Method of detecting specific nucleic acid sequences

• US 5,814,447 A
• Filed: 11/29/1995
• Issued: 09/29/1998
• Est. Priority Date: 12/01/1994
• Status: Expired due to Term

• Current Assignee: Tosoh Corporation
• Original Assignee: Tosoh Corporation
• Inventors: Inoue, Teruhiko, Otsuka, Masami, Yawata, Hideo, Ishiguro, Takahiko, Sugiura, Yukio
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/564,650
• Time in Patent Office: 1,035 Days
• Field of Search: 435/6, 435/91.2, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]

Abstract

A labeled nucleic acid probe comprising a single-stranded oligonucleotide probe having a nucleic acid sequence complementary to a specific nucleic acid sequence in a target nucleic acid and an intercalating fluorochrome bound to said probe is used to detect the target nucleic acid by a convenient, single-stage method in a homogeneous system. The formation of a complementary bond between the probe and the target nucleic acid can be detected and the amount of the resulting complementary binding product determined without requiring any extra step such as for removing the excess probe which has not participated in the complementary binding.

Claims

1. In a method of assaying a target nucleic acid, or a nucleic acid in a sample of interest that has at least one specific nucleic acid sequence, said method using as a probe a single-stranded oligonucleotide having a nucleic acid sequence complementary to said specific nucleic acid sequence in the target nucleic acid and including the step of adding the probe to the sample such as to form a complementary bond with the target nucleic acid, the improvement wherein said probe is a single-stranded oligonucleotide labeled with an intercalating fluorochrome which is to be intercalated into the complementary binding portion between the target nucleic acid and the single-stranded oligonucleotide probe, wherein said probe and said target nucleic acid are not bound to a solid support.

3. A method according to claim 1, wherein said intercalating fluorochrome has such a property that a fluorescence characteristic thereof will change when it is intercalated into the complementary binding portion between the target nucleic acid and the single-stranded oligonucleotide probe.

2. In a method of assaying a target nucleic acid, or a double-stranded nucleic acid in a sample of interest that has at least one specific nucleic acid sequence, said method using as a probe a single-stranded oligonucleotide having a nucleic acid sequence complementary to said specific nucleic acid sequence in the target nucleic acid and including the step of adding the probe to the sample such as to form a triple-stranded nucleic acid with the target nucleic acid, the improvement wherein said probe is a single-stranded oligonucleotide labeled with an intercalating fluorochrome which is to be intercalated into the triple-stranded portion formed from the target nucleic acid and the single-stranded oligonucleotide probe, wherein said probe and said target nucleic acid are not bound to a solid support.

4. A method according to claim 2, wherein said intercalating fluorochrome has such a property that a fluorescence characteristic thereof will change when it is intercalated into the triple-stranded portion formed from the double-stranded target nucleic acid and the improvement wherein said probe is a single-stranded oligonucleotide labeled with an intercalating fluorochrome which is to be intercalated into the triple-stranded portion formed from the target nucleic acid and the single-stranded oligonucleotide probe.

5. In a method of assaying a target nucleic acid, or a nucleic acid in a sample of interest that has at least one specific nucleic acid sequence, which method includes the step of amplifying at least said specific nucleic acid sequence portion, said method using as a probe a single-stranded oligonucleotide having a nucleic acid sequence complementary to said specific nucleic acid sequence in the target nucleic acid and further including the step of adding the probe to the sample such as to form a complementary bond with the target nucleic acid, the improvement wherein said probe is a single-stranded oligonucleotide labeled with an intercalating fluorochrome which is to be intercalated into the complementary binding portion between the target nucleic acid and the single-stranded oligonucleotide probe, wherein said probe and said target nucleic acid are in solution not bound to a solid support.

7. A method according to claim 5 or 6, wherein the single-stranded oligonucleotide probe is added to the sample prior to the step of amplifying the specific nucleic acid sequence portion.

8. A method according to claim 5 or 6, wherein the step of amplifying the specific nucleic acid sequence portion is performed by a PCR (polymerase chain reaction) technique.

6. In a method of assaying a target nucleic acid, or a double-stranded nucleic acid in a sample of interest that has at least one specific nucleic acid sequence, which method includes the step of amplifying at least said specific nucleic acid sequence portion, said method using as a probe a single-stranded oligonucleotide having a nucleic acid sequence complementary to said specific nucleic acid sequence in the target nucleic acid and further including the step of adding the probe to the sample such as to form a complementary bond with the target nucleic acid, the improvement wherein said probe is a single-stranded oligonucleotide labeled with an intercalating fluorochrome which is to be intercalated into the triple-stranded portion formed from the target nucleic acid and the single-stranded oligonucleotide probe, wherein said probe and said target nucleic acid are not bound to a solid support.