Genomic DNA Amplification Primers for Direct Sequencing Libraries
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Solution Overview
Problem
Current whole-genome amplification methods are not compatible with second-generation sequencing technologies, requiring separate library construction and often result in poor sequencing yields due to complications and inefficiencies.
Innovation Solution
A method involving a specific primer structure and thermal cycle program for amplifying genomic DNA, using first and third primers with variable sequences and a nucleic acid polymerase, allowing direct library formation and high-quality sequencing results without loop or dimer formation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional PCR methods are used for DNA amplification, then DNA amplification can be achieved, but the process requires multiple temperature cycles and multiple reagent additions which increases complexity and time
Solution Approach 1:
The patent combines multiple PCR steps (denaturation, annealing, extension) and multiple reagent additions into a single isothermal amplification reaction. The master mix contains all necessary components (polymerase, nucleotides, buffers, and temperature-sensitive modifiers) that are normally added separately at different temperatures, thereby merging multiple operations into one simple step.
Solution Approach 2:
The patent changes the temperature parameter from cyclic variations to a constant isothermal condition. By using temperature-sensitive modifiers that change conformation or activity at the specific isothermal temperature, the method achieves temperature-dependent control without actual temperature cycling, simplifying the process while maintaining amplification efficiency.
2Productivity
If conventional PCR methods are used, then DNA amplification is achieved, but the time required for multiple temperature cycles is excessive
Solution Approach 1:
The patent eliminates periodic temperature cycling and replaces it with a single continuous isothermal reaction. The temperature-sensitive modifiers provide the necessary periodic-like control (through their temperature-dependent conformational changes) without requiring actual periodic temperature changes, thereby reducing total reaction time while maintaining amplification functionality.
Solution Approach 2:
The patent performs preliminary preparation of all reaction components at the target isothermal temperature. The master mix is prepared with all necessary ingredients and the temperature-sensitive modifiers are pre-configured to become active at the specific isothermal temperature, eliminating the need for subsequent temperature adjustments or additional reagent additions during the reaction.
3Manufacturing precision
If multiple reagents are added at different temperatures in conventional PCR, then specific amplification is achieved, but the risk of contamination increases
Solution Approach 1:
The patent merges all reagents into a single master mix that is prepared and added in one step. This eliminates multiple separate reagent addition steps that would otherwise require opening the reaction system multiple times, thereby reducing contamination risk while maintaining amplification specificity through the included temperature-sensitive modifiers.
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 direct library construction from amplified genomic DNA, reducing time and improving sequencing efficiency by minimizing loop and dimer formation, and ensuring compatibility with second-generation sequencing technologies.
Implementation Method 1
a temperature-sensitive modifier that changes conformation or activity in response to temperature changes
Implementation Method 2
DNA amplification using a polymerase and a pair of primers
Implementation Method 3
DNA amplification using a polymerase and a pair of primers
Data Source
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AI summary
The present application relates to a method of amplifying genomic DNA, comprising: (a) providing a first reaction mixture, wherein said first reaction mixture comprises a sample containing genomic DNA, a first primer, a mixture of nucleotide monomers, and a nucleic acid polymerase, wherein the first primer comprises, in a 5' to 3' orientation, a common sequence and a first variable sequence comprising a first random sequence; (b) placing the first reaction mixture in a first thermal cycle program to obtain a pre-amplification product; (c) providing a second reaction mixture, wherein said second reaction mixture comprises a pre-amplification product, a second primer, a mixture of nucleotide monomers, and a nucleic acid polymerase, wherein the second primer comprises or consists of, in a 5' to 3' orientation, a specific sequence and the common sequence; (d) placing the second reaction mixture in a second thermal cycle program for amplification, to obtain an amplification product. The present application also relates to a kit for amplifying genomic DNA.