Multiplex PCR Target Enrichment for Short cfDNA Fragments
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Solution Overview
Problem
Conventional methods, such as Ampliseq, are ineffective in enriching target regions in cellular free DNA due to short fragment lengths and require time-consuming PCR processes, limiting their application in high-throughput sequencing.
Innovation Solution
A method involving linker connection, PCR amplification, single-strand capture, and exponential amplification using specific primers and affinity labels allows for efficient enrichment of target regions in short DNA fragments, enabling pooling of samples before enrichment and reducing sequencing costs and time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional Ampliseq multiplex PCR is used for target region enrichment, then the enrichment process is simplified and time is reduced, but it cannot enrich target regions in cellular free DNA due to short fragment lengths
Solution Approach 1:
The patent modifies the PCR primer design to have different structural characteristics at different regions: the 5' end contains a universal binding site for amplification, while the 3' end contains sample-specific or target-specific sequences. This local differentiation allows the same primer to function both in amplifying short fragments and in specifically enriching target regions, resolving the contradiction between general amplification capability and specific target enrichment.
Solution Approach 2:
The enrichment process is divided into multiple sequential stages: initial multiplex PCR amplification of all fragments, followed by selective enrichment of target regions using tagged primers, and finally exponential amplification of enriched targets. This segmentation allows each stage to optimize for its specific function - the first stage handles short fragments efficiently, while subsequent stages provide target-specific enrichment.
2Manufacturing precision
If PCR amplification is performed before sample pooling to achieve region capture, then target regions can be enriched, but the process becomes time-consuming
Solution Approach 1:
The patent performs preliminary multiplex PCR amplification on all samples simultaneously before pooling, using universal primers that amplify all DNA fragments regardless of origin. This preliminary action creates sufficient material for subsequent target enrichment while maintaining all samples in an identical state, enabling efficient pooling and reducing overall processing time compared to individual sample processing.
Solution Approach 2:
The universal primers used in the initial PCR step can amplify any DNA fragment containing the linker sequence, making them applicable to all samples simultaneously. This universal amplification capability allows multiple samples to be processed in parallel before pooling and target enrichment, significantly reducing total processing time while maintaining capture accuracy.
3Quantity of substance
If high-throughput sequencing is performed on whole genome, then comprehensive coverage is achieved, but the cost becomes prohibitively high
Solution Approach 1:
The patent extracts and enriches only the specific target regions of interest from the entire genome using tagged primers that specifically bind to target sequences. By isolating and amplifying only these relevant regions before sequencing, the method reduces the total amount of DNA that needs to be sequenced, thereby lowering sequencing costs while maintaining comprehensive coverage of the target regions.
Solution Approach 2:
The patent changes the concentration and specificity parameters of primers during different amplification stages. In the initial multiplex PCR, low-specificity universal primers are used to amplify all fragments. In the enrichment stage, high-specificity tagged primers are used to selectively amplify only target regions. This parameter change enables cost-effective sequencing by focusing resources on only the relevant genomic regions.
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 effectively captures target regions in cellular free DNA, reduces sequencing costs, and increases throughput by enabling sample pooling prior to enrichment, while distinguishing between molecules with low mutation rates in high-depth sequencing.
Implementation Method 1
connecting a first linker and a second linker respectively to a terminal end of a nucleic acid fragment comprising a target region to be enriched under the action of a ligase
Implementation Method 2
PCR amplifying the linker-connected product with a first primer that specifically binds to the fixed sequence of the first linker and a second primer that specifically binds to the second linker
Implementation Method 3
capturing a single strand of the amplification product harboring the first affinity label via a solid-phase support, wherein the solid-phase support carries a second affinity label capable of affinity binding to the first affinity label
Implementation Method 4
performing single-primer linear amplification with a third primer using the captured single strand as template
Implementation Method 5
performing exponential amplification with the third primer and the first primer using a linear amplification product as template to obtain a product comprising the target region
Data Source
AI summary
Provided are a target region enrichment method based on multiplex PCR, and a reagent, the method comprising: connecting a first linker and a second linker respectively at two ends of a nucleic acid segment containing target regions to be enriched so as to obtain a linker-connected product; performing a PCR amplification on the linker-connected product using a first primer specifically bound to the first linker and a second primer specifically bound to the second linker to obtain an amplified product, the first primer or the second primer having a first affinity label; capturing a single strand having the first affinity label in the amplified product using a solid phase carrier; performing single primer linear amplification using a third primer with the captured single strand as a template; performing exponential amplification using the third primer and the first primer, with the linearly amplified product as the template, to obtain a product containing the target regions.