Method and Apparatus for Melting Curve Analysis of Nucleic Acids in Microarray Format

Abstract

A method and apparatus for performing melting curve analyses of nucleic acids on a microarray is described. The present method includes varying the temperature of a fluid on a microarray to dissociate and remove target DNA, scanning the mircoarray for fluorescence, collecting the target DNA removed from the microarray, and reusing the collected target DNA and the microarray. The apparatus of the present disclosure includes a microarray stage, a light source and detector, and a temperature controller, wherein the temperature controller is configured to adjust the temperature of a fluid within a sample chamber on the microarray such that the temperature of the fluid is varied during the analysis such that target DNA is dissociated from the microarray, and wherein the light source is directed to the microarray and the resulting fluorescence is perceived by the detector.

We claim: 
     
         1 . An apparatus for conducting a melting curve analysis of nucleic acids on a microarray by exciting a predefined portion of the microarray with a light beam and detecting emissions of fluorescent light comprising:
 an illuminator assembly, including
 a light source configured to produce the light beam, the light beam comprising electromagnetic radiation having one or more wavelengths, 
 a band pass filter configured to eliminate wavelengths close to the wavelength of fluorescent light emitted from the nucleic acids, 
 a mirror configured to reflect the light beam and to allow the fluorescent light emission to pass through, and 
 an objective lens configured to focus the light beam on a predefined portion of the microarray; 
   an emissions detector configured to sense emissions of fluorescent light from the predefined portion of the microarray;   a fluid circulation system, including
 a microarray cassette assembly including
 a frame including
 an opening configured to receive a microarray slide, the opening having a spacer disposed about an inside perimeter, 
 a fluid inlet port, and 
 a fluid outlet port, 
 
 an optical window attached to the frame, 
 a seal disposed on the frame opposite the optical window, 
 wherein when a microarray slide is received within the opening, the spacer maintains the microarray slide separate from the optical window, the space between the microarray slide and the optical window defining a microarray chamber configured to allow the flow of fluid from the fluid inlet port to the fluid outlet port across the microarray slide, 
 
 a pump configured to receive fluid from a fluid reservoir and to circulate the fluid through a fluid supply line coupled to the fluid inlet port, 
   a fluid return line coupled to the fluid outlet port; and   a temperature control assembly including a temperature controller, and fluid heater;   wherein the light beam and the micro array are movable relative to each other.   
     
     
         2 . The apparatus of  claim 1 , wherein the microarray is movable in a first direction and the light beam is movable in a second direction, the second direction being substantially perpendicular to the first direction. 
     
     
         3 . The apparatus of  claim 1 , wherein the light beam is fixed and the microarray is movable in a first direction and wherein the microarray is also movable in a second direction, wherein the second direction is substantially perpendicular to the second direction. 
     
     
         4 . The apparatus of  claim 3  further including:
 a frame movably attached to a base and configured to receive the microarray cassette, 
 a first frame positioner attached to the base and configured to directionally move the frame along a first axis, and 
 a second frame positioner attached to the base and configured to directionally move the frame along a second axis, the second axis being perpendicular to the first axis. 
 
     
     
         5 . The apparatus of  claim 1  wherein the emissions detector further comprises a dichroic filter positioned such that the fluorescent light emitted from the nucleic acids passes through the dichroic filter before entering the emissions detector. 
     
     
         6 . The apparatus of  claim 1  wherein the emissions detector is selected from the group consisting of a photomultiplier tube, an avalanche photodiode, a photo sensor, and a digital camera. 
     
     
         7 . The apparatus of  claim 1  wherein the fluid heating unit comprises an electrical heating element. 
     
     
         8 . The apparatus of  claim 1  wherein the fluid heating unit is configured for the transfer of thermal energy with the fluid circulation system. 
     
     
         9 . The apparatus of  claim 8  wherein the thermal heating unit is configured for the transfer of thermal energy with the microarray chamber. 
     
     
         10 . The apparatus of  claim 9  wherein the thermal heating unit comprises a thermoelectric Peltier device. 
     
     
         11 . The apparatus of  claim 1  further comprising a waste line in fluid communication with one of the fluid supply line and the fluid return line. 
     
     
         12 . The apparatus of  claim 11  further including a waste reservoir in fluid communication with the waste line configured to collect waste fluid from the fluid circulation system. 
     
     
         13 . The apparatus of  claim 1  wherein the fluid reservoir includes a preheater. 
     
     
         14 . The apparatus of  claim 1  wherein the fluid reservoir comprises a plurality of chambers configured to contain a plurality of distinct fluids for introduction to the fluid circulation system. 
     
     
         15 . The apparatus of  claim 14  further comprising a selector configured to introduce a specific fluid into the fluid circulation system from the plurality of distinct fluids. 
     
     
         16 . A microarray cassette assembly for use in an apparatus for conducting a melting curve analysis of nucleic acids, the microarray cassette assembly comprising:
 a frame, having an opening including an inside perimeter, the opening being configured to receive a microarray slide and a spacer disposed about an inside perimeter of the opening;   an optical window attached to the frame and disposed on one side of the opening;   a fluid inlet port,   a fluid outlet port; and   a seal attached to the frame opposite the optical window,   wherein the spacer is configured such that when the microarray slide is disposed within the opening, the microarray slide is spaced apart from the optical window, the space between the microarray slide and the optical window defining a chamber, and   wherein the chamber is configured to receive a fluid through the inlet port, the fluid having a flow that is distributed across the microarray slide, and discharged from the microarray chamber through the outlet port.   
     
     
         17 . An apparatus for conducting a melting curve analysis of nucleic acids on a microarray by exciting a predefined portion of the microarray with a light beam and detecting emissions of fluorescent light comprising:
 an illuminator assembly, including a light source configured to produce the light beam, the light beam comprising electromagnetic radiation having one or more wavelengths,   an emissions detector configured to sense emissions of fluorescent light from the predefined portion of the microarray;   a fluid circulation system, including
 a microarray cassette assembly defining a microarray chamber, the micro array cassette assembly including a fluid inlet port and a fluid outlet port, 
   a pump configured to receive fluid from a fluid reservoir and to discharge the fluid through a fluid inlet line coupled to the fluid inlet port,   a fluid outlet line coupled to the fluid outlet port, and   a temperature control assembly including a temperature controller configured to regulate the temperature of a fluid within the fluid circulation system, and;   a microarray positioner, including
 a microarray cassette support movably attached to a base and configured to receive the microarray cassette, 
   a first support positioner attached to the base and configured to directionally move the microarray cassette support along a first axis, and   a second support positioner attached to the base and configured to directionally move the microarray cassette support along a second axis, the second axis being perpendicular to the first axis.