Multiplexing and quantification in PCR with reduced hardware and requirements
Methods and algorithms for a multiplexed single detection channel amplification process and quantification of generated amplicons is presented. Various mathematical approaches for quantifying and verifying the amplicons in a reaction are presented. Usage of such methods and approaches allow upgrading of existing single and multiple channel instruments for further multiplexing capabilities.
1. A method for performing a Polymerase Chain Reaction (PCR) comprising:
performing a multiplexed PCR in a single channel using a thermal cycler, using a limited set of primers to control plateau levels of a plurality of different amplicons, thereby obtaining different plateau levels for different amplicons, and without performing an end-point melting curve analysis;
detecting, using a detector, an amplicon of the plurality of different amplicons, the plurality of different amplicons generated during the multiplexed PCR; and
associating, using a processor controlled analyzer, a cycle threshold (CT) value to an amplicon of the plurality of different amplicons generated during said multiplexed PCR, wherein the associating step comprises:
(i) receiving a digital representation of a sum amplitude signal, wherein said sum amplitude signal comprises a sum amplitude curve corresponding to a total detected intensity generated by the plurality of different amplicons of said multiplexed PCR, wherein said total detected intensity comprises the signal intensity over a plurality of different wavelengths detected in the single detection channel;
(ii) measuring an amount of asymmetry in a derivative of said sum amplitude curve;
(iii) determining a relative position of any two CT values corresponding to any two amplicons from the plurality of different amplicons by using the measured asymmetry; and
(iv) associating a CT value to an amplicon of said plurality of amplicons, wherein associating the CT value is performed using a characteristic of said sum amplitude signal without determining melting temperatures (Tm) for each of said plurality of different amplicons; and
wherein the processor controlled analyzer is connected to the thermal cycler for temperature measuring and modulation.
2. The method of claim 1 , wherein (ii), (iii), or (iv) is performed using mathematical analysis of the digital representation of the sum amplitude signal, the mathematical analysis being implemented in software and/or firmware code and executed on the processor controlled analyzer.
3. The method of claim 1 , wherein the multiplexed PCR further comprises using a plurality of excitation sources.
4. The method of claim 1 , wherein the detecting comprises detecting, with a bandwidth filter, a plurality of amplicon signals emitting at different wavelength peaks.
5. The method of claim 1 , wherein said characteristic comprises a slope or shape of a portion of said sum amplitude signal.
6. The method of claim 1 , wherein a first reporter for a first amplicon and a second reporter for a second amplicon of the plurality of amplicons emit at different wavelength peaks.
7. The method of claim 1 , wherein a first reporter for a first amplicon and a second reporter for a second amplicon of the plurality of amplicons emit at a same wavelength peak.