Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-06
2003-07-01
Riley, Jezia (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S022100, C536S023100, C536S024300
Reexamination Certificate
active
06586178
ABSTRACT:
NUCLEIC ACID PROBE
The present invention relates to a nucleic acid probe useful in the field of clinical diagnosis such as gene diagnosis and in the field of exploration of unknown genes and a method for qualitative or quantitative assay of a DNA or RNA containing a specific nucleic acid sequence as a target nucleic acid using the nucleic acid probe.
Qualitative or quantitative assays of a target nucleic acid containing a specific nucleic acid sequence (anticipated) in samples require high specificity, like assays of biogenic components. In assays specific for a target nucleic acid, a single-stranded oligonucleotide (a nucleic acid probe) which is complementary to a specific nucleic acid sequence in the target nucleic acid and sequence-specifically binds to the regional specific nucleic acid sequence in the target nucleic acid is used.
In assays of a target nucleic acid using a nucleic acid probe, it is common to link the nucleic acid probe to a spectroscopically detectable label which enables detection of the hybrid sequence-specifically formed between the nucleic acid probe and the target nucleic acid. Assays of a target nucleic acid using a nucleic acid probe also require high detection sensitivity. Especially, for example, in clinical samples for diagnoses of infectious diseases such as blood, target nucleic acids from viruses such as HCV and HIV are usually present in trace amounts. Therefore, use of a nucleic acid probe labeled with an enzyme together with a chemiluminescent substrate of the enzyme and use of a nucleic acid probe linked via a linker to a fluorescent intercalative dye which intercalates into base pairs of a double-stranded nucleic acid have been proposed.
In recent years, it is common to amplify a target nucleic acid by PCR (polymerase chain reaction) or the like in advance and then hybridize a nucleic acid probe with the amplified target nucleic acid with a view to increasing the sensitivity. Specifically, because a fluorescent intercalative dye-labeled nucleic acid probe as mentioned above characteristically obviates the need to use a support for separation of the unhybridized target nucleic acid from a mixture of a target nucleic acid and a nucleic acid probe in conventional assays of the target nucleic acid, the presence of such a fluorescent intercalative dye-labeled nucleic acid probe during an amplification reaction such as PCR makes it possible to measure a target nucleic acid during or after the amplification in a sealed vessel without addition of any reagents during a series of operations from amplification to measurement and obviate the possibility of contamination of other samples by aerosol generated from a sample in hand (JP-A-8-211050, Japanese Patent Application JP10-186434, EP-A-714986).
The above-mentioned amplification by PCR or the like in the presence of a nucleic acid probe produces an excellent effect of enabling assays in sealed vessels, which were impossible by conventional methods. However, customary amplification in the presence of a nucleic acid probe is accompanied by elongation of the nucleic acid probe from the 3′ end, which can lead to a recognizable signal from the label even in the absence of the target nucleic acid.
For DNA amplification by PCR, it is known to introduce one or two bases in the 3′ end of a nucleic acid probe uncomplementary to a target nucleic acid (JP-A-8-211050). But in the case of amplification of an RNA as the target nucleic acid comprising synthesis of a DNA complementary to the RNA using a primer and a reverse transcriptase along the RNA as the template, DNA elongation from a promoter primer partly complementary to the resulting complementary DNA triggered by their hybridization and subsequent large scale production of the target RNA by the action of an RNA polymerase on the resulting double-stranded DNA, it has been found that use of such a nucleic acid probe can result in a signal, though weak, from the label due to DNA elongation from the 3′ end of the nucleic acid probe during the reverse transcription.
The object of the present invention is to provide a nucleic acid probe which does not undergo DNA elongation from the 3′ end during nucleic acid amplification involving synthesis of a complementary nucleic acid using a target nucleic acid as the template such as the above-mentioned DNA and RNA amplification. Another object of the present invention is to realize nucleic acid amplification in the presence of a nucleic acid probe by using the nucleic acid probe.
In order to achieve the above-mentioned objects, according to claim
1
of the present application, the present invention provides a nucleic acid probe which is a single-stranded nucleic acid complementary to a specific nucleic acid sequence and is labeled so as to give off a measurable fluorescent signal on hybridization with a nucleic acid containing the specific acid sequence, wherein the 3′ end of the probe is modified as represented by formula (1).
In order to achieve the above-mentioned objects, according to claim
2
of the present application, the present invention provides a method for qualitative or quantitative assay of a target nucleic acid containing a specific base sequence, which comprises amplifying the target nucleic acid in the presence of a single-stranded nucleic acid probe complementary to the specific nucleic acid sequence which is labeled so as to give off a measurable signal on hybridization with a nucleic acid containing the specific nucleic acid sequence and measuring the amplified target nucleic acid hybridized with the nucleic acid probe during and/or after the amplification, wherein the 3′ end of the single-stranded nucleic acid probe is modified as represented by formula (2).
REFERENCES:
patent: 5814447 (1998-09-01), Ishiguro et al.
patent: 6211354 (2001-04-01), Horie et al.
patent: 0714907 (1996-05-01), None
Matthews et al. “Review. Analytica;l Strategies for the Use of DNA Probes” Analytical Biochemistry, 169, pp. 1-25, 1988.*
Gait et al. Journal of Chemical Society 1979, pp. 1389-1394.*
Greenberg et al. Tetrahedron vol. 51, No: 1, pp. 29-38, 1995.*
Takahiko Ishiguro et al., Fluorescence detection of specific sequence of nucleic acids by oxazole yellow-linked oligonucleotides. Homogeneous quantitative monitoring of in vitro transcription,Nucleic Acids Research,vol. 24, No. 24, pp. 49893-4997 (1996).
Ryuichi Horie et al., “Intercalator-linked DNA probe having stereoregular 2-aminoethylphosphonatediester linkage,”Nucleic Acids Symposium SeriesNo. 39, pp. 39-40, (1998).
Horie Ryuichi
Ishiguro Takahiko
Saitoh Juichi
Taya Toshitaka
Tokunaga Takumi
Jacobson & Holman PLLC
Riley Jezia
Tosoh Corporation
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