Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-01-11
2004-06-01
Riley, Jezia (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C008S636000, C008S648000, C536S024300, C536S026600
Reexamination Certificate
active
06743588
ABSTRACT:
The present invention relates to a novel fluorescent dye, a method of its production, a nucleic acid probe linked to the dye by a chemical bond and a method of measuring a nucleic acid using the probe. In particular, the present invention relates to a novel fluorescent dye which shows a large Stokes shift and enhance its fluorescence intensity in the presence of a double-stranded nucleic acid, a method of producing it, a nucleic acid probe obtained by chemically linking the dye, and a method of measuring a nucleic acid characterized by the use of it.
The method of the present invention relates to a method of qualitative or quantitative assay of a target RNA containing a specific base sequence anticipated in a gene mixture such as DNA and RNA. The method of the present invention is useful in gene diagnosis and other areas of clinical diagnostics and in identification or quantification of microorganisms in food and in the environment such as in rooms, soil, rivers and sea.
Generally, assays of biogenic components require high specificity and sensitivity. Assays of a specific nucleic acid having a specific base sequence (target nucleic acid) utilizes sequence-specific hybridizability of the nucleic acid with a complementary nucleic acid (a nucleic acid probe).
Generation of a measurable signal corresponding to the amount to the hybridization product is essential for quantification of a target nucleic acid having a specific base sequence. In quantification of a target nucleic acid for clinical diagnosis, because samples may contain the target nucleic acid only in trace amounts, the signal generation has to involve amplification of a trace amount of the nucleic acid.
Especially, in diagnosis of virus infections, for sensitive and reproducible assay of the target nucleic acid (viral nucleic acid) which is usually found in trace amounts in clinical samples, preamplification of the target nucleic acid by polymerase chain reaction (PCR) has been proposed to increase the sensitivity through signal enhancement. For RNA amplification, techniques called NASBA (Patent No. 2650159) and 3SR (EP-A-373960) are known.
A nucleic acid probe having a nucleic acid sequence complementary to a specific base sequence in a target nucleic acid, which is so labeled with a fluorescent intercalative dye as to give off a measurable fluorescent signal upon binding to the target nucleic acid (Japanese Unexamined Patent Publication JP-A-8-211050) gives off a measurable fluorescent signal upon hybridization with the target acid. The probe makes it possible to detect hybridization and quantify the hybridization product without the need to separate the unhybridized probe from the reaction system and has the advantage that it does not give false positive results attributable to carryover of the amplification product because sampling from reaction vessels is no longer needed after amplification.
Further, support-free isothermal assay of a target RNA in a closed system during amplification of the target RNA which is characterized in that isothermal amplification of an RNA having a specific nucleotide sequence by the action of nucleic acid primers and nucleic acid polymerases is carried out in the presence of the nucleic acid probe, has been developed (Japanese Patent Application JP12-144000).
Intercalative fluorescent dyes intercalate into double-stranded nucleic acids and changes their fluorescence characteristics as they lose freedom upon intercalation. As compound having such characteristics, ethidium bromide, oxazole yellow, thiazol orange and the like are known. Further, dimers of these compounds obtained by linking their molecules via a linker such as ethidium diner, YOYO and the like are also known to show fluorescent enhancement upon intercalation into double-stranded nucleic acids. Therefore, if two or more fluorescent intercalative dyes which change their fluorescent characteristics distinguishably are so used as to enable simultaneous measurement of multiple nucleic acids during their amplification, there are numerous possible applications of industrial significance. For example, it is possible to detect multiple target nucleic acids simultaneously, or check if a target nucleic acid is being amplified successfully or quantify a target acid by amplifying a known amount of a standard nucleic acid together with the target nucleic acid.
However, the number of intercalative dyes is not infinite, and the spectra of the fluorescent radiations from them have maximum values at certain wavelengths from which they spread to both sides, and their fluorescent quantum yields are generally different. Therefore, with fluorescent intercalative dyes having overlapping spectra, precise fluorescence measurement at a certain wavelength is difficult. Besides, because the types of lasers and light emitting diodes available as radiation sources to excite florescent intercalative dyes are limited, it has been very tough to select two or more fluorescent intercalative dyes that do not overlapping fluorescent spectra in view of optimum combination of sources of excitation radiations. For example, although cyanine dyes such as oxazole yellow and thiazole orange are known as fluorescent intercalative dyes, the difference between the maximum fluorescence wavelengths of two cyanine dyes has to be at least about 100 nm in order to avoid a spectral overlap between them. However, the difference between the maximum emission wavelength and the maximum excitation wavelength of a cyanine fluorescent substance is only from 20 to 40 nm. However, because fluorometric excitation of two dyes requires two radiation sources, the need for two radiation sources restricts the fluorometric use of these dyes.
The first object of the present invention is to provide a compound as a novel fluorescent intercalative dye which shows a large fluorescent enhancement upon intercalation into a double-stranded nucleic acid when used in detection of the nucleic acid, shows a great difference between the excitation wavelength and the emission wavelength (i.e., has a large Stokes shift) and does not have a fluorescent spectrum that overlaps with those of conventionally known fluorescent intercalative dyes. The second object of the present invention is to provide a novel nucleic acid probe having the fluorescent dye chemically linked. The third object of the present invention is to provides a method of measuring (identifying or quantifying) a nucleic acid using the nucleic acid probe which comprises amplifying at least one target nucleic acid and measuring the amplification product in a closed vessel without any separation operation to detect and quantify the target nucleic acid with great precision, especially a method of measuring at least two target nucleic acids simultaneously.
It is widely known that when one of two neighboring compounds which meet specific conditions is excited, the energy transfers to the other and is emitted as fluorescence from the compound which receives the energy (the energy acceptor). The present inventors conducted extensive research on substances which efficiently transfer energy in the molecule and found out a novel fluorescent dye which shows a large enhancement in fluorescent intensity upon intercalation into a double stranded nucleic acid in the detection of a nucleic acid and shows a large difference between its excitation wavelength and emission wavelength (i.e. a large Stokes shift). Thus, the present invention is accomplished to attain the above-mentioned objects. According to a first aspect of the invention, the present invention provides a novel compound represented by the formula 1, or a salt, hydrate, solvate or stereoisomer thereof whose structure, synthesis or fluorescent characteristics have not been known (wherein R
1
is a lower alkyl group, each of A and D, which may be the same or different, is a group represented by the formula CHR
2
—(wherein R
2
is a hydrogen atom, a lower alkyl group or a lower alkyl group substituted with a halogen atom), a group represented by the formula —NR
3
—(wherein R
3
is a hydrogen ato
Horie Ryuichi
Ishiguro Takahiko
Tokunaga Takumi
Riley Jezia
Tosoh Corporation
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