Method for determining a concentration of target nucleic acid molecules, nucleic acid probes for the method, and method for analyzing data obtained by the method

• US 6,492,121 B2
• Filed: 11/29/2000
• Issued: 12/10/2002
• Est. Priority Date: 04/20/1999
• Status: Expired due to Term

• Current Assignee: National Institute of Advanced Industrial Science and Technology (Government of Japan), Nippon Steel Kankyo Engineering Company Limited (Government of Japan)
• Original Assignee: Agency of Industrial Science and Technology, Japan Bioindustry Association, Kankyou Engineering Co., Ltd.
• Inventors: Kurata, Shinya, Koyama, Osamu, Kanagawa, Takahiro, Yamada, Kazutaka, Furusho, Kenta, Yokomaku, Toyokazu, Kurane, Ryuichiro, Kamagata, Yoichi
• Primary Examiner(s): Fredman, Jeffrey
• Application Number:
  US09/725,265
• Publication Number:
  US 20010000175A1
• Time in Patent Office: 741 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 536/23.1, 536/24.3, 536/24.31, 536/24.33
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2565/549 characterised by the capture oligonucleotide being a reporter labelled capture oligonucleotide

Abstract

A method is provided for determining a concentration of a target nucleic acid by using a nucleic acid probe labeled with a fluorescent dye. The method comprises:

providing, as the probe, a nucleic acid probe capable of reducing fluorescence emission from the fluorescent dye when hybridized with the target nucleic acid;

hybridizing the probe to the target nucleic acid; and measuring a decrease in fluorescence emission from the fluorescent dye after the hybridization relative to fluorescence emission from the fluorescent dye before the hybridization.

Claims

1. A method for determining a concentration of a target nucleic acid by using a nucleic acid probe labeled with only a fluorescent dye, which comprises:

providing, as said probe, a nucleic acid probe capable of reducing fluorescence emission from said fluorescent dye when hybridized with said target nucleic acid;

hybridizing said probe to said target nucleic acid;

measuring a decrease in fluorescence emission from said fluorescent dye after said hybridization relative to fluorescence emission from said fluorescent dye before said hybridization, wherein only said nucleic acid probe is provided as a nucleic acid probe in the reaction system of the hybridization; and

correlating a rate of said decrease in fluorescence emission from said fluorescent dye after said hybridization to the concentration of the target nucleic acid.

2. The method according to claim 1, wherein said probe has G or C as a 3′

end base and is labeled at said 3′

end thereof with said fluorescent dye.

3. The method according to claim 1, wherein the nucleic acid probe has G or C as a 5′

end base and is labeled at said 5′

end thereof with said fluorescent dye.

4. The method according to claim 1, wherein a hydroxyl group of a 2′

or 3′

carbon of a ribose or a 3′

carbon of a deoxyribose at 3′

end of said probe has been phosphorylated.

5. The method according to claim 1, wherein an oligoribonucleotide of said probe is a chemically-modified nucleic acid.

6. The method according to claim 1, wherein an oligonucleotide of said probe is a chimeric oligonucleotide comprising a ribonucleotide and a deoxyribonucleotide.

7. The method according to claim 6, wherein said ribonucleotide is a 2′

-O-methyloligoribonucleotide.