Multiplex PCR Probes Using Distinct Melting Profiles
Solution Overview
Problem
Current multiplex real-time PCR methods are limited by low multiplexity and sensitivity, as they can only detect and quantify up to four or five fluorescence dyes simultaneously, requiring large differences in amplicon sizes for distinction, which restricts the ability to analyze multiple target sequences effectively.
Innovation Solution
The method employs probes with distinct melting properties and emission changes based on their internal double-stranded portions, allowing for the simultaneous amplification and detection of multiple target nucleic acid sequences by measuring melting profiles and consumption of probes with different signature melting temperatures, even when using the same or similar labels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescence dyes are used for detection in multiplex PCR, then real-time quantification is enabled, but the multiplexity is limited to four or five dyes simultaneously
Solution Approach 1:
The patent changes the detection parameter from relying solely on fluorescence dye emission spectra to utilizing melting temperature (Tm) as an additional distinguishing parameter. Probes are designed with different Tm values (e.g., 65°C, 70°C, 75°C) while using the same or similar fluorescence labels, enabling multiplex detection beyond the limit of distinguishable dye spectra.
Solution Approach 2:
The invention adds a new dimension to probe differentiation by incorporating melting temperature as a second distinguishing feature alongside fluorescence label. This transforms the detection space from one-dimensional (fluorescence wavelength only) to two-dimensional (fluorescence label + melting temperature), allowing more probes to be distinguished simultaneously.
2Adaptability or versatility
If probes with different fluorescence labels are used to increase multiplexity, then more targets can be detected, but the complexity of distinguishing overlapping emission spectra increases
Solution Approach 1:
The patent introduces melting temperature as an additional discrimination parameter to reduce reliance on spectral distinction. By designing probes with distinct Tm values (through variations in probe length, GC content, or sequence composition), the system can differentiate probes even when they share the same or overlapping fluorescence labels, thereby reducing spectral complexity requirements.
3Measurement precision
If amplicon size differences are used to distinguish targets, then detection is enabled, but large size differences are required which restricts analysis of multiple targets
Solution Approach 1:
The patent shifts the distinction parameter from amplicon physical size to probe melting temperature. By designing probes with different Tm values that hybridize to targets within the same amplicon or similarly sized amplicons, the method enables multiplex detection without requiring large amplicon size differences, thereby increasing versatility.
4Ease of manufacture
If the same fluorescence label is used for multiple probes, then cost is reduced and detection is simplified, but the probes cannot be distinguished without additional features
Solution Approach 1:
The patent introduces melting temperature as a distinguishing parameter that does not affect probe cost or detection simplicity. Probes with the same fluorescence label can be differentiated by their Tm values, which are determined by probe sequence and length during synthesis. This maintains cost-effectiveness while enabling probe distinction through thermal properties rather than requiring additional expensive labels.
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 enables the detection and quantification of a large number of different target sequences in a single PCR reaction with enhanced multiplexity and sensitivity, overcoming the limitations of existing methods by distinguishing probes with the same labels through unique melting profiles.
Implementation Method 1
each probe with double-stranded portion has a signature melting temperature
Implementation Method 2
each probe in the set comprises a detectable label or detectable combination of labels which is/are capable of producing a changeable signal
Data Source
AI summary
The invention relates to the field of multiplex amplification. In particular, the invention relates to methods for assaying a sample for one or more nucleic acid targets in a single reaction based on the distinct melting temperatures or melting profiles of primers and/or probes. The invention also provides probes and kits for use in such methods.