Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-07-31
2001-04-24
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S174000, C536S063000, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06221590
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for the quantitative determination of telomerase activity.
BACKGROUND ART
Telomerase is known to be an enzyme which catalyzes the extension of the telomere terminal end (terminal portion of a linear chromosome) and many studies thereon have been conducted: Greider C. W. and Blackburn E. H., (1987) Cell, 51, 887-898; Morin G. B. (1989) Cell, 59, 521-529.
The activity of telomerase is not detected in normal cells except in certain cells such as hemopoietic stem cells, while strong telomerase activity can be detected in most cancer cells. Telomerase is considered to be associated with the maintenance of infinite proliferation of cancer cells. Thus, the detection and quantitative determination of the telomerase activity is important in the diagnosis of cancer. Further, an inhibitor therefor may be expected to be an anti-cancer agent with little side-effect on normal cells: Counter C. M. et al., (1989) EMBO J., 11, 1921-1929; Counter C. M. et al., (1994) Proc. Natl. Acad. Sci. USA, 91, 2900-2904; Chadeneau C. et al., (1995) Cancer Res., 55, 2533-2536;
Hiyama E. et al., (1995) Nature Med., 1, 249-255, Shay J. W. et al., (1995) Mol. Cell. Biol., 15, 425-432.
Recently, the detection of telomerase has been studied by using a ciliate Tetrahymena (Greider and Blackburn, 1985; 1987; 1989). In these studies, telomerase was detected in a single primer extension assay system. Substrates required for such an assay are the single-stranded telomere oligonucleotide sequence (TTGGGG)
3
, dGTP, dTTP and dGTP labelled with
32
P. The repeat of TTGGGG which is the telomere sequence of Tetrahymena is added to the 3′ end of the oligonucleotide primer during short incubation at room temperature. Then, the reaction product is detected by electrophoresis and autoradiography. This assay has enabled the study on the kinetics and primer specificity of telomerase derived from Tetrahymena and other ciliates, and the cloning of telomerase RNA (Romero and Blackburn, 1991; Lee and Blackburn, 1993, Collins and Greider, 1993; Autexier and Greider, 1994).
On the other hand, the number of human telomeres is as small as 92 and it has been considered that it is impossible to detect the telomerase activity. However, the telomerase activity was found in the telomerase assay using extracts of Hela cell line derived from human cervical cancer and it was elucidated that the enzyme adds a number of TTAGGG repeat sequences to the 3′ end of telomere (Morin, 1989). It has been suggested that telomerase is an enzyme conserved in all eucaryotic cells and is markedly activated in human tumor cells (Counter et al., 1992; 1994).
In the conventional assay systems, however, there are problems in the sensitivity, time period required for detection, quantitativeness and dealing of a large amount of sample (Counter et al., 1994).
Recently, the sensitivity and detection time have been improved by the means based on the polymerase chain reaction called as TRAP (telomeric repeat amplification protocol): Kim N. W. et al., (1994) Science, 206, 2011-2015; Piatyszek M. A. et al., (1995) Meth. Cell Sci., 17, 1-15.
This technique involves the telomerase reaction and polymerase chain reaction followed by the detection of reaction products by polyacrylamide gel electrophoresis and autoradiography. The quantitative determination of telomerase activity is performed by measuring the intensities of appearing bands with a densitometer or other means and comparing them with known amounts of, e.g., Hela cell extracts, used as a control.
This method has improved the sensitivity, enabling the detection of telomerase activity even in a small number of cells, such as 100 cells. That is to say, the sensitivity of detection of telomerase activity can be increased 10
4
times as compared with conventional assay methods; the sensitivity has been greatly improved as compared with the conventional techniques.
Even in this TRAP method, however, analysis of
32
P- or fluorescence-labelled reaction products by polyacrylamide gel electrophoresis, HPLC or other means is still required, so that the number of samples to be measured is limited. Further, in the case of
32
P, its handling has problems such as treatment or disposal of gel or a large amount of waste liquid. In addition, it will take a long time to conduct a series of operations such as preparation of the gel, electrophoretic separation (analysis) and exposure or detection (usually 2 to 48 hours). Subsequently, the intensities of the detected bands must be measured by a densitometer or other means. Thus, there has been a problem of delay in obtaining the results.
These problems are very inconvenient, in particular in the diagnosis of progression and prognosis of cancer which requires real time analysis, and are also inconvenient in the analysis of a large amount of samples.
DISCLOSURE OF INVENTION
It is an object of the present invention to provide a method enabling rapid detection and quantitative determination of telomerase activity with a high sensitivity.
The present inventors have eagerly studied the above problems and, as a result, found that telomerase activity can be rapidly detected and quantitatively determined with a high sensitivity by constructing a system comprising the DNA synthetic reaction with teromerase in combination with the scintillation proximity assay technology (SPA) developed by Amersham (Bothworth N. and Towers P. (1989) Nature, 341, 167-168), leading to the completion of the present invention.
That is to say, the present invention is a method for the quantitative determination of telomerase activity comprising: amplifying an oligonucleotide sequence which has been synthesized by a DNA synthesis reaction with a telomerase, using a primer modified with either of two materials capable of mutually binding to each other, in the presence of a radioisotope element; making the resulting sequence to bind to a fine particle which has been previously coated with the other of said two materials capable of mutually binding to each other; and measuring scintillation generated from the fine particle due to said binding to quantitatively determine the telomerase activity.
Further, the present invention is a method for the quantitative determination of telomerase activity comprising: carrying out a DNA synthesis reaction with a telomerase, using a primer modified with either of two materials capable of mutually binding to each other, in the presence of a radioisotope element; making the resulting reaction product to bind to a fine particle which has been previously coated with the other of said two materials capable of mutually binding to each other; and measuring scintillation generated from the fine particle due to said binding to quantitatively determine the telomerase activity.
The two materials capable of mutually binding to each other as used herein include biotin and avidin.
Hereinafter the present invention will be described in detail.
The present invention is a method for detecting and quantitatively determining telomerase activity in cell extracts. The present invention involves a method for amplifying an oligonucleotide sequence (telomere repeat sequence) synthesized by a DNA synthesis reaction with a telomerase, using a primer modified with either one of two materials capable of mutually binding to each other, in the presence of a radioisotope. However, a method for reacting without amplification of the telomere repeat sequence is also included in the present invention. Then, the resulting reaction product is allowed to bind to a fine particle which has been previously coated with the other of said two materials capable of mutually binding to each other and scintillation generated from the fine particle due to said binding is measured to quantitatively determine the telomerase activity.
(1) Amplification of Telomere Repeat Sequence by Cell Extracts
The telomere repeat sequence may be amplified by, for example, the polymerase chain reaction method. The polymerase chain reaction provides detection results with a high s
Akamatsu Ken-ichi
Savoysky Ericka
Tsuchiya Masayuki
Chugai Seiyaku Kabushiki Kaisha
Davidson Davidson & Kappel LLC
Fredman Jeffrey
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