Nucleic hybridization assay method and device using a cleavage technique responsive to the complementary double strand or the single strand of nucleic acids or oligonucleotides

Abstract

A cleavable signal element applicable to quantitative and qualitative assay devices, using a cleavable technique specifically responsive to a complementary double strand or single strand of nucleic acids, and a nucleic acid hybridization assay method and device using the cleavable signal element are provided. Using the cleavable technique responsive to the complementary double strand or single strand of nucleic acids, detection sensitivity to a target nucleic acid can be increased, and diagnosis and detection reliability can be improved twice through in-situ determinations. Through simultaneous single nucleotide polymorphism (SNP) detection and expression profile determination, more accurate diagnosis for many diseases can be achieved. The assay device can be easily modified to be suitable for detection with general laser-based detection systems such as CD-ROM readers. Information read from the assay device is digitized as software and transmitted to and received by doctors and patients through a computer network or wirelessly, which enables construction of remote diagnosis systems.

What is claimed is:  
     
         1 . A cleavable signal element comprising: 
 a restriction probe of a single strand having a particular sequence cleavable by a restriction enzyme specific to a double strand of a particular sequence; and    a capture probe of a single strand having a complementary sequence to a target nucleic acid for diagnosis or assay to form a double strand through hybridization to the target nucleic acid,    wherein one end of the restriction probe is attached to a solid support substrate, and the other end of the restriction probe is ligated to the capture probe, thus forming a single-stranded, cleavable capture probe.    
     
     
         2 . The cleavable signal element of  claim 1 , wherein when the capture probe contacts a sample including the target nucleic acid of the complementary sequence, the capture probe is double-stranded through hybridization to the target nucleic acid, the restriction probe is double-stranded through DNA extension using the target nucleic acid hybridized to the capture probe as a primer with the addition of a DNA polymerization solution, the double-stranded restriction probe is cleaved by the restriction enzyme, and the cleaved cleavable capture probe is removed from the solid support substrate through washing, thus resulting in a cleaved signal element; and 
 when the capture probe contacts a sample not including the target nucleic acid of the complementary sequence, the single-stranded, cleavable capture probe remains attached to the solid support substrate even after additions of the DNA polymerization solution and the restriction enzyme and washing, thus resulting in an uncleaved signal element.    
     
     
         3 . The cleavable signal element of  claim 2 , wherein the DNA polymerization solution comprises a solution of four dNTPs and a DNA polymerase solution.  
     
     
         4 . A cleavable signal element comprising a capture probe of a single strand having a complementary sequence to a target nucleic acid for diagnosis or assay to form a double strand through hybridization to the target nucleic acid, wherein one end of the capture probe is attached to a solid support substrate, and the capture probe itself forms a single-stranded, cleavable capture probe which is cleavable by a cleavage enzyme specifically responsive to a double strand or single strand of nucleic acids.  
     
     
         5 . The cleavable signal element of  claim 4 , wherein when the capture probe contacts a sample including the target nucleic acid of the complementary sequence, the capture probe is double-stranded through hybridization to the target nucleic acid, the double-stranded capture probe is cleaved by the cleavage enzyme specifically responsive to the double strand of nucleic acids, and the cleaved cleavable capture probe is removed from the solid support substrate through washing, thus resulting in a cleaved signal element; and 
 when the capture probe contacts a sample not including the target nucleic acid of the complementary sequence, the single-stranded, cleavable capture probe remains attached to the solid support substrate even after the addition of the cleavage enzyme and washing, thus resulting in an uncleaved signal element.    
     
     
         6 . The cleavable signal element of  claim 5 , wherein the cleavage enzyme is a DNAse.  
     
     
         7 . The cleavable signal element of  claim 4 , wherein when the capture probe contacts a sample not containing the target nucleic acid of the complementary sequence, the capture probe remains as a single strand without hybridization, the single-stranded capture probe is cleaved by the cleavage enzyme specifically responsive to the single strand of nucleic acids, and the cleaved cleavable capture probe is removed from the solid support substrate through washing, thus resulting in a cleaved signal element; and 
 when the capture probe contacts a sample including the target nucleic acid of the complementary sequence, the capture probe is double-stranded through hybridization to the target nucleic acid, and the double-stranded, cleavable capture probe remains attached to the solid support substrate even after the addition of the cleavage enzyme and washing, thus resulting in an uncleaved signal element.    
     
     
         8 . The cleavable signal element of  claim 7 , wherein the cleavage enzyme is a nuclease.  
     
     
         9 . The cleavable signal element of  claim 8 , wherein the nuclease is derived from mung bean.  
     
     
         10 . The cleavable signal element of  claim 1  or  4 , wherein the solid support substrate is a plastic substrate, a glass substrate, a silicon substrate, or a gold substrate.  
     
     
         11 . The cleavable signal element of  claim 1  or  4 , wherein the solid support substrate has a self-assembled monolayer (SAM) on the surface.  
     
     
         12 . The cleavable signal element of  claim 1  or  4 , wherein the capture probe has a length ranging from about 5- to about 30-mers.  
     
     
         13 . The cleavable signal element of  claim 1  or  4 , wherein a label is attached to one end of the cleavable capture probe to form a label-attached cleavable capture probe structure or to one end or side of an uncleaved probe to form a label-attached uncleaved probe structure, to increase detection sensitivity for an uncleaved signal element.  
     
     
         14 . The cleavable signal element of  claim 13 , wherein the label comprises a metal microsphere, a conducting polymer, a fluorescent dye, a magnetic microsphere, and a streptavidin-labeled microsphere.  
     
     
         15 . The cleavable signal element of  claim 14 , wherein the metal microsphere is formed of a metal selected from the group consisting of gold, silver, nickel, platinum, chromium, and copper.  
     
     
         16 . The cleavable signal element of  claim 15 , wherein a gold microsphere has a diameter ranging from about 1 nm to about 10 μm.  
     
     
         17 . The cleavable signal element of  claim 13 , wherein the streptavidin-labeled microsphere is attached to the cleavable capture probe via biotin.  
     
     
         18 . A nucleic acid hybridization assay device comprising: 
 a solid support substrate;    a plurality of cleavable signal elements according to any of claims  1  through  17  attached to the solid support substrate; and    an internal or external detector which detects a uncleaved signal element and a cleaved signal element from the plurality of cleavable signal elements.    
     
     
         19 . The nucleic acid hybridization assay device of  claim 18 , wherein the detector comprises an optical device, an electrochemical device, a mass measurement device, or a capacitance and impedance measurement device.  
     
     
         20 . The nucleic acid hybridization assay device of  claim 19 , wherein the optical device detects fluorescence of the uncleaved signal element and cleaved signal element.  
     
     
         21 . The nucleic acid hybridization assay device of  claim 19 , wherein the detector detects a differential reflective signal or a is differential conductive signal of the uncleaved signal element and the cleaved signal element.  
     
     
         22 . The nucleic acid hybridization assay device of  claim 21 , wherein the detector detects the differential reflective signal by measuring the reflectance, absorbance, or scattering of light or a laser beam incident on the uncleaved signal element and the cleaved signal element.  
     
     
         23 . The nucleic acid hybridization assay device of  claim 21 , wherein the detector detects the differential conductive signal by measuring the capacitance and impedance of the uncleaved signal element and the cleaved signal element.  
     
     
         24 . The nucleic acid hybridization assay device of  claim 23 , wherein the capacitance and impedance measurement device measures the frequency response characteristics of the uncleaved signal element and the cleaved signal element.  
     
     
         25 . The nucleic acid hybridization assay device of  claim 23 , wherein the capacitance and impedance measurement device comprises interdigitated array electrodes having at least one digit and arranged on the solid support substrate.  
     
     
         26 . The nucleic acid hybridization assay device of  claim 25 , wherein the interdigitated array electrodes have an input port to check for the frequency response characteristics, and the input port is connected to an electronic control device which generates a frequency signal of a constant bandwidth.  
     
     
         27 . The nucleic acid hybridization assay device of  claim 25 , wherein a plurality of cleavable signal elements are deposited on the interdigitated array electrodes.  
     
     
         28 . The nucleic acid hybridization assay device of  claim 25 , wherein the plurality of cleavable signal elements are deposited only in the space between the interdigitated array electrodes.  
     
     
         29 . The nucleic acid hybridization device of  claim 25 , wherein the interdigitated array electrodes are substantially formed of gold.  
     
     
         30 . The nucleic acid hybridization device of  claim 18 , wherein a label-attached uncleaved probe structure is formed on the solid support substrate by attaching a label to the uncleaved signal element to increase the sensitivity of the detector.  
     
     
         31 . The nucleic acid hybridization device of  claim 18 , wherein the plurality of cleavable signal elements are deposited on the solid support substrate in a spatially-addressable pattern.  
     
     
         32 . The nucleic acid hybridization device of  claim 31 , wherein the spatially-addressable pattern enables a single-sample assay for multiple analytes, a multiple-sample assay for a single analyte, or a multiple sample assay for multiple analytes.  
     
     
         33 . The nucleic acid hybridization device of  claim 31 , wherein the solid support substrate is a plastic substrate formed of a material selected from the group consisting of polypropylenes, polyacrylates, polyvinyl alcohols, polyethylenes, polymethylmethacrylates, and polycarbonates.  
     
     
         34 . The nucleic acid hybridization device of  claim 33 , wherein the solid support substrate is formed of polycarbonates.  
     
     
         35 . The nucleic acid hybridization device of  claim 31 , wherein the solid support substrate is formed of a circular disk.  
     
     
         36 . The nucleic acid hybridization device of  claim 31 , wherein the solid support substrate is formed of a rectangular disk.  
     
     
         37 . The nucleic acid hybridization device of  claim 35 , wherein the circular disk has a diameter of approximately 120 mm and a thickness of approximately 1.2 mm.  
     
     
         38 . The nucleic acid hybridization device of  claim 35 , wherein the circular disk comprises: 
 a central void to engage a rotational drive means;    a sample injection port through which a sample is injected; and    an annular and/or a spiral track in which the plurality of cleavable signal elements are deposited in the spatially-addressable pattern.    
     
     
         39 . The nucleic acid hybridization device of  claim 38 , wherein an address pattern that provides coded address information is formed on the circular disk.  
     
     
         40 . The nucleic acid hybridization device of  claim 35 , wherein the circular disk comprises: 
 a central void to engage a rotational drive means;    a sample injection port through which a sample is injected; and    a radial assay sector in which the plurality of cleavable signal elements are deposited in the spatially-addressable pattern.    
     
     
         41 . The nucleic acid hybridization device of  claim 40 , wherein the circular disk comprises a plurality of assay sectors.  
     
     
         42 . The nucleic acid hybridization device of  claim 41 , wherein the plurality of assay sectors are connected to respective separate sample injection ports.  
     
     
         43 . The nucleic acid hybridization device of  claim 41 , wherein the plurality of assay sectors are connected to a common sample injection port.  
     
     
         44 . The nucleic acid hybridization device of  claim 41 , wherein the plurality of cleavable signal elements are deposited in each of the plurality of assay sectors in an appropriate pattern for a single-analyte assay or a multiple-analyte assay.  
     
     
         45 . The nucleic acid hybridization device of  claim 35 , comprising a plurality of circular disks.  
     
     
         46 . The nucleic acid hybridization device of  claim 35 , wherein the circular disk includes in a central track a database associated with bioinformatics required for diagnosis and assay interpretation, and telephone numbers, web link information and software required for remote diagnosis.  
     
     
         47 . The nucleic acid hybridization device of  claim 35 , wherein a detector is mounted on the circular disk.  
     
     
         48 . The nucleic acid hybridization device of  claim 47 , wherein the detector comprises a non-contact interface through which information read from the cleaved signal element and the uncleaved signal element is transmitted to an external central controller or storage device.  
     
     
         49 . The nucleic acid hybridization assay device of  claim 48 , wherein the non-contact interface comprises an infrared interface and an optical interface.  
     
     
         50 . The nucleic acid hybridization assay device of  claim 49 , wherein the infrared interface is an infrared sensor, and the optical interface is a photosensor.  
     
     
         51 . The nucleic acid hybridization assay device of  claim 35 , wherein the circular disk simultaneously comprises at least one SNP (single nucleotide polymorphism) assay sector for SNP detection and at least one expression assay sector for expression profile analysis.  
     
     
         52 . The nucleic acid hybridization assay device of  claim 51 , wherein the SNP assay sector and the expression assay sector are arranged separate in an angular direction or in a radial direction.  
     
     
         53 . A bio-driver apparatus comprising: 
 a rotary disk receiver onto which the nucleic acid assay device according to any of claims  18  through  52  is to be loaded;    a motor driver which rotates the disk;    a rotary connector which connects the motor driver to a central void portion of the disk such that the disk is rotatable; and    an optical device to write data in or to read data from the disk.    
     
     
         54 . The bio-driver apparatus of  claim 53 , further comprising a central controller which transmits information read from the disk by the optical device to an external storage unit, transmits information to be written to the optical device, and generates and outputs a variety of control signals for the motor driver and the other elements.  
     
     
         55 . The bio-driver apparatus of  claim 53 , wherein the rotary connector comprises an upper rotor and/or a lower rotor, the upper and lower rotors being pushed close to the top and bottom surfaces, respectively, of the central void portion when the disk begins to rotate.  
     
     
         56 . The bio-driver apparatus of  claim 53 , wherein the optical device detects a differential reflective signal by measuring the reflectance, absorbance, or scattering of incident light or an incident laser beam.  
     
     
         57 . The bio-driver apparatus of  claim 53 , wherein the optical device detects fluorescence.  
     
     
         58 . A bio-driver apparatus comprising: 
 a rotary disk receiver onto which the nucleic acid assay device according to any of claims  18  through  52  is to be loaded;    a motor driver which rotates the disk;    a rotary connector which rotatably connects the motor driver to a central void portion of the disk;    an external power connector which powers and/or supplies a control signal to a detector mounted on the disk; and    a non-contact interface through which information read by the detector is transmitted.    
     
     
         59 . The bio-driver apparatus of  claim 58 , further comprising a central controller which transmits information read from the disk by the detector to an external storage unit and generates and outputs a variety of control signals for the motor driver and the other elements.  
     
     
         60 . The bio-driver apparatus of  claim 58 , further comprising an optical device to write data in or to read data from the disk.  
     
     
         61 . The bio-driver apparatus of  claim 58 , wherein the detector detects a differential conductive signal by measuring capacitance and impedance.  
     
     
         62 . The bio-driver apparatus of  claim 58 , wherein the rotary connector comprises an upper rotor and/or a lower rotor, the upper and lower rotors being pushed close to the top and bottom surfaces, respectively, of the central void portion when the disk begins to rotate.  
     
     
         63 . The bio-driver apparatus of  claim 58 , wherein the power connector comprises a brush that frictionally contacts the upper and/or lower rotors in connection with an external power supply unit, and each of the upper and lower rotors comprises an annular electrode plate frictionally contacting the brush.  
     
     
         64 . The bio-driver apparatus of  claim 63 , wherein the annular electrode plate comprises at least one conductive arm connected thereto, and the central void portion of the disk comprises a hole to engage the at least one conductive arm and a circuit pattern connected to the hole to power the detector mounted on the disk and/or supply the control signal to the detector.  
     
     
         65 . The bio-driver apparatus of  claim 64 , wherein the at least one conductive arm has a spring at its one end that is connected to the annular electrode plate.  
     
     
         66 . The bio-driver apparatus of  claim 58 , wherein the power connector comprises an electromagnet attached to the rotary disk receiver in connection with the external power supply unit, and the electromagnet induces an AC voltage to a wound coil on the disk so that the detector is powered in a non-contact manner.  
     
     
         67 . The bio-driver apparatus of  claim 66 , wherein the disk further comprises a rectifier for rectifying the AC voltage induced to the wound coil.  
     
     
         68 . A remote diagnostic system comprising: 
 the nucleic acid hybridization assay device according to any of claims  18  through  52 ;    an existing communication network; and    a computer in which software capable of controlling access to the existing communication network and digitizing information read from the nucleic acid hybridization assay device is installed,    wherein the digitized information from the nucleic acid assay hybridization assay device is transmitted to a doctor or a hospital, and a patient is provided with a prescription, through the existing communication network.    
     
     
         69 . The remote diagnostic system of  claim 68 , wherein the computer comprises assay interpretive algorithms, bioinformatics information, and self-diagnostics related software.  
     
     
         70 . The remote diagnostic system of  claim 68 , wherein the computer comprises software capable of uploading diagnostic information to remote locations and device drivers.  
     
     
         71 . The remote diagnostic system of  claim 70 , wherein the software comprises educational information for patients on clinical assays, a variety of wet sites and links enabling a patient to directly communicate with a doctor or hospital based on his/her diagnosis result.  
     
     
         72 . The remote diagnostic system of  claim 68 , wherein the computer comprises a camera and a microphone for viewing a patient's face and listening to his/her voice.  
     
     
         73 . The remote diagnostic system of  claim 68 , wherein the diagnostic data based on the digitized information are displayed on a computer monitor, the computer automatically or manually transmits the diagnostic data to a specialist through the existing communication network, and the patient waits for a prescription from the specialist.  
     
     
         74 . A nucleic acid hybridization assay method comprising: 
 hybridizing a capture probe to a target nucleic acid present in a liquid sample by contacting the nucleic acid hybridization assay device according to any of claims  18  through  52  with the liquid sample;    contacting the cleavable capture probe with a restriction enzyme or cleavage enzyme which is specifically responsive to a cleavable signal element depending on whether the capture probe and the target nucleic acid are hybridized or not;    washing the nucleic acid hybridization assay device to remove the cleavable signal element cleaved by the restriction enzyme or cleavage enzyme; and    detecting whether the uncleaved signal element or the cleaved signal element exists on the solid support substrate.    
     
     
         75 . The nucleic acid hybridization assay method of  claim 74 , further comprising contacting the cleavage capture probe with a DNA polymerization solution before contact with the restriction enzyme.  
     
     
         76 . The nucleic acid hybridization assay method of  claim 75 , further comprising contacting the cleavage capture probe with a 3′-5′ exonuclease solution before contact with the DNA polymerization solution.  
     
     
         77 . The nucleic acid hybridization assay method of  claim 74 , further comprising attaching a label to the cleavable signal element before contacting the capture probe with the liquid sample, or to an uncleaved signal element after contacting the cleavable capture probe with the restriction enzyme or cleavage enzyme.  
     
     
         78 . The nucleic acid hybridization assay method of  claim 74 , further comprising at least one wash step.  
     
     
         79 . The nucleic acid hybridization assay method of  claim 74 , wherein washing is performing by rotating the nucleic acid hybridization assay device with or without addition of a detergent solution, or by applying an external electric field.  
     
     
         80 . A nucleic acid hybridization assay method comprising: 
 (a) injecting a sample into a sample injection port disposed near the center of a disk in a nucleic acid hybridization assay device;    (b) rotating the disk and stopping the rotation of the disk when the sample reaches an outer edge of the disk;    (c) incubating the disk in a stationary state at room temperature for hybridization;    (d) adding a buffer solution as a washing solution while rotating the disk at a high speed, to wash the disk;    (e) adding a DNA polymerization solution containing a mixed solution of four dNTPs and a DNA polymerase and incubating the disk in a stationary state for DNA extension;    (f) adding a solution of a restriction enzyme specifically responsive to a double strand of a particular sequence and incubating the disk in a stationary state, to cleave the double strand;    (g) washing the disk by rotating the disk at a high speed with the addition of a buffer solution or by applying an external electric or magnetic field; and    (h) drying the disk and reading information from the disk using a detector which is programmed to detect a predetermined assay site on which a cleavable signal element is deposited and comprises an optical device, an electrochemical device, or a capacitance and impedance measurement device.    
     
     
         81 . The nucleic acid hybridization assay method of  claim 80 , further comprising adding a 3′-5′ exonuclease solution before step (e) of DNA extension.  
     
     
         82 . A nucleic acid hybridization assay method comprising: 
 (a) injecting a sample into a sample injection port disposed near the center of a disk in a nucleic acid hybridization assay device;    (b) rotating the disk and stopping the rotation of the disk when the sample reaches an outer edge of the disk;    (c) incubating the disk in a stationary state at room temperature for hybridization;    (d) adding a buffer solution as a washing solution while rotating the disk at a high speed, to wash the disk;    (e) adding a solution of a cleavage enzyme specifically responsive to a double strand or single strand of nucleic acids and incubating the disk in a stationary state, to cleave the double strand or single strand;    (f) washing the disk by rotating the disk at a high speed with the addition of a buffer solution or by applying an external electric or magnetic field; and    (g) drying the disk and reading information from the disk using a detector which is programmed to detect a predetermined assay site on which a cleavable signal element is deposited and comprises an optical device, an electrochemical device, or a capacitance and impedance measurement device.    
     
     
         83 . The nucleic acid hybridization assay method of  claim 80  or  82 , further comprising attaching a label to the cleavable signal element before sample injection, or to an uncleaved signal element after strand cleavage, to increase detection sensitivity.