Nucleic Acid Library Construction via DOP-PCR Amplification
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Solution Overview
Problem
Current Next-Generation sequencing technologies face challenges in constructing nucleic acid libraries from single cells or trace amounts of nucleic acid samples, requiring significant initial DNA/RNA amounts and hindering applications such as single-cell or trace-sample sequencing.
Innovation Solution
A method involving DOP-PCR amplification followed by a second PCR using a DOP-Amp primer and adaptor-ligation PCR to construct a nucleic acid library, which includes using a kit containing DOP, DOP-Amp, and adaptor-ligation primers to facilitate library construction and sequencing, enabling the use of high-throughput sequencing platforms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional library construction methods are used, then sequencing can be performed with sufficient DNA/RNA amounts, but single cell or trace amount samples cannot be directly subjected to library construction
Solution Approach 1:
The patent applies preliminary action by performing DOP-PCR amplification before library construction to pre-amplify trace nucleic acid samples. This preliminary amplification step increases the quantity of template DNA/RNA from single-cell or trace levels to amounts sufficient for conventional library construction methods, thereby enabling sequencing applications that were previously impossible with such limited samples
Solution Approach 2:
The patent uses DOP-PCR amplification as an intermediary process between trace nucleic acid samples and library construction. The amplification step acts as a mediator that transforms incompatible sample quantities into compatible ones, allowing trace samples to be converted into sufficient quantities for standard library preparation protocols
2Quantity of substance
If DOP-PCR amplification is performed, then trace nucleic acid samples can be amplified, but the library construction procedure becomes more complex
Solution Approach 1:
The patent merges the DOP-PCR amplification step with the library construction process by using the same reaction体系和primers for both amplification and adapter ligation. This combination reduces the number of separate steps and simplifies the overall procedure, preventing the complexity that would normally arise from adding a separate amplification step
Solution Approach 2:
The patent applies universality by designing primers that serve multiple functions: they perform both the amplification of trace nucleic acid samples and the ligation of sequencing adapters simultaneously. This multi-functionality eliminates the need for separate amplification and adapter ligation steps, maintaining procedural simplicity while achieving both sample amplification and library construction
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 allows for the effective construction and sequencing of nucleic acid libraries from single cells or trace amounts, offering low-cost, high-sensitivity, high-accuracy, and repeatable results, suitable for various nucleic acid samples including single cells and trace DNA/RNA.
Implementation Method 1
subjecting a nucleic acid sample to a DOP-PCR amplification, to obtain a first PCR amplification product
Implementation Method 2
subjecting the second PCR amplification product to an adaptor-ligation PCR, to obtain a third PCR amplification product
Data Source
Figure 1
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Figure 4A~4D
AI summary
Provided are a method for preparing a nucleic acid library, a method for determining the nucleic acid sequence of a nucleic acid sample, and kits thereof. The method for preparing a nucleic acid library includes the following steps: amplifying a nucleic acid sample by DOP-PCR to obtain the first PCR amplification products; amplifying the PCR first amplification products secondly using DOP-Amp primers to obtain the second PCR amplification products; amplifying the second PCR amplification products by adaptor-ligation PCR to obtain the third PCR amplification products, which form the nucleic acid library.