Two-Round Tandem PCR for Multiplex Gene Quantification

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Solution Overview

Problem

Current methods for multiplex PCR face challenges in accurately amplifying and quantifying multiple gene expression products due to competition for reaction components, leading to difficulties in diagnosing, forensic analysis, and research applications, especially with small or degraded samples.

Innovation Solution

A two-round tandem PCR protocol that limits multiplex amplification to prevent competition by using outer primers in the first round and inner primers in the second round, along with SYBR-green or fluorogenic probes for detection, allowing for the amplification and quantification of multiple nucleic acid sequences from small samples.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If multiplex PCR amplification is performed to analyze multiple gene expression products simultaneously, then the productivity and efficiency of gene analysis is improved, but the accuracy of amplification and quantification deteriorates due to competition for reaction components

Engineering Contradiction:

Improvenumber of gene expression products analyzed simultaneouslyVSAvoidaccuracy of amplification and quantification

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent divides the multiplex PCR process into two sequential rounds. The first round uses a limited number of primer pairs (2-6 pairs) to amplify target sequences with high efficiency. The second round then analyzes multiple gene expression products from the first round products. This segmentation reduces competition for reaction components in each round while maintaining the ability to analyze multiple genes, thereby resolving the contradiction between productivity and measurement precision.

Inventive Principle:

Principle #1Segmentation

2Adaptability or versatility

If the number of genes analyzed in multiplex PCR is increased, then the versatility and diagnostic value are improved, but the reliability of quantification deteriorates due to increased competition and variation between replicates

Engineering Contradiction:

Improvenumber of genes analyzedVSAvoidconsistency of quantification between replicates

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The two-round approach segments the analysis so that the first round focuses on amplifying target sequences from multiple genes with minimal competition, while the second round performs quantification with high reliability. This allows the system to maintain versatility (analyzing many genes) while ensuring reliability (consistent quantification) by separating these functions into distinct phases.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The first round of multiplex PCR performs preliminary amplification of target sequences from multiple genes before the quantification step. By pre-amplifying the templates in the first round with optimized primer concentrations and cycle numbers, the system ensures that sufficient material is available for accurate quantification in the second round, thereby maintaining both versatility and reliability.

Inventive Principle:

Principle #10Preliminary action

3Measurement precision

If conventional qPCR is used to quantify PCR products, then the measurement capability is improved, but the application is limited to single-gene analysis due to competition in multiplex reactions

Engineering Contradiction:

Improvequantification capabilityVSAvoidnumber of genes that can be quantified

Core Design Contradiction:

Measurement precisionVSAdaptability or versatility

Solution Approach 1:

The patent segments the quantification process into two rounds: the first round performs multiplex amplification of multiple gene targets, and the second round applies qPCR quantification to the amplified products. This segmentation enables the system to maintain the measurement precision of qPCR while extending its versatility to multiple genes by performing the amplification phase in multiplex format first.

Inventive Principle:

Principle #1Segmentation

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 enables accurate and efficient amplification and quantification of up to 150 gene expression products, reducing competition and increasing sensitivity, making it suitable for diagnostic, forensic, and research applications with small or degraded samples.

Implementation Method 1

along with SYBR-green or fluorogenic probes for detection

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS8962250B2Methods for the amplification, quantitation and identification of nucleic

Publication Date: 2015.02.24 AUSDIAGNOSTICS

AI summary

The invention relates to improved methods of amplifying and optionally quantifying and/or identifying a plurality of selected nucleic acid molecules from a pool of nucleic acid molecules. A first round of multiplex amplification used where the amplification reaction is allowed to proceed to a point prior to that at which significant competition between amplicons for reaction components has occurred. This is the followed by a second round of amplification that typically includes a fluorescent reporter to allow for each of the selected nucleic acid sequences to be quantified. The methods are useful for the amplification and quantification of nucleic acids from a variety of sources, such as gene expression products, whereby many such products may be amplified and quantified from very limited samples and from degraded archival samples.