Systems and Methods for Calibration Using Dye Signal Amplification

Abstract

The present teachings relate to a method of generating calibration information during a real-time polymerase chain reaction (RT-PCR) or other amplification reaction. A sample well plate or other support can contain one or more dyes or other reference materials that are subjected to the same RT-PCR thermal cycles or other conditions used to conduct amplification or other reactions on a biological sample. A set of maxima values and a set of minimum values, and/or other calibration information useful for adjusting emission data for sample dyes can be recorded, for example, for 10 cycles, 20 cycles, or each cycle of a complete RT-PCR run. Such testing of dye response under realistic operating conditions can enable more accurate characterization of plate, dye, filter, or instrument response and therefore more accurate calibration corrections and other and/or adjustments.

What is claimed is: 
     
         1 . A method of generating calibration information for an amplification reaction, comprising:
 performing a first amplification reaction on at least one reference material in a sample support of an analytical instrument   performing a second amplification reaction on a sample in a sample support of the analytical instrument;   receiving first emission data generated by the at least one reference material during the first amplification reaction;   receiving second emission data generated by the sample during the second amplification reaction;   generating calibration information based on the received first emission data; and   adjusting the received second emission data as a function of the calibration information.   
     
     
         2 . The method of  claim 1 , wherein the at least one reference material comprises at least one dye. 
     
     
         3 . The method of  claim 1 , wherein the at least one reference material comprises at least one fluorescent dye. 
     
     
         4 . The method of  claim 1 , wherein the first and second amplification reactions, each comprise a polymerase chain reaction. 
     
     
         5 . The method of  claim 1 , wherein the calibration information generated comprises time-varying information corresponding to multiple cycles of the first amplification reaction. 
     
     
         6 . The method of  claim 1 , wherein the calibration information generated comprises at least one of a set of maximum values of the emission data, a set of minimum values of the emission data, and spectral response information derived from the first emission data. 
     
     
         7 . The method of  claim 1 , wherein the first amplification reaction is performed simultaneously with the second amplification reaction. 
     
     
         8 . The method of  claim 1 , wherein the first amplification reaction is performed before the second amplification reaction. 
     
     
         9 . A system for generating calibration information, comprising:
 an input unit, configured to receive first emission data generated by at least one reference material during an amplification reaction and configured to receive second emission data generated by at least one sample during an amplification reaction; and   a processor communicating with the input unit and configured to generate calibration information based on the first emission data and to adjust the second emission data based on the calibration information.   
     
     
         10 . The system of  claim 9 , wherein the at least one reference material comprises at least one dye. 
     
     
         11 . The system of  claim 9 , further comprising at least one sample support and at least one reference material, wherein the at least one reference material comprises a dye and the dye is disposed in the sample support. 
     
     
         12 . The system of  claim 9 , wherein the input unit is configured to receive first emission data generated by at least one reference material during a polymerase chain reaction. 
     
     
         13 . The system of  claim 9 , wherein the processor is configured to generate calibration information comprising time-varying information corresponding to multiple cycles of an amplification reaction. 
     
     
         14 . The system of  claim 9 , wherein the processor is configured to generate calibration information comprising at least one of a set of maximum values of the first emission data, a set of minimum values of the first emission data, and spectral response information derived from the first emission data. 
     
     
         15 . A set of calibration information, the set of calibration information being generated by a method comprising:
 disposing at least one amplifiable reference material into a sample support;   performing multiple cycles of an amplification reaction on the at least one reference material in the sample support;   receiving emission data generated by the at least one reference material during the multiple cycles of the amplification reaction; and   generating a set of calibration information based on the received emission data.   
     
     
         16 . The set of calibration information of  claim 15 , wherein the emission data comprises fluorescent intensity data generated during at least 10 cycles of polymerase chain reaction. 
     
     
         17 . The set of calibration information of  claim 15 , wherein the emission data comprises emission data generated during a polymerase chain reaction. 
     
     
         18 . The set of calibration information of  claim 15 , comprising time-varying information corresponding to at least 10 cycles of the amplification reaction. 
     
     
         19 . The set of calibration information of  claim 15 , comprising at least one of a set of maximum values of the emission data, a set of minimum values of the emission data, and spectral response information derived from the emission data. 
     
     
         20 . The set of calibration information of  claim 15 , comprising at least one set of maximum values of time-varying information corresponding to at least 10 cycles of the amplification reaction and at least one set of minimum values of time-varying information corresponding to at least 10 cycles of the amplification reaction.