Detection system

Abstract

A method for detecting the presence of a target nucleic acid sequence in a sample, said method comprising: (a) adding to a sample suspected of containing said target nucleic acid sequence, a probe specific for said target sequence and DNA duplex binding agent, said probe comprising a reactive molecule able to absorb fluorescence from or donate fluorescent energy to said DNA duplex binding agent, (b) subjecting the thus formed mixture to an amplification reaction in which target nucleic acid is amplified, (c) subjecting said sample to conditions under which the said probe hybridizes to the target sequence, and (d) monitoring fluorescence from said sample. This method can be used for example to monitor amplification reactions such as PCR reactions, such that the amount of target sequence present in the sample may be determined. Additionally or alternatively, it may be used to generate duplex destabilization data such as melt hysteresis information for amplification monitoring or for detection and quantitation of polymorphisms or allelic variation, and so is useful in genetic diagnosis.

1 . A method for determining a characteristic of a sequence, said method comprising: 
 (a) adding to a sample suspected of containing said sequence, a probe specific for said target sequence DNA duplex binding agent, said probe comprising a reactive molecule able to absorb fluorescence from or donate fluorescent energy to said DNA duplex binding agent;    (b) subjecting said sample to conditions under which the said probe hybridises to the said sequence;    (c) monitoring fluorescence from said sample and determining a particular reaction condition, characteristic of said sequence, at which fluorescence changes as a result of the hybridisation of the probe to the sample of destabilisation of the duplex formed between the probe and the target nucleic acid sequence.    
     
     
         2 . A method according to  claim 1  wherein the reaction condition characteristic of said sequence is temperature, electrochemical potential, or reaction with an enzyme or chemical.  
     
     
         3 . A method according to  claim 2  wherein the condition is temperature.  
     
     
         4 . A method according to any of  claims 1  to  3  wherein the results obtained from two sequences are compared in order to determine the presence of polymorphisms or variations there between.  
     
     
         5 . A method according to any one of  claims 1  to  3  wherein the DNA duplex binding agent is an intercalating dye.  
     
     
         6 . A method according to  claim 5  wherein the intercalating dye is selected from the group consisting of SYBRGreen, SYBRGreen I, SYBRGold, ethidium bromide and YOPRO-1.  
     
     
         7 . A method according to any one of  claims 1  to  3  wherein the reactive molecule is an acceptor molecule.  
     
     
         8 . A method according to  claim 7  wherein the reactive molecule is fluorescein.  
     
     
         9 . A method according to any one of  claims 1  to  3  wherein the probe is immobilised on a solid support.  
     
     
         10 . A method according to any one of  claims 1  to  3  wherein the product is immobilised on a solid support.  
     
     
         11 . A kit comprises a probe specific for a target nucleotide sequence which contains an reactive molecule, and a DNA duplex binding agent which is compatible with said reactive molecule.  
     
     
         12 . A kit according to  claim 11  wherein the DNA duplex binding agent is an intercalating dye.  
     
     
         13 . A kit according to any one of claims  11  or  12  which further comprises one or more reagents used in an amplification reaction.  
     
     
         14 . A probe which comprises a sequence which will hybridise with a target nucleotide sequence and a reactive molecule.  
     
     
         15 . The method of  claim 1  wherein said characteristic is genetic diagnosis.  
     
     
         16 . The method of  claim 1  wherein said characteristic is DNA melting curve analysis.  
     
     
         17 . A method according to  claim 4  wherein the DNA duplex binding agent is an intercalating dye.  
     
     
         18 . A method according to  claim 17  wherein the intercalating dye is selected from the group consisting of SYBRGreen, SYBRGreen I, SYBRGold, ethidium bromide and YOPRO-1.  
     
     
         19 . A method according to  claim 4  wherein the reactive molecule is an acceptor molecule.  
     
     
         20 . A method according to  claim 4  wherein the probe is immobilised on a solid support.  
     
     
         21 . A method according to  claim 4  wherein the product is immobilised on a solid support.