Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-01-31
2003-01-14
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C436S094000, C540S001000
Reexamination Certificate
active
06506567
ABSTRACT:
This application claims Paris Convention priority of Japanese Patent Application No. 2000-022181 filed Jan. 31, 2000, the complete disclosure of which is hereby incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to a fluorescent intercalator compound which is favorably employable in a procedure of analyzing oligonucleotides or polynucleotides such as DNA fragments by fluorometry.
BACKGROUND OF THE INVENTION
In the gene analysis in the fields of biochemistry and clinical test, the detection of a DNA or its fragment having a specific base sequence is performed by way of a hybridization method, particularly Southern hybridization method (Southern blotting method). Southern hybridization is performed using a radioisotope (RI) label. The conventional analytical methods using radioisotope label such as Southern hybridization method appear to be disadvantageous in that they need troublesome radioisotopes.
Recently, the hybridization is generally performed using a DNA chip in which a group of probe molecules of nucleotide derivatives or their analogues (such as PNA) are fixed on a substrate.
A Southern hybridization method using a fluorescent label in place of a radioisotope label is also known. This method appears to be superior to the method using RI in safety and rapidness in obtaining analytical results. Therefore, DNA chips comprising a substrate such as a slide glass or a silicone plate and a great number of oligonucleotide or polynucleotide molecules (i.e., probe molecules) fixed onto the substrate are now commercially available for the use in the fluorescence detection systems.
In the well known fluorescence detection system, a fluorescent label is attached to a target DNA fragment (i.e., DNA fragment to be analyzed in its base sequence). Thus labeled target DNA fragment is brought into contact with probe molecule of a DNA chip. If the target DNA fragment is complementary to the probe molecule, the target DNA fragment is combined with the probe molecule and hence fixed to the DNA chip. The DNA chip to which the target DNA fragment having fluorescent label is fixed is detected by fluorometry. This means that the question on whether or not the target DNA fragment is complementary to the probe molecule of the DNA chip is answered.
Recently, a new fluorescence detection system in which a fluorescent intercalator is utilized is developed. The new system is performed by the process including the following steps:
bringing a group of probe molecules fixed on a substrate into contact with oligonucleotide samples or polynucleotide samples in an aqueous medium in the presence of a fluorescent intercalator so as to form by hybridization a complex of the group of probe molecules and the oligonucleotide samples or polynucleotide samples on the substrate in which the fluorescent intercalator compound is intercalated; and
detecting a fluorescence emitted from the fluorescent intercalator compound intercalated into the hybridized complex on the substrate.
Bull. Chem. Soc. Jpn., 72, 327-337 (1999) indicates a compound having two fluorescent acridine dye such as N,N-bis{3-[4-(3-(6-chloro-9-imino-2-methoxyacridinyl)-propyl)-1,4-diazacyclohexyl]propyl}naphthalenediimide which is represented by the below-illustrated formula is employable as the fluorescent intercalator.
According to the study of the present inventors, it has been confirmed that when a fluorescent intercalator is brought in contact with a hybrid structure in an aqueous medium, a portion of the intercalator is intercalated into the hybrid structure and other portion remains in the aqueous medium without being intercalated into the hybrid structure In other words, equilibrium is present when an intercalator is kept in contact with a hybrid structure in an aqueous medium.
Accordingly, not only the fluorescent intercalator intercalated into the hybrid structure but also the fluorescent intercalator remaining in the aqueous medium emit fluorescence, and hence it is not easy to fluorometrically differentiate fluorescence emitted by the intercalator intercalated into the hybrid structure from fluorescence emitted by the intercalator dissolved in the aqueous medium. Therefore, in the presence of the aqueous medium containing the fluorescent intercalator, the hybridization is not clearly detected. In other words, S/N (signal
oise) ratio is not satisfactory high, so long as the above-mentioned fluorescent intercalator compound is employed.
Accordingly, it is an object of the present invention to provide a new fluorescent intercalator compound which is favorably employable in processes for fluorometrically detecting oligonucleotide samples or polynucleotide samples complementary to a group of probe molecules of nucleotide derivatives or their analogues fixed onto a substrate.
SUMMARY OF THE INVENTION
The present invention resides in a water-soluble fluorescent intercalator compound having the formula (I):
F—La—X (I)
in which F is a fluorescent moiety, X is a cyclic group, and La is a linking group, in which at least one of X and La has a site imparting water solubility to the compound or a site that is convertible into a site imparting water solubility to the compound.
In the formula (I), X preferably has a substituent group represented by the formula of —Lb—Z, in which Z is a non-fluorescent moiety and Lb is a linking group.
The present invention further resides in a process for fluorometrically detecting oligonucleotide samples or polynucleotide samples complementary to a group of probe molecules of nucleotide derivatives or their analogues fixed onto a substrate, which comprises the steps of:
bringing the group of probe molecules into contact with oligonucleotide samples or polynucleotide samples in an aqueous medium in the presence of a fluorescent intercalator compound of the formula (I) so as to form by hybridization a complex of the group of probe molecules and the oligonucleotide samples or polynucleotide samples on the substrate in which the fluorescent intercalator compound is intercalated; and
detecting a fluorescence emitted from the fluorescent intercalator compound intercalated into the hybridized complex on the substrate.
Furthermore, the invention resides in a process for fluorometrically detecting oligonucleotide samples or polynucleotide samples complementary to a group of probe molecules of nucleotide derivatives or their analogues fixed onto a substrate, which comprises the steps of:
bringing the group of probe molecules into contact with oligonucleotide samples or polynucleotide samples in an aqueous medium so as to form by hybridization a complex of the group of probe molecules and the oligonucleotide samples or polynucleotide samples on the substrate;
bringing a fluorescent intercalator compound of the formula (I) into contact with the hybridized complex on the substrate, whereby the fluorescent intercalator compound is intercalated into the hybridized complex; and
detecting a fluorescence emitted from the fluorescent intercalator compound intercalated into the hybridized complex on the substrate.
REFERENCES:
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patent: 5698408 (1997-12-01), Rokos
patent: 5731148 (1998-03-01), Becker et al.
patent: 6261780 (2001-07-01), Ogawa et al.
patent: 6294670 (2001-09-01), Takenaka
Makino Yoshihiko
Ogawa Masashi
Takagi Makoto
Takenaka Shigeori
Fuji Photo Film Co. , Ltd.
Horlick Kenneth R.
Reed Smith LLP
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