Process for the measurement of nucleic acids

• US 5,543,292 A
• Filed: 06/14/1993
• Issued: 08/06/1996
• Est. Priority Date: 06/16/1992
• Status: Expired due to Term

• Current Assignee: Hitachi, Ltd.
• Original Assignee: Hitachi, Ltd.
• Inventors: Imai, Kazumichi, Nomura, Yasushi, Imai, Kyoko
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): CAMPBELL, EGGERTON A
• Application Number:
  US08/076,165
• Time in Patent Office: 1,149 Days
• Field of Search: 435/6, 935/77, 935/78
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2537/162 Helper probe
• CPC Class Codes: G01N 15/1409 Handling samples, e.g. injecting samples
• CPC Class Codes: G01N 2035/00485 combined with sample carriers
• CPC Class Codes: G01N 35/025 having a carousel or turntable for reaction cells or cuvettes

Abstract

A process for detecting the existence of at least one sequence of oligonucleotide in a nucleic acid sample includes at least one performance of a step of mixing the sample with

a labeled common polynucleotide, and

a polynucleotide probe comprising a sequence complementary to at least a part of the sequence of oligonucleotide and a sequence complementary to at least a part of the labeled common polynucleotide. The existence of a sequence of oligonucleotide which is an analyte existing in a nucleic acid sample can be detected by the use of a common reagent and an unattached polynucleotide having a base sequence specific for the analyte to be measured.

Claims

1. A process for detecting the existence of at least two target polynucleotide sequences of oligonucleotide analytes in a nucleic acid sample, comprising the steps of;

(a) mixing said nucleic acid sample with a labeled first common polynucleotide, and a second polynucleotide as a probe which is hybridizable with a first target polynucleotide sequence of oligonucleotide and with the labeled first common polynucleotide, to detect the existence of the first target polynucleotide sequence of oligonucleotide; and

(b) mixing said nucleic acid sample with the labeled first common polynucleotide used in step (a) for detecting said first target polynucleotide sequence of oligonucleotide, and a third polynucleotide as a probe which is hybridizable with a second target polynucleotide sequence of oligonucleotide and with the labeled first common polynucleotide, to detect the existence of the second target polynucleotide sequence of oligonucleotide.

2. A process according to claim 1, which further comprises a step of (c) cleaving or dissociating a double-stranded portion formed by the labeled first common polynucleotide and the third polynucleotide, by the action of a restriction enzyme recognizing said portion, and a step of (d) detecting a signal derived from a label liberated by the cleavage.

6. A process according to claim 1, which further comprises a step of (c) cleaving or dissociating a double-stranded portion formed by the labeled first common polynucleotide and the second polynucleotide, by the action of a restriction enzyme recognizing said portion, and a step of (d) detecting a signal derived from a label liberated by the cleavage.

9. A process according to claim 1, wherein the labeled first common polynucleotide is labeled with particles by which the labeled first common polynucleotide is distinguished for detection of a target polynucleotide sequence of oligonucleotide hybridized thereto.

10. A process according to claim 2, wherein the labeled first common polynucleotide is labeled with particles by which the labeled first common polynucleotide is distinguished for detection of a target polynucleotide sequence of oligonucleotide hybridized thereto.

3. A process for detecting the existence of at least two target polynucleotide sequences of oligonucleotide analytes in a nucleic acid sample, comprising the steps of:

(a) mixing said nucleic acid sample with a labeled first common polynucleotide, a second polynucleotide as a probe which is hybridizable with a first target polynucleotide sequence of oligonucleotide and with the labeled first common polynucleotide, a third common polynucleotide attached to a solid phase and which is selected without regard to said first target polynucleotide sequence of oligonucleotide, and a fourth polynucleotide as a probe which is hybridizable with said first target polynucleotide sequence of oligonucleotide and with the third common polynucleotide attached to the solid phase, to detect the existence of the first target polynucleotide sequence of oligonucleotide; and

(b) mixing said nucleic acid sample with the labeled first common polynucleotide used in step (a) for detecting said first target polynucleotide sequence of oligonucleotide, a fifth polynucleotide as a probe which is hybridizable with a second target polynucleotide sequence of oligonucleotide and with the labeled first common polynucleotide, the third common polynucleotide attached to the solid phase and used in step (a), and a sixth polynucleotide as a probe which is hybridizable with said second target polynucleotide sequence of oligonucleotide and with the third common polynucleotide attached to the solid phase, to detect the existence of the second target polynucleotide sequence of oligonucleotide.

4. A process according to claim 3, which further comprises a step of (c) cleaving or dissociating a double-stranded portion formed by the labeled first common polynucleotide and the fifth polynucleotide, by the action of a restriction enzyme recognizing said portion, and a step of (d) detecting a signal derived from a label liberated by the cleavage.

5. A process according to claim 3, which further comprises a step of (c) cleaving or dissociating a double-stranded portion formed by the third common polynucleotide and the sixth polynucleotide, by the action of a restriction enzyme recognizing said portion, and a step of (d) detecting a signal derived from a label liberated by the cleavage.

7. A process according to claim 3, which further comprises a step of (c) cleaving or dissociating a double-stranded portion formed by the labeled first common polynucleotide and the second polynucleotide, by the action of a restriction enzyme recognizing said portion, and a step of (d) detecting a signal derived from a label liberated by the cleavage.

8. A process according to claim 3, which further comprises a step of (c) cleaving or dissociating a double-stranded portion formed by the third common polynucleotide and the fourth polynucleotide, by the action of a restriction enzyme recognizing said portion, and a step of (d) detecting a signal derived from a label liberated by the cleavage.

11. A process according to claim 3, wherein the labeled first common polynucleotide is labeled with particles by which the labeled first common polynucleotide is distinguished for detection of a target polynucleotide sequence of oligonucleotide hybridized thereto.

12. A process according to claim 4, wherein the labeled first common polynucleotide is labeled with particles by which the labeled first common polynucleotide is distinguished for detection of a target polynucleotide sequence of oligonucleotide hybridized thereto.

13. A process according to claim 5, wherein the labeled first common polynucleotide is labeled with particles by which the labeled first common polynucleotide is distinguished for detection of a target polynucleotide sequence of oligonucleotide hybridized thereto.