qPCR Detection Using Intercalating Dye and Sequence-Specific Probe
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Solution Overview
Problem
Current quantitative real-time polymerase chain reaction (qPCR) methods face challenges in discriminating between specific and non-specific products, and in determining the cause of suboptimal PCR performance, as they either lack specificity or fail to provide comprehensive information on PCR amplicons and library quality.
Innovation Solution
A combination of an intercalating dye that preferentially binds to double-stranded DNA and a sequence-specific fluorophore-labeled oligonucleotide probe is used, allowing simultaneous detection and quantification of target nucleic acid molecules, enabling the assessment of PCR performance and library quality through fluorescence measurement.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If intercalating dyes are used for real-time detection of PCR products, then detection sensitivity is improved, but specificity is worsened due to inability to discriminate between correct products and non-specific products
Solution Approach 1:
The patent combines two detection methods (intercalating dye and probe-based detection) into a single qPCR assay. The intercalating dye provides sensitive detection of total dsDNA amplification, while the sequence-specific probe provides discriminatory power to identify correct amplicons. This merging allows simultaneous measurement of both sensitivity and specificity parameters.
Solution Approach 2:
The dual-detection system serves multiple functions: the intercalating dye monitors overall PCR efficiency and detects all dsDNA products, while the probe specifically identifies target sequences. This multi-functionality enables comprehensive quality control including detection of primer dimers, non-specific amplification, and verification of correct product formation.
2Reliability
If probe-based detection is used for sequence-specific quantification, then specificity is improved, but comprehensive information on PCR amplicons is lost
Solution Approach 1:
The patent merges probe-based specific detection with intercalating dye-based total amplification monitoring. The probe signal provides sequence-specific quantification while the intercalating dye signal provides information on total dsDNA formed, including non-specific products and primer dimers. Together they deliver comprehensive amplicon information.
Solution Approach 2:
The intercalating dye acts as an intermediary that monitors overall PCR reaction health and efficiency, while the probe specifically identifies target sequences. The combination allows differentiation between successful specific amplification versus failed or non-specific amplification events.
3Loss of information
If dual-dye system is used for comprehensive detection, then information completeness is improved, but measurement precision is worsened due to signal overlap
Solution Approach 1:
The patent employs dyes with spectrally distinct properties - the intercalating dye (SYBR Green) emits in the green spectrum while the probe fluorophore (FAM) emits in the blue-green spectrum. This local spectral differentiation allows independent measurement of each signal channel without significant overlap, maintaining measurement precision while achieving information completeness.
Solution Approach 2:
The patent resolves signal overlap by utilizing the spectral dimension - measuring fluorescence intensity at different wavelengths corresponding to each dye's emission maximum. This dimensional separation in the optical spectrum allows simultaneous quantification of both signals with high precision.
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 provides comprehensive information on PCR amplicons and library quality, differentiates between specific and non-specific products, and optimizes PCR conditions by identifying the cause of suboptimal performance, enhancing the accuracy and efficiency of nucleic acid quantification and amplification assays.
Implementation Method 1
an intercalating dye recognizing double-stranded DNA molecules with higher affinity than single-stranded DNA molecules and said intercalating dye has increasing fluorescence upon intercalation with double-stranded nucleic acid molecules
Implementation Method 2
said intercalating dye has increasing fluorescence upon intercalation with double-stranded nucleic acid molecules
Implementation Method 3
at least one fluorophore-labeled oligonucleotide-probe being sequence specific for the target nucleic acid molecule, and said at least one fluorophore-labeled oligonucleotide-probe undergoes a detectable change in fluorescence upon amplification of said one or more target nucleic acid molecules
Implementation Method 4
said at least one fluorophore-labeled oligonucleotide-probe undergoes a detectable change in fluorescence
Data Source
Figure 1A~1B
Figure 1C~1D
Figure 2A~2B
AI summary
The present invention relates to methods and uses for the detection or quantification of newly-synthesized double-stranded target nucleic acid molecules in a sample during quantitative real-time polymerase chain reaction (qPCR) amplification. According to the invention, an intercalating dye recognizing double-stranded DNA molecules with higher affinity than single-stranded DNA molecules and a fluorophore-labeled oligonucleotide-probe being sequence specific for a target nucleic acid molecule are simultaneously employed, thus enabling quantification a specific target and total amount of a mixed nucleic acid population, and enabling assessing the cause of suboptimal PCR performance.