Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-10-22
2002-07-23
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S912000, C435S094000, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06423490
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for measuring the concentration of a polynucleotide such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in a sample using agglutination of an agent, and to a kit and apparatus for use in such a method.
BACKGROUND OF THE INVENTION
Recent innovation in the field of genetic engineering has been remarkable. In particular, the development of a polymerase chain reaction (PCR) method has enabled the mass replication of target DNA. The PCR method is based on the principle that DNA polymerase does not function without a primer. The PCR method is used to amplify DNA in large amounts by repeating the following cycle: (1) DNA in a sample is heat denaturated resulting in single stranded DNA; (2) a primer is bonded to the DNA under reduced temperature; and (3) the DNA is formed by thermo-stable DNA polymerase under this condition. Consequently, target DNA can be prepared in large amounts by using a specific primer prepared by chemical synthesis or otherwise. In this method, the replication of target DNA is typically monitored by electrophoresis, a dot blotting method, a PCR-SSCP (Single Strand Conformation Polymorphism) method, a PCR-fluorescence method or the like.
In gene analysis, electrophoresis is a generally used method for detecting the mobility of polynucleotide in a gel in the presence of an electric field. The dot blotting method is used to judge whether or not the amount of analyte has increased or has decreased by the following steps: (1) the polynucleotide extracted from a sample is gradually diluted and the equivalent amount is spotted to a nitrocellulose filter or a nylon film; (2) DNA, RNA and the like labeled by the radioisotope (
32
P) are hybridized with the polynucleotide; and (3) the polynucleotide is exposed to an X-ray film and is analyzed, or the spot portion of film is cut off after hybridization and measured by a scintillation counter. The PCR-SSCP method is a method for detecting the position of a band by autoradiography by the following steps: (1) sample DNA is amplified by the PCR method using the primer labeled by the radioisotope (
32
P); (2) the labeled DNA fragment obtained is heat denaturated resulting in single stranded DNA; and (3) single stranded DNA is separated by electrophoresis using a neutral polyacrylamide gel. The PCR-fluorescence method comprises a first step of obtaining the initial amount of the target nucleic acid by following the change in the fluorescence intensity after PCR has been carried out in the presence of an intercalating fluorescence material (Progress in Medical, Vol. 173, No. 12, Jun. 17, 1995, Pages 959-963).
However, these analytical methods suffer the drawback that special apparatus is needed, the manipulation is complicated and takes a long time. Moreover, the type of polynucleotide which may be analyzed is limited with respect to the chosen analytical method.
More specifically, electrophoresis is a complex and lengthy technique in which it is necessary to prepare a gel as a carrier and to pre-select a gel (size) for which PCR was carried out with regard to the size of DNA. Typically, a sample takes about 75 minutes to electrophorese and it is therefore not a rapid technique.
In the dot blotting method and the PCR-SSCP method, there is normally a safety factor because of the use of a radioisotope. In the method in which no radioisotope is used, the DNA probe must be labeled with a fluorescent substance or a luminescent material. This makes the method complicated and it takes a long time for the manipulation of film transfer. Furthermore, in the PCR-fluorescence method a fluorophotometer is needed and it is difficult to detect single stranded DNA. The conventional analytical methods have in common the problem that the analysis can not be carried out in the presence of materials other than polynucleotide. Therefore, a sample for which PCR has been carried out (a PCR product) should be purified prior to analysis. This makes the overall manipulation lengthy.
It is an object of the present invention to seek to solve these problems and others in the known techniques for measuring polynucleotide concentration in a sample.
SUMMARY OF THE INVENTION
Thus viewed from one aspect the present invention provides a method for measuring the presence of polynucleotide in a sample comprising the steps of: (1) preparing a sample, an agglutinative agent and an agglutination promoter capable of binding to polynucleotide; (2) mixing said sample, said agent and said promoter; and (3) measuring the degree of agglutination of the agent. Preferably the method is used to determine the concentration of said polynucleotide.
The present invention is based on the principle that agglutination of the agent is not promoted by the promoter when the promoter is bound to a polynucleotide. Therefore, by measuring the degree of agglutination, it is possible to confirm the presence of polynucleotide in the sample and in a preferred embodiment to measure its concentration. The measurement of the degree of agglutination may be readily carried out by measuring the absorbance with a spectrophotometer or simply by visual observation. In this invention, mixing the sample with the agent and the promoter is a simple step. Moreover, the method of the invention as a whole may be carried out in a short period of time, since the agglutination of the agent typically occurs in e.g. 1-2 seconds and the measurement of the degree of agglutination may be equally carried out in a short time. Furthermore, the concentration of polynucleotide in a sample may be measured by the method of the invention, even if the materials other than polynucleotide are included in the sample e.g. PCR product that has not been purified.
In the present invention, the expression “an agglutination promoter capable of binding to polynucleotide” refers to material which has the characteristics of bonding to polynucleotide and of promoting the agglutination of the agglutinative agent and which can not promote the agglutination of the agent when bound to polynucleotide. The term “an agglutinative agent” in the present invention refers to material that agglutinates rapidly in the presence of the promoter.
The promoter and the agent may be added to the sample at the same time, or consecutively e.g. the promoter may be added before the agent.
The polynucleotide under investigation may be, for example, single stranded DNA, double stranded DNA, RNA, a complex of RNA and DNA or PNA (Peptide nucleic acid), etc. The method of the present invention is effective on a sample which has been subjected to the PCR method (PCR product) or on a DNA sample. As mentioned above, the PCR method is used for amplifying target DNA. If target DNA is not present in the sample, trace amounts of DNA would be found in the PCR product. Therefore, by measuring the degree of agglutination in accordance with the method of the present invention, it may be determined simply and rapidly whether or not target DNA has been amplified. Further, the amount of amplified DNA can be measured provided a calibration curve relating the degree of agglutination and the amount of DNA has been prepared beforehand.
In addition, the method of the present invention may usefully be applied to samples in which the amplification of DNA is carried out by the Strand displacement amplification method (SDA method) or by the Ligase chain reaction (LCR method) and in which the amplification of RNA is carried out using the Q&bgr; replicase (Q&bgr; method). The SDA method includes the method described in Nucleic Acids Research, Vol.20. No.7 1691-1696. The LCR method is that method in which thermo-stable DNA ligase which is not denaturated by heat (even at 94° C.) is used. This method is based on the principle that for normal DNA samples having no mismatches, the two kinds of oligonucleotide used are bonded by the DNA ligase, resulting in them functioning as a substrate in the next reaction cycle and DNA amplification. Where the DNA sample has mismatches, the reaction stops, since the two kinds of olig
Fredman Jeffrey
Kyoto Dai-ichi Kagaku Co. Ltd.
Merchant & Gould P,C,
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