DNA-based fluourescent sensor

Abstract

The fluorescent detection of target DNA is enhanced by amplifying the DNA in a sample by initiating a polymerase chain reaction using a first pair of primers and an appropriate polymerase. The DNA is then reamplified, this time using a second pair of nested primers which bracket DNA base sequences within the region bracketed by the first set of primers. At least one of the second pair of primers is bound to a fluorescent reporter molecule and at least one of the second pair of primers has bound thereto a specific binding site for a double-stranded DNA-binding protein. The thus amplified sample is then contacted with a fluorescent sensor having the appropriate double-stranded DNA-binding protein attached thereto. Then, the fluorescent reporter molecule is excited by light of the appropriate wavelength. The present invention provides greater sensitivity, adaptability and ease of use than previously available.

What is claimed is: 
     
       1. A method for the fluorescent detection of target DNA, comprising the steps of: a) performing a first amplification on a sample suspected of containing said target DNA by initiating a polymerase chain reaction with a first pair of primers, which delimit therebetween a first region of DNA base sequences of said target DNA to be amplified by said amplification, and a polymerase which catalyzes said polymerase chain reaction, said amplification being sufficiently great that the amount of DNA other than target DNA in the amplified sample is negligible in comparison to the amount of target DNA in the amplified sample if target DNA were present in the unamplified sample;   b) performing a second amplification on the amplified sample using a second pair of primers, said second pair of delimiting therebetween a second region of DNA base sequences within the first region of DNA base sequences bracketed by said first pair of primers and selected for amplification by said first amplification using said first pair of primers, at least one member of said second pair of primers having a fluorescent reporter molecule bound thereto and at least one member of said second pair of primers having a specific binding site for a double-stranded DNA-binding protein bound thereof, thereby producing an amount of double stranded, fluorescent reporter molecule-bound and double stranded DNA-binding protein site-bound, twice amplified DNA which, when excited at an excitation frequency of said reporter molecule, emits a detectable level of fluorescence if target DNA was present in the unamplified sample;   c) contacting at least an aliquot of said twice amplified sample with a fluorescent sensor comprising a fiber waveguide having attached to its exposed surface a double stranded CNA-binding protein which binds to said double stranded DNA-binding protein site on said twice amplified DNA;   d) exciting the fluorescent reporter molecule bound to any of said twice amplified DNA bound to said sensor via said double stranded CNA-binding protein with light of an excitation wavelength for said reporter molecule;   e) detecting any fluorescent emission from said excited fluorescent reporter molecule.   
     
     
       2. The method of claim 1, wherein said bound reporter molecule is excited with said light in the form of an evanescent wave along said sensor, and wherein the energy emitted by said bound reporter molecule is coupled back into said sensor. 
     
     
       3. The method of claim 1, wherein said reporter molecule is selected from the group consisting of fluorescein isothiocyanate, tetramethyl rhodamine isothiocyanate, rhodamine and texas red. 
     
     
       4. The method of claim 1, wherein said sensor is a glass optical fiber.