Nucleic acid hybridization assay method

• US 8,101,356 B2
• Filed: 09/16/2009
• Issued: 01/24/2012
• Est. Priority Date: 01/27/2001
• Status: Expired due to Fees

• Current Assignee: Jae Chern Yoo
• Original Assignee: Jae Chern Yoo
• Inventors: Yoo, Jae Chern
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US12/585,513
• Publication Number:
  US 20100129812A1
• Time in Patent Office: 860 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2565/625 being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

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.

Claims

1. A nucleic acid hybridization assay method comprising:

contacting a nucleic acid hybridization assay device with a liquid sample including a target nucleic acid, wherein the nucleic acid hybridization assay device comprises a cleavable signal element comprising a capture probe of a single strand having a complementary sequence to the target nucleic acid; and

a single stranded restriction probe not being complementary to the target nucleic acid, wherein one end of the single stranded restriction probe is ligated to the capture probe; and

a solid support substrate attached to the other end of the single stranded restriction probe;

hybridizing the cleavable signal element to the target nucleic acid present in the liquid sample;

making the single stranded restriction probe into a double stranded restriction probe by DNA extension in the presence of a DNA polymerization solution using the target nucleic acid hybridized to the capture probe as a primer;

cleaving the double stranded restriction probe by a restriction enzyme and/or the double stranded cleavable signal element by a cleavage enzyme, wherein the double stranded restriction probe is cleaved by the restriction enzyme specifically responsive to a double strand with a particular sequence and/or the cleavage enzyme is specifically responsive to double stranded nucleic acids;

washing the nucleic acid hybridization assay device to remove the cleavable signal element cleaved by the restriction enzyme and/or the cleavage enzyme; and

detecting whether the uncleaved signal element or the cleaved signal element exists on the solid support substrate.

3. The nucleic acid hybridization assay method of claim 1 or 2, wherein the cleavage enzyme is a DNase or a nuclease.

4. The nucleic acid hybridization assay method of claim 1 or 2, further comprising contacting the cleavable signal element with a 3′

-5′

exonuclease solution before contacting with the DNA polymerization solution.

5. The nucleic acid hybridization assay method of claim 1 or 2, further comprising attaching a label to one end of the capture probe, or to the uncleaved signal element.

6. The nucleic acid hybridization assay method of claim 5, the label is one or more selected from the group consisting of a metal microsphere, a conducting polymer, a fluorescent dye, a magnetic microsphere, and a streptavidin-labeled microsphere.

7. The nucleic acid hybridization assay method of claim 1 or 2, wherein whether the uncleaved signal element or the cleaved signal element exists on the solid support substrate is detected by a detector, which is one or more selected from the group consisting of an optical device, an electrochemical device, a mass measurement device, or a capacitance and impedance measurement device.

8. The nucleic acid hybridization assay method of claim 1 or 2, wherein the solid support substrate has the shape of a circular disk.

9. The nucleic acid hybridization assay method of claim 8, wherein washing is performing by rotating the solid support substrate, or by applying an external electric field.

10. The nucleic acid hybridization assay method of claim 1 or 2, further comprising diagnosing remotely a patient by

a communication network;

a computer to which the nucleic acid hybridization assay device is connected; and

a software installed in the computer, wherein the software is capable of controlling access to the communication network and digitizing information read from the nucleic acid hybridization assay device, wherein the digitized information is transmitted to a doctor or a hospital, and the patient is provided with a prescription, through the communication network.

2. A nucleic acid hybridization assay method comprising:

contacting a nucleic acid hybridization assay device with a liquid sample including a target nucleic acid, wherein the nucleic acid hybridization assay device comprises a cleavable signal element comprising a capture probe of a single strand having a complementary sequence to the target nucleic acid; and

a single stranded restriction probe not being complementary to the target nucleic acid, wherein one end of the single stranded restriction probe is ligated to the capture probe; and

a solid support substrate attached to the other end of the single stranded restriction probe;

hybridizing the cleavable signal element to the target nucleic acid present in the liquid sample;

maintaining the single stranded restriction probe remain as a single strand after DNA extension except when the single stranded restriction probe is changed into a double stranded restriction probe by the DNA extension, where the DNA extension is done in the presence of a DNA polymerization solution using the target nucleic acid hybridized to the cleavable signal element as a primer;

cleaving the single stranded restriction probe by a cleavage enzyme after the DNA extension, wherein the cleavage enzyme is specifically responsive to the single stranded nucleic acids;

washing the nucleic acid hybridization assay device to remove the cleavable signal element cleaved by the cleavage enzyme; and

detecting whether the uncleaved signal element or the cleaved signal element exists on the solid support substrate.