Method for determining of nucleic acid analytes

• US 20050106587A1
• Filed: 06/21/2004
• Published: 05/19/2005
• Est. Priority Date: 12/21/2001
• Status: Abandoned Application

• Current Assignee: Micronas Gmbh (TDK Corporation)
• Original Assignee: Micronas Gmbh (TDK Corporation)
• Inventors: Klapproth, Holger, Lehmann, Mirko
• Application Number:
  US10/870,986
• Publication Number:
  US 20050106587A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00655 the compounds being bound to magnets embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 27/414 Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS

Abstract

The present invention concerns generally a method as well as an apparatus for the determination of nucleic acid analytes. In particular, the present invention concerns the detection of the presence of such analytes without the conventional use of optically detectable marker substances.

Claims

1. A method for the determination of a nucleic acid analyte by hybridizing the analyte on a suitable nucleic acid probe immobilized on a solid phase, wherein

(a) the nucleic acid analyte is incubated with the nucleic acid probe under suitable hybridization conditions to form a hybridization complex, and

(b) the analyte is determined on the basis of chemical/physical measurement data, which is specifically associated with a mass increase or decrease in the hybridization complex that is due to an individual enzymatic step,

whereby the measurement of the data is performed within a few seconds or minutes by at least one sensor, the sensor being an integral component of the solid phase and being selected from the group including electrode structures, field effect transistors, magnetic sensors, optical sensors, pH sensors, and combinations of the same.

2. The method recited in claim 1 wherein the steps (a) and (b) are carried out continuously (flow through).

3. The method as recited in claim 1, wherein the enzyme that effects the mass increase or decrease in the hybridization complex is selected from the group that includes polymerases, ligases, ribozymes, quasi-catalytic nucleic acids, DNases, RNases, and RNase H.

4. The method as recited in claim 1, wherein polymerase is selected as the enzyme.

5. The method as recited in claim 4, wherein the polymerase exhibits 5′

- and/or 3′

-exonuclease activity.

6. An apparatus for performing the method as recited claim 1, comprising a minimum of one solid phase, a minimum of one nucleic acid probe either directly or indirectly immobilized thereon, as well as a minimum of one sensor for recording the chemical/physical measurement data, wherein the sensor is an integral component of the solid phase.

7. The apparatus as recited in claim 6, wherein the minimum of one sensor is selected from the group including electrode structures, field effect transistors, magnetic sensors, optical sensors, and pH sensors, and combinations thereof.

8. The apparatus as recited in claim 6, wherein a plurality of different nucleic acid probes are arranged in a raster configuration forming a microarray.

9. The apparatus recited in claim 6, wherein each immobilized nucleic acid probe is associated with a minimum of one sensor.

10. The apparatus as recited in claim 6, comprising in addition a minimum of one heating element that is associated with a minimum of one sensor.