Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1992-11-12
2001-10-09
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007900, C435S007920, C536S023100, C536S024300, C536S024310, C536S024320, C702S019000, C702S020000
Reexamination Certificate
active
06300058
ABSTRACT:
BACKGROUND OF THE INVENTION
2. Field of the Invention
This invention is related to a method for measuring messenger RNA (mRNA) and polynucleotide-immobilized supports useful in this method. Specifically, this invention is related to measuring methods and reagents in which various types of mRNA can be measured at the same time. In one embodiment of this invention, the mRNA need not be purified from the cell, and the mRNA can be rapidly quantified with high sensitivity.
2. Description of Previous Techniques
Previously well-known techniques for measuring mRNA levels include the Northern Blot Method (See, e.g., J. Sambrook, et al., Molecular Cloning, 2nd ed., Cold spring Harbor Laboratory Press, 1989 hereafter referred to as “Molecular Cloning”), the Dot Blot Method (See, e.g., Molecular Cloning), Ribonuclease Protection Assay (See, e.g., Molecular Cloning), and the Reverse PCR Method (H. A. Erlich (ed.), PCR Technology-Principles and Applications for DNA Amplification, Stockton Press, New York, N.Y., 1989). The disclosures of all of these previous references are hereby incorporated by reference.
The Northern Blot Method detects mRNA which is separated by molecular weight using electrophoresis. The separated molecules are immobilized on a membrane and mRNA levels are determined by hybridization of marker DNA probes. With this method, the molecular weight of the subject mRNA must generally be known. By using this method a complex between a labeled probe, mRNA and an insoluble membrane is formed. The amount of label in the complex at the appropriate site on the membrane is then measured colorimetrically or by radioactive emissions.
The Dot Blot Method detects mRNA by hybridization with a labeled probe after purified mRNA is immobilized as a spot on a membrane. This method can be used to rapidly examine many samples at one time. However, both the Dot Blot and the Northern Blot methods require the use of a specific labeled probe for each mRNA which is desired to be detected.
The Ribonuclease Protection Assay method is used to detect mRNA after it is initially purified. The purified mRNA is hybridized with a labeled RNA probe, and then treated with a ribonuclease that specifically digests single stranded RNA. The labeled RNA probe will form a duplex in the solution and be protected from digestion. This is then immobilized on a membrane after separation of the double-stranded RNA/probe complex.
The reverse PCR method is used where cDNA is synthesized using reverse transcriptase and purified mRNA. This cDNA can be immobilized to a membrane. The synthesized cDNA is then amplified using standard PCR (Polymerase Chain Reaction) methods. After separating cDNA according to its molecular weight by electrophoresis, it becomes a single band. The DNA is then immobilized on a membrane, and detected by hybridization with a labeled DNA probe. However, after PCR, no quantitative information remains due to the amplification of the original message.
All of the foregoing methods take extended periods of time to perform since the mRNA used must be in at least a partially purified form. Also, electrophoresis of the samples and immobilization to a membrane is necessary. Both of these steps reduce the reliability of quantification of the mRNA.
Thus, there remains a need to resolve the above problems, and to provide a method of measuring for rapidly determining messenger RNA levels and a measuring reagent useful in such a method.
SUMMARY OF THE INVENTION
In the mRNA assay methods of the present invention, a first and a second polynucleotide probe can be used. The inventors have discovered that immobilization of probes (a first polynucleotide) rather than samples, on a water-insoluble support can provide a basis for the rapid quantification of multiple unpurified samples containing mRNA. Applicants have provided an efficient method of identifying appropriate specific probes for this purpose. In preferred forms of the present invention, the first polynucleotide probe has a nucleotide sequence complementary to a region which is unique in the sample to the mRNA and does not bind other portions in the target mRNA. The inventors also discovered that a single kind of second polynucleotide probe can be used for simultaneous quantification of various mRNAs. However, any of a variety of probes that bind to a different region of the mRNA than the first probe can be used for this purpose. In this regard, the inventors have found that a second polynucleotide probe containing sequences complementary to a polyadenylic acid tail of mRNA can be used in such a simultaneous assay method. Based on these and other discoveries, the inventors established the present invention.
One embodiment of the present invention is a highly sensitive, quantitative and rapid method for detecting and quantifying mRNA in a sample without the need to purify mRNA from cells. In this embodiment of the invention, the method comprises the following steps:
(a) identifying a polynucleotide sequence that is unique to the mRNA;
(b) immobilizing a first polynucleotide to an insoluble support, the first polynucleotide having a first sequence complementary to the sequence unique to the mRNA;
(c) incubating the sample with the insoluble support under conditions wherein the unique sequence will hybridize with the first polynucleotide, thereby immobilizing mRNA present in the sample to the insoluble support;
(d) washing non-immobilized components of the sample from the insoluble support;
(e) labeling mRNA on the support in a manner that label is incorporated onto the support related to the amount of mRNA on the support; and
(f) measuring the amount of label immobilized on the support.
In a preferred embodiment, the above step of labeling mRNA on the support involves incubating a labeled probe, most preferably poly-d(T), with the insoluble support under conditions wherein the labeled probe will hybridize with mRNA on the support, and the labeled probe bears a label and is complementary to a part of the mRNA other than a part of the mRNA complementary to the first probe, thereby immobilizing to the insoluble support the label on the labeled probe which has hybridized with the mRNA; and thereafter washing any non-immobilized labeled probe from the insoluble support.
In addition, the above method of labeling nucleic acids in step (e) can preferably involve labeling the oligonucleotides with a nucleic acid stain, most preferably from the group consisting of ethidium bromide, yoyo-1 and toto-1.
In a further preferred embodiment, the above method can utilize a label, wherein the label comprises a radionuclide, or alternatively biotin. In the method using biotin, the incubation can preferably be with enzyme-conjugated streptavidin; followed by measurement of the amount of enzyme bound to the insoluble support. In this embodiment step (f) can additionally comprise adding streptavidin bound to alkaline phosphatase to the insoluble support.
In still another embodiment of the preferred method, the mRNA can code for a &bgr; receptor, and wherein the first polynucleotide comprises the sequence of SEQ ID NO:6. An alternative embodiment of the above method includes mRNA coding for the &agr; subunit of Gs protein, wherein the first polynucleotide comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NO:1, 145-146, 209-212, 268-287, 705 and 708.
Yet another alternative mRNA sequence that can be used with the present invention method codes for the Gi-2 protein, wherein the first polynucleotide comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NO:703, 706, 142, 143, 187-197 and
238-253. The present method can also preferably involve the use mRNA coding for substance P receptor, wherein the first polynucleotide comprises the sequence of SEQ ID NO:
5.
mRNA encoding Gi-3 protein can also be used in the present method, wherein the first polynucleotide comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NO:704, 707, 144, 198-208 and 254-267. Still another embo
Akitaya Tatsuo
Cooper Allan
Mitsuhashi Masato
Guzo David
Hitachi Chemical Research Center Inc.
Knobbe Martens Olsen & Bear LLP
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