Multiplex PCR Channel Expansion Through Crosstalk Compensation
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Solution Overview
Problem
The number of practically usable channels in multiplex PCR is limited due to signal crosstalk, particularly in TAGS-PCR, despite theoretical increases through color and temperature channels, as practical compensation is often not feasible.
Innovation Solution
A method involving labeling probe oligonucleotides with specific labels that emit signals in multiple channels, including crosstalk channels, and measuring these signals during PCR amplification to determine target polynucleotides based on measured values.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple color and temperature channels are used in TAGS-PCR to increase the number of detection channels, then the theoretical number of usable channels increases, but signal crosstalk between channels increases, making reliable compensation difficult and reducing the number of practically usable channels
Solution Approach 1:
The patent converts the harmful crosstalk signals into useful information by measuring them and using them for compensation. Instead of avoiding crosstalk, the method deliberately measures crosstalk signals in multiple channels and uses mathematical compensation based on these measurements to eliminate their harmful effects, thereby enabling the use of additional channels.
Solution Approach 2:
The patent implements feedback by measuring the actual crosstalk signals during the PCR reaction and using these measurements to adjust and compensate for the crosstalk effects. The system continuously monitors signal values in multiple channels and uses this feedback information to calculate compensation factors that correct the signal interference.
2Adaptability or versatility
If more probe oligonucleotides with the same label are differentiated by melting points of tags, then the number of temperature channels increases, but the proportionality of signal bleeding over is not maintained, reducing the number of reliably compensatable channels
Solution Approach 1:
The patent applies preliminary action by measuring signal values in multiple channels during the PCR amplification process before final determination. By capturing the signal behavior throughout the amplification cycles, the system can calculate compensation factors in advance that account for signal bleeding, enabling reliable compensation even when proportionality is not perfectly maintained.
Solution Approach 2:
The patent changes the parameter approach by measuring signal values across multiple channels and using these parameter changes to calculate compensation factors. Instead of relying on fixed proportional relationships, the method dynamically adjusts compensation based on actual measured signal behavior, thereby maintaining reliability across different temperature channels.
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
Enhances the number of usable channels in multiplex PCR by effectively compensating for crosstalk signals, allowing for more accurate and reliable detection of multiple target polynucleotides.
Implementation Method 1
said first probe oligonucleotide is labeled with a first label (label-1) emitting a label-1 signal in a first channel
Implementation Method 2
amplifying said target polynucleotides by PCR
Data Source
Figure 1A~1C
Figure 2A~2C
Figure 3A~3B
AI summary
The present invention relates to a method for performing a multiplex PCR to determine at least a first target polynucleotide and a second target polynucleotide in a sample, comprising (i) contacting said sample in a single container with at least a first probe oligonucleotide for the first target polynucleotide and with a second probe oligonucleotide for the second target polynucleotide; wherein said first probe oligonucleotide is labeled with a first label (label-1) emitting a label-1 signal in a first channel, a first label-1 crosstalk signal in a second channel, and a second label-1 crosstalk signal in a third channel, and wherein said second probe oligonucleotide is labeled with a second label (label-2) emitting a label-2 signal in said third channel; (ii) amplifying said target polynucleotides by PCR; (iii) measuring a multitude of values in the second channel and the third channel over at least part of the amplification in step (ii); and (iv) determining the first target polynucleotide based on the values measured in step (iii). The present invention also relates to systems, computer-readable storage media, and uses related to said method.