Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-30
2001-05-08
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06228592
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method for the detection of a target nucleic acid in the cytoplasm of a living cell. More specifically, it relates to a method for the detection of a target nucleic acid existing in the cytoplasm of a living cell through hybridization by using probes labeled with fluorescent dyes.
2. Related Background Art
Hybridization is known as one of the methods to detect a nucleic acid having a specified base sequence (hereafter referred to as “target nucleic acid”). This method employs an oligonucleotide probe having a base sequence capable of hybridizing to the target nucleic acid as a detection probe to form a hybrid, and performs detection of the target nucleic acid by detecting the hybrid through various detection means.
Hybridization methods in the prior art, however, suffer from the drawbacks described below. Thus it will be difficult to apply them to detecting a target nucleic acid in the cytoplasm of a living cell. In other words, when a detection probe is introduced into the cytoplasm, it will rapidly move to the nucleus. This makes it difficult to allow the probe to form a hybrid with the target nucleic acid existing in the cytoplasm. In addition, the detection probe, which has been introduced into the cytoplasm, or the hybrid between the detection probe and the target nucleic acid is rapidly digested by various kinds of nuclease existing in the cytoplasm, which renders the detection of the target nucleic acid difficult.
SUMMARY OF THE INVENTION
The present inventors have discovered that a detection probe having a specified structure does not rapidly move to nucleus when introduced into the cytoplasm of a living cell and that it is not easily digested by nuclease, and thus have accomplished this invention. Specifically, when the detection probe having the specified structure is introduced into the cytoplasm, the probe does not readily move into the nucleus; nor is it readily digested by the nuclease. The probe forms a hybrid with a target nucleic acid existing in the cytoplasm, and the hybrid will be detectable without being subjected to digestion by the nuclease. Accordingly, when such a detection probe is used, the method for the detection of a target nucleic acid existing in the cytoplasm of a living cell (hereafter referred to as “the detection method of this invention) will be attained.
Specifically, the detection probe to be used in the method of this invention has the following characteristics: it is an oligonucleotide probe having a base sequence capable of hybridizing to the specified base sequence of a target nucleic acid; it is provided with a molecule that prevents the movement of the oligonucleotide into nucleus through nuclear membrane pores, and preferably, blocks the digestion of the oliginucleotide by nuclease; and it is provided with a fluorescent label that only allows the detection of the hybrid with the target nucleic acid.
Preferably, the detection probe to be used in the detection method of this invention is a pair of probes comprising two types of oligonucleotide probe which are respectively bound to an energy donor fluorescent dye (also referred to as “donor fluorescent dye” or simply as “donor” hereafter) and an energy acceptor fluorescent dye (also referred to as “acceptor fluorescent dye” or simply as “acceptor” hereafter) such that fluorescence resonance energy transfer (also referred to as “FRET” hereafter) can take place to allow the detection of only the hybrid with the target nucleic acid; each of the probes is an oligonucleotide probe having a base sequence capable of hybridizing to the target nucleic acid adjacently with each other with the result of forming the hybrid.
Specifically, this invention is characterized in that it employs the detection probe having such a specified structure, and the following detection method is provided in accordance with the invention:
A method for detecting a target nucleic acid existing in cytoplasm of a living cell, the method comprising:
introducing into the cytoplasm, a detection probe bound to a nuclear membrane unpermeable molecule via a linker and labeled with a fluorescent dye, the probe having a base sequence capable of hybridizing to the target nucleic acid;
forming a hybrid between the target nucleic acid and the probe; and
determining any change in fluorescence of the fluorescent dye due to formation of the hybrid.
According to this invention, there is further provided:
The method for detecting a target nucleic acid existing in the cytoplasm of a living cell as described above, wherein the detection probe comprises a first probe member and a second probe member, the first and second probe members have base sequences capable of hybridizing to the target nucleic acid adjacently with each other, the first probe member is labeled with an energy donor fluorescent dye and the second probe member is labeled with an energy acceptor fluorescent dye, and the change in fluorescence of the fluorescent dyes is fluorescence resonance energy transfer from the fluorescent dye of the first probe member to the fluorescent dye of the second probe member.
This invention provides the detection method as described above wherein the nuclear membrane unpermeable molecule is preferably at least one member selected from the group consisting of proteins, sugars, beads, and metal particles that have sizes sufficient so as not to pass through the nuclear membrane pores. More preferably, the protein includes streptavidin and avidine. Also, the sugar includes dextran.
In the detection method described above, the detection probe may further contain a molecule that blocks digestion of an oligonucleotid by nuclease (hereafter referred to as “nuclease-blocking molecule” and that is bound to the detection probe via a linker. Here, it is preferred that the nuclease-blocking molecule be identical with the nuclear membrane unpermeable molecule. This means that the nuclear membrane unpermeable molecule for use in the invention is provided with the function of a nuclease-blocking molecule.
This invention provides the detection method as described above wherein the nuclear membrane unpermeable molecule is characterized in that it is preferably at least one member selected from the group consisting of proteins, sugars, beads, and metal particles. More preferably, the protein includes streptavidin and avidine. Also, the sugar includes dextran.
In the detection method described above, the detection probe is preferably an oligonucleotide comprising from 10 to 20 bases.
Further, in the detection method described above the target nucleic acid to be detected is preferably messenger RNA (mRNA).
Since the detection method of the invention is highly specific, it will become possible to detect only the target nucleic acid with high sensitivity despite that a large number of nucleic acids of other kinds are present in a living cell.
The present invention will be more fully understood from the detailed description given hereinbelow and the accompanying drawings, which are given by way of illustration only and are not to be considered as limiting the present invention.
Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description.
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patent: 4996143 (1991-02-01), Heller et al.
patent: 5225326 (1993-07-01), Bresser et al.
patent: 5728527 (1998-03-01), Singer et al.
patent: 5985549 (1999-11-01), Singer et al.
patent: WO93/23570 (1993-11-01), None
patent: WO98/13524 (1998-04-01), None
patent: WO98/1324 (1998-08-01), None
Sixou et al., Nucleic Acids Research 22 : 662-668 (1994).*
Zobel et al., Antisense & Nucleic Acid Drug Development 7 :483-493 (1997).*
J. R. Lakow
Hirano Masahiko
Ishibashi Kaname
Koshimoto Hiroyuki
Tsuji Akihiko
Laboratory of Molecular Biophotonics
Leydig , Voit & Mayer, Ltd.
Whisenant Ethan
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