Detection of nucleic acid hybridization by fluorescence polarization

Abstract

Methods, systems and assays are provided for FP detection of nucleic acid hybridization.

1 . A system comprising: 
 a container comprising a duplexed nucleic acid disposed in the container, wherein at least one strand of the nucleic acid duplex comprises a neutral or positively charged fluorescent label;    a polarized light source positioned to shine plane polarized light through a portion of the container, thereby exciting the fluorescent label during operation of the system; and,    a detector that detects resultant polarization of light emitted by the fluorescent label.    
     
     
         2 . The system of  claim 1 , wherein the container comprises a microfluidic device which contains the duplexed nucleic acid in one or more channels or chambers of the device.  
     
     
         3 . The system of  claim 2 , the microfluidic device comprising a body structure, the body structure having two or more intersecting microchannels disposed therein, the microfluidic device further comprising a source of the first nucleic acid and a source of a second nucleic acid, which sources are in fluid communication with the at least two intersecting microchannels, wherein, during operation of the device, the first nucleic acid is flowed from the source of the first nucleic acid into at least one of the at least two intersecting channels and the second nucleic acid is flowed from the source of the second nucleic acid into the at least one channel, whereby the first and second nucleic acids are mixed in the at least one channel.  
     
     
         4 . The system of  claim 3 , wherein the detector is proximal to the at least one channel.  
     
     
         5 . The system of  claim 1 , wherein the fluorescence polarization is increased by less than about 50% by the addition of polylysine to the duplexed nucleic acid in the container.  
     
     
         6 . The system of  claim 1 , wherein the duplexed nucleic acid is present in a composition which is substantially free of polyion.  
     
     
         7 . The system of  claim 6 , wherein the composition comprises less than 1 μM polyion.  
     
     
         8 . The system of  claim 1 , wherein a rotational diffusion rate of the duplexed nucleic acid is less than the rotational diffusion rate of a first or second strand of the duplexed nucleic acid.  
     
     
         9 . The system of  claim 1 , wherein the first or second nucleic acid comprises one or more of: DNA, RNA, a DNA analogue, an RNA analogue or a PNA.  
     
     
         10 . The system of  claim 1 , wherein one or more of the nucleic acids is nuclease resistant.  
     
     
         11 . The system of  claim 1 , wherein the fluorescent label comprises rhodamine or BODIPY.  
     
     
         12 . The system of  claim 1 , wherein the first nucleic acid is a DNA and the second nucleic acid is a PNA which comprises a rhodamine label.  
     
     
         13 . A microfluidic fluorescent polarization nucleic acid analysis system comprising: 
 a microfluidic device comprising a body structure having at least two microfluidic channels disposed therein;    a source of a first nucleic acid;    a source of a second labeled nucleic acid, which second nucleic acid comprises a neutral or positively charged fluorescent label;    a source of plane polarized light, which source is positioned to illuminate a portion of at least one of the at least two microchannels; and,    a fluorescence polarization detector positioned to detect plane polarized light emitted from the microfluidic device.    
     
     
         14 . In an assay system for quantifying a nucleic acid hybridization parameter, which assay system comprises providing a first nucleic acid composition, which nucleic acid composition comprises a first nucleic acid having a positive or neutral fluorescent label, introducing a second nucleic acid into the first nucleic acid composition to produce a second nucleic acid composition, the second nucleic acid reacting with the first nucleic acid to produce a fluorescently labeled product having a substantially different rotation rate than the first nucleic acid, a computer implemented process, comprising the steps of: 
 determining a first level of fluorescence polarization of the first nucleic acid composition;    determining a second level of fluorescence polarization of the second nucleic acid composition;    comparing the first and second levels of fluorescent polarization; and,    calculating the nucleic acid hybridization parameter.