Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-12-29
2001-04-24
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091210, C435S091500, C435S183000, C435S174000, C435S194000, C536S024320, C536S024330
Reexamination Certificate
active
06221584
ABSTRACT:
The present invention concerns a method for the detection of telomerase activity as well as reagents suitable therefor.
Telomeres are specific structures at the ends of chromosomes and in the case of eukaryotic organisms they are composed of an accumulation of repeated defined nucleotide sequences (repeats) which for example contain the sequence TTAGGG in humans. In somatic cells each replication of the cell inevitably leads to a shortening of the telomere ends and once the telomere falls below a certain length this finally leads to cell death.
In contrast virus-transformed or immortalized cells do not exhibit a reduction in their telomere length. This is due to the activity of an endogenous ribo-nucleoprotein in these cells which is denoted telomerase and can counteract the telomere shortening in a reaction similar to reverse transcriptase.
Since according to previous findings the expression of telomerase is confined to tumour cells, germ cells and immortalized cells, this protein is a very promising parameter for the diagnosis of tumours and is also a target for tumour therapy (cf. e.g. the review articles by Greider, 1994, Curr. Oppin. Gen. Dev. 4, 203-211; Counter et al., 1994, Proc. Natl. Acad. Sci. USA 91, 2900-2904 and Hiyama et al., 1995, Nature Med. 1, 249-255).
The methods described in the literature for detecting telomerase activity are all based on an in vitro detection of the enzyme activity. At present it is not possible to immunologically detect the human enzyme since its protein sequence is not yet known. Only the sequence of the telomerase from tetrahymena was recently described (Collins et al., 1995, Cell 81, 677-686).
In the detection methods described in the literature one differentiates between two principle methods. The first method is based on a synthetic oligonucleotide derived from the telomere sequence which serves as a primer. This primer is added together with unlabelled dideoxynucleotides and a radioactively labelled deoxynucleotide to a sample e.g. a cell extract containing telomerase whereby the primer is specifically elongated by the telomerase and the product of synthesis is radioactively labelled in this process. The reaction mixture is subsequently separated by gel electrophoresis and the pattern of bands is visualized by exposure of an X-ray film with subsequent development (Morin, 1989, Cell 59, 521-529; Nilsson et al., 1993, Oncogene 9, 3043-3048).
A telomerase-specific elongation product is also firstly produced in the other detection method. However, this is amplified in a subsequent polymerase chain reaction (PCR) and simultaneously labelled by the addition of radioactive deoxynucleotides. The labelled PCR products are detected by gel electrophoresis (Kim et al., 1994, Science 266, 2011-2015).
WO 95/13381 describes a method for the detection of telomerase activity in which a cell extract to be tested is contacted with a primer which contains no telomere repeat sequences wherein the telomerase can catalyse an extension of the primer by attaching telomere repeat sequences. Subsequently an amplification step is carried out with addition of a second primer. The telomerase activity is finally detected by gel electrophoretic separation of the resulting amplification products.
However, these detection methods of the state of the art have some disadvantages. Thus the sensitivity of a detection method without an amplification step is too low for routine applications since quantities of extracts containing 10
6
to 10
7
cells have to be used. Therefore this method cannot be used to examine primary tumour material which is only available in a small amount. In addition the exposure time of the gels is in the range of two to seven days which is also due to the low sensitivity. The detection method comprising an amplification step does not have this disadvantage since only 10
5
cell equivalents per test have to be used routinely and 10
3
cell equivalents can be reproducibly detected. However, the exposure times for the gels are also still at least one day for this method (Kim et al., 1994, Supra; Chadeneau et al., 1995, Cancer Res. 55, 2533-2536).
Both of the detection methods described in the literature have the disadvantage that the labelling of the elongation product or of the PCR product has to be achieved with radioactive labelling groups in order to obtain satisfactory results. This leads to long exposure times and to the undesired formation of radioactive waste. Furthermore, the gel electrophoretic separation of the reaction mixture followed by subsequent exposure and development of an X-ray film is very labour intensive.
Moreover neither of the said methods allows a high sample throughput. They are not suitable for automation as is necessary for example for routine analysis or for an effector screening.
Thus the object of the present invention was to at least partially eliminate the disadvantages of the methods of the state of the art. This object is achieved by a method for the detection of telomerase activity characterized in that
(a) a sample to be tested is provided,
(b) a first primer suitable as a telomerase substrate and nucleoside triphosphates are added and the reaction mixture is incubated under conditions under which a primer extension by the telomerase can take place,
(c) an amplification of the extension product produced by the telomerase is carried out,
(d) the amplification product produced in step (c) is immobilized on a solid phase and
(e) the immobilized amplification product is detected qualitatively or/and quantitatively.
surprisingly it was found that the specificity of the telomerase reaction is retained by immobilizing the amplification product i.e. a positive signal can be reproducibly attributed to a telomerase activity. Hence the gel electrophoretic separation of the reaction mixture required by the state of the art is superfluous. In addition a very high sensitivity is achieved. In certain test formats it is even possible to achieve a gain in sensitivity by the method according to the invention compared to the methods of the state of the art. In addition the use of non-radioactive labels is possible and preferred in the method according to the invention which avoids the difficulties which occur when handling radioactive substances. These advantages make the method according to the invention very well suited to routine applications in automated detection instruments.
The method according to the invention enables a specific detection of amplified telomerase extension products without requiring a separation of the reaction products. This could not have been simply expected since all amplification mixtures also contain unspecific byproducts such as e.g. primer dimers, repeat sequences in addition to the telomerase extension products. Thus one has to expect that these unspecific products would also be detected in the test using a capture or detection probe directed towards the repeat sequence. In addition it was to be expected that when an internal standard was added this would always be co-detected. However, surprisingly it was found that by selecting suitable hybridization conditions and optionally by the addition of unlabelled oligonucleotides which are complementary to the primer sequences a highly specific detection of telomerase extension products without a separation step is achieved.
The telomerase activity is preferably detected by using labelling groups in particular non-radioactive labelling groups. All known labelling groups can be used as non-radioactive labelling groups e.g. immunologically reactive groups, e.g. nucleotide analogues or haptens which can react with a detection antibody, enzymes such as peroxidase, galactosidase or alkaline phosphatase, fluorescent or luminescent groups e.g. electrochemi-luminescent groups or other detection groups such as NMR-active labelling groups or electron-dense groups.
Immunologically reactive groups are preferred such as nucleotide analogues e.g. halogen-derivatized nucleotides such as Br-dUTP or nucleotides derivatized with organic residues that contain at least
Emrich Thomas
Hinzpeter Matthias
Karl Gerlinde
Leying Hermann
Arent Fox Kintner Plotkin & Kahn
Myers Carla J.
Roche Diagnostics GmbH
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