Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-23
2003-04-01
Horlick, Kenneth R. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S091210, C435S091500, C435S091510, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06541205
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for qualitatively or quantitatively analyzing a target RNA having a specific nucleotide sequence, which is considered to be contained in a gene mixture of DNA, RNA, or the like, and the method is useful in the field of clinical diagnoses, such as gene diagnosis and the like. Also, the present invention relates to a method for qualitatively or quantitatively analyzing microorganisms in environments, such as foods, indoors, soils, rivers, ocean, and the like.
2. Discussion of the Background
Generally, a high specificity and a high sensitivity are required for an assay of biological components. An assay of a nucleic acid having a specific nucleotide sequence (target nucleic acid) can use a property that the nucleic acid sequence-specifically forms a complex with another nucleic acid having a sequence complementary to the specific nucleotide sequence (nucleic acid probe).
When the target nucleic acid having a specific nucleotide sequence is assayed, a means for obtaining a measurable signal related to the amount of the formed complex is important. Furthermore, the amount of the target nucleic acid which can be present in a sample is extremely small for a purpose of clinical diagnosis etc. so that such a means requires a step for amplifying the extremely small amount of the nucleic acid.
In diagnosis of viral infection, since the amount of a target nucleic acid (viral nucleic acid) in a clinical sample is often extremely small, a polymerase chain reaction (PCR), particularly a competitive PCR, is known as a means for obtaining a high sensitivity by improving the signal strength in order to realize the measurement with high sensitivity and good reproducibility. In this method, PCR is carried out by adding a competitor (a different nucleic acid sequence having a primer recognizing region common to the target nucleic acid) having a known concentration to a sample, and the concentration of the target nucleic acid in the sample is estimated by comparing amplified degrees between the competitor and the target nucleic acid. More specifically, a nucleic acid having a sequence complementary to a primer on its terminus and can be distinguished from the amplified product of the target nucleic acid by a separation means, such as electrophoresis or the like (e.g., based on a different chain length) is prepared, this nucleic acid is added to a sample to give respective concentrations, and PCR of these mixtures is simultaneously carried out.
Additionally, an assay method in a homogenous system has been proposed as a method for assaying a target nucleic acid using PCR. For example, an assay method has been proposed, in which PCR is carried out in the presence of an intercalating fluorochrome, the fluorescence of the reaction solution is measured in each PCR cycle, and the initial amount of the target nucleic acid is determined based on its changes (JP-A-5-237000 (the term “JP-A” as used herein means an “unexamined published Japanese patent application”);
Igaku
-
no Ayumi,
173(12): 959-963 (1995);
Analytical Biochemistry,
229: 207-213 (1995)). In this assay method, the amplified product by PCR is a double-stranded DNA so that an intercalating fluorochrome having a property to change its fluorescence characteristic, such as an increase in the fluorescence intensity by intercalation into the double-stranded nucleic acid, is used, the fluorochrome is added to the reaction solution in advance before amplification operation by PCR, the fluorescence intensity in the reaction solution is measured with time, and the initial amount of the target nucleic acid is determined, for example, based on its build up cycle etc.
On the other hand, NASBA method and 3SR method are also known as RNA amplification methods. In these methods, a double-stranded DNA fragment containing a promoter sequence is synthesized for a target RNA using a primer containing the promoter sequence, a reverse transcriptase and ribonuclease H, an RNA containing a specific nucleotide sequence of the target RNA is synthesized in the presence of an RNA polymerase, and then a chain reaction is carried out in which the RNA is subsequently used as the template for the synthesis of the double-stranded DNA containing the promoter sequence. Thereafter, the reaction is completed when the RNA is amplified, and the amplified RNA is determined by an electrophoresis method or a hybridization method using a labeled nucleic acid probe.
In the hybridization method using a labeled nucleic acid probe, a nucleic acid probe labeled in such a manner that it generates a measurable signal, such as visible light, fluorescence, emission, or the like, forms a complex with a target nucleic acid, and then unreacted nucleic acid probe is washed or degraded, and the label is measured to assay the target nucleic acid. A method called sandwich assay is generally known, which uses two probes each comprising a sequence capable of forming a complementary bond with a specified sequence at different region in a specified nucleic acid. In this method, a first probe is immobilized on an insoluble carrier, and a part of a second probe is labeled with a dye having a color in the visible region, a fluorescent material, or an enzyme capable of forming them. Then, these probes are added to a sample, a specified nucleic acid in the sample is complementary bound to the first and second probes, and a complex composed of these three materials is formed on the insoluble carrier. Subsequently, the resultant supernatant in the sample reaction solution is separated from the insoluble carrier to separate the free second probe (B/F separation step). Thereafter, the presence or absence and amount of the specified nucleic acid in the sample are determined by measuring the label in the complex on the insoluble carrier. Also, when an enzyme which can form a dye having a color in the visible region or a fluorescent material is used as the second probe, the free second probe is removed after the complex forming step, an enzyme substrate as the precursor is added to the sample reaction solution, and then the presence or absence and amount of the target nucleic acid in the sample are determined by, measuring the dye or fluorescent material which is the reaction product.
Since the sandwich assay uses an insoluble carrier in the reaction solution, the second probe is nonspecifically adsorbed on the insoluble carrier. Accordingly, when the label in the complex on the insoluble carrier is measured, an error occurs in the measured results due to the presence of the label of the second probe nonspecifically adsorbed on the insoluble carrier. Thus, a problem is occurred when the presence or absence and amount of the specified nucleic acid in the sample are determined. Particularly, since the diagnosis of viral infection requires detection of an extremely small amount of viral nucleic acid in a clinical sample with a good reproducibility and a high sensitivity, the problem caused by nonspecific adsorption is an important problem to be solved.
In order to avoid this problem, various attempts are made, such as hydrophilic treatment of the surface of the insoluble carrier, blocking of adsorption points of the carrier surface with a protein etc., sufficiently washing of the insoluble carrier after the B/F separation step, and the like.
However, in the chemical hydrophilic treatment of the carrier surface, its result depends on the material of the carrier, and it is not always easy technically. Also, in the method in which the carrier surface is coated with a protein in order to block adsorption points on the carrier surface in advance, there is a possibility that the protein interacts with the nucleic acid moiety or label of the second probe to cause additional nonspecific adsorption on the carrier. Additionally, in the B/F separation step, there is an operational limitation in increasing the number of times of washing. Thus, for example, when a surfactant is added to the washing solution, degradation of the complex
Ishiguro Takahiko
Saitoh Juichi
Yokoyama Akihiro
Auerbach Jeffrey I.
Horlick Kenneth R.
Liniak Berenato & White
Tosoh Corporation
Wilder Cynthia
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