Chromophore-based characterization and detection methods

Abstract

This disclosure provides methods, compositions and kits for the detection of a plurality of analytes in a sample. In some examples, this disclosure provides methods, compositions, and kits for detecting analytes, genetic variations, monitoring reaction process, and monitoring analyte-analyte interactions by measuring signals. In some examples, the presence of signals or changes in signals may be used to construct signal profiles which can be used to detect analytes.

1 . A method of detecting at least one nucleic acid target in a sample, comprising:
 a) performing at least one cycle of a polymerase chain reaction (PCR) with a first nucleic acid target, a first forward (FWD) primer and a first rewind (RWD) primer, wherein a first quencher is ligated to the 5′ end of the first FWD primer, a first fluorophore is ligated to the 5′ end of the first RWD primer, and the first FWD primer and the first RWD primer are specific for the first nucleic acid target; and   b) measuring a first fluorescent signal generated by the first fluorophore and quencher after each cycle of the PCR;   wherein an attenuation of the first fluorescent signal indicates the presence of the first nucleic acid target.   
     
     
         2 . The method of  claim 1 , wherein the first nucleic acid target is a DNA target. 
     
     
         3 . A method of detecting multiple nucleic acid targets of differing lengths comprising:
 a) contacting the multiple nucleic acid targets with a plurality of forward (FWD) and rewind (RWD) primers, wherein each of the FWD and RWD primers are specific for a single nucleic acid target, the FWD primers comprise a quencher and the RWD primers comprise a fluorophore;   b) conducting a polymerase chain reaction (PCR); and   c) measuring an endpoint fluorescence of each target, wherein the endpoint fluorescence for each target is different.   
     
     
         4 . The method of  claim 3 , wherein the RWD primers comprise the same fluorophore. 
     
     
         5 . A method of detecting the morphology of a polymer, comprising:
 a) providing a sample containing the polymer, wherein the polymer comprises multiple fluorophores and a quencher;   b) measuring a first fluorescence from the sample at a first temperature;   c) measuring a second fluorescence from the sample at a second temperature; and   d) using the first and second fluorescences to detect the morphology of the polymer.   
     
     
         6 . The method of  claim 5 , wherein the polymer is a protein or a DNA. 
     
     
         7 . The method of  claim 6 , wherein the polymer is a DNA. 
     
     
         8 . The method of  claim 7 , wherein the DNA comprises three different fluorophores. 
     
     
         9 . The method of  claim 7 , wherein the DNA comprises a quencher at its 3′ end. 
     
     
         10 . The method of  claim 5 , wherein the first fluorescence is a first fluorescence signature. 
     
     
         11 . The method of  claim 5 , wherein the second fluorescence is a second fluorescence signature. 
     
     
         12 . The method of  claim 1 , wherein:
 step a) further comprises performing at least one cycle of a polymerase chain reaction (PCR) with a second nucleic acid target, a second forward (FWD) primer and a second rewind (RWD) primer, wherein a second quencher is ligated to the 5′ end of the second FWD primer, a second fluorophore is ligated to the 5′ end of the second RWD primer, and the second FWD primer and the second RWD primer are specific for the second nucleic acid target; and   step b) further comprises measuring a second fluorescent signal generated by the second fluorophore and quencher after each cycle of the PCR;   wherein an attenuation of the second fluorescent signal indicates the presence of the second nucleic acid target.   
     
     
         13 . The method of  claim 12 , wherein the first and second fluorophores are different. 
     
     
         14 . The method of  claim 3 , wherein the RWD primers comprise the fluorophore at its 5′ end. 
     
     
         15 . The method of  claim 3 , wherein the FWD primers comprise the quencher at its 5′ end. 
     
     
         16 . A method of detecting a nucleic acid target in a sample, comprising:
 a) combining the nucleic acid target with a fluorophore and a quencher, wherein the fluorophore and the quencher are each incorporated into a separate primer that is complementary to a region within the nucleic acid target;   b) performing at least one cycle of a polymerase chain reaction (PCR) to incorporate the fluorophore and the quencher into products of the PCR; and   c) detecting a fluorescent signal from the fluorophore and the quencher at a first timepoint and a second timepoint, wherein the second timepoint is later than the first timepoint and wherein a decrease in fluorescent signal at the second timepoint relative to the first timepoint is indicative of the presence of the nucleic acid target.   
     
     
         17 . The method of  claim 16 , wherein the first timepoint is after step a) and the second timepoint is after step b). 
     
     
         18 . The method of  claim 16 , wherein the products of the at least one cycle of PCR each comprise a first nucleic acid strand and a second nucleic acid strand, wherein the first nucleic acid strand and the second nucleic acid strand are complementary. 
     
     
         19 . The method of  claim 18 , wherein the fluorophore is incorporated into the first nucleic acid strand and the quencher is incorporated into the second nucleic acid strand. 
     
     
         20 . The method of  claim 19 , wherein the fluorophore is incorporated at the 5′ end of the first nucleic acid strand and the quencher is incorporated at the 5′ end of the second nucleic acid strand. 
     
     
         21 . The method of  claim 16 , wherein the nucleic acid target is a DNA target.