.
Solution Approach 2:
The patent implements continuous linear amplification through multiple cycles of LAMP, where each cycle continuously produces linear amounts of product. This continuous action over multiple cycles accumulates sufficient yield while maintaining uniformity, as each cycle operates independently with the same linear kinetics rather than compounding exponential growth.
2Quantity of substance
If PCR amplification cycles are increased to improve yield, then quantity of amplicons is improved, but error accumulation increases
Solution Approach 1:
The patent changes the amplification parameter from exponential to linear, which fundamentally alters error propagation. In linear amplification, errors do not compound exponentially as they do in PCR, thereby maintaining sequence accuracy even as yield increases through multiple cycles.
Solution Approach 2:
The patent uses continuous linear amplification over multiple cycles, where each cycle independently produces linear amounts of product. This continuous linear process maintains error rates at acceptable levels while achieving sufficient yield, unlike PCR where errors accumulate exponentially with each cycle.
3Manufacturing precision
If isothermal linear amplification is used to reduce bias, then amplification uniformity is improved, but amplification speed decreases
Solution Approach 1:
The patent employs continuous linear amplification through multiple cycles, where each cycle continuously produces linear amounts of product. This continuous operation maintains uniformity while achieving sufficient yield, and the process can be optimized for speed through enzyme selection and condition optimization.
Solution Approach 2:
The patent changes the amplification parameter to linear mode with optimized reaction conditions, allowing the process to achieve both uniformity and acceptable speed. The linear mechanism inherently provides uniformity, and speed is optimized through parameter selection in the linear amplification protocol.
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 approach results in less biased and more accurate nucleic acid amplification with reduced error accumulation, achieving high fidelity and uniform amplification yields comparable to PCR but with fewer cycles, thus improving the reliability of sequencing data.
Implementation Method 1
contacting each target in the sample with a nucleic acid polymerase and a primer
Implementation Method 2
The primer may comprise a precursor of a quadruplex-forming sequence
Implementation Method 3
a 5'-3'-duplex dependent exonuclease
Data Source
AI summary
The present invention relates to a kit and a method of linear amplification of a least one nucleic acid target in a sample, said method comprising: (a) contacting each target in the sample with a nucleic acid polymerase and a primer comprising a component preventing copying of the primer by the nucleic acid polymerase; and at least one nuclease blocking nucleotide; (b) generating a primer extension product; (c) preventing priming by the 3′-end of the primer extension product, and (d) repeating steps b) and c) at least once.