Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-12-23
2002-12-03
Zitomer, Stephanie (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091520, C536S024330, C536S024300, C536S025320
Reexamination Certificate
active
06489097
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for the detection of telomerase activity, a method for the detection of cancer cells, a method for the diagnosis of cancer, as well as a kit for the detection of cancer cells and/or the diagnosis of cancer.
BACKGROUND ART
Human somatic cells have 22 pairs of autosomes and a pair of sex chromosomes. A total of 46 chromosomes are present in a cell. These chromosomes are independent of each other and rarely associated with each other. Telomeres are responsible for this important function and telomerase plays a role in maintaining the presence of telomeres. Telomeres are located in the terminal end of chromosomes and, in human, have a characteristic sequence of a few hundreds of repeated 6 bases 5′-TTAGGG-3′. The replication of DNA is essential to proliferation of cells by division and, based on the mechanism of the DNA replication, the terminal telomere of a chromosome is shortened once DNA has been replicated. Whenever the cell division is repeated, the telomeric sequence is shortened. Deletion of this portion induces association of chromosomes which causes adverse effects on cells. Further, other gene abnormalities may also occur and cells are led to death (senescence and death of cells). Telomeric sequences have an important role in the senescence and death of cells, that is, alternation of generations of cells. However, it is considered that if telomeric sequences of some cell populations which should naturally die (aged cell populations in which gene abnormalities have been accumulated) are still continuously added by the action of telomerase, a part of the populations may be immortalized and eventually cancerized. Accordingly, the detection of telomerase activity is very useful in the diagnosis of cancer or in monitoring the prognosis of treatment.
Recently, TRAP (telomeric repeat amplification protocol) for detecting the telomerase activity with a high sensitivity by using PCR (polymerase chain reaction) has been developed: Kim N. W. et al., (1994) Science, 206, 2011-2015; Piatyszek M. A. et al., (1995) Meth. Cell Sci., 17, 1-15. This method involves the detection of telomerase by a single primer extension assay system and is roughly divided in three steps. First, telomerase is extracted from cells. Then, extension reaction of TTAGGG chain by the telomerase is carried out and the reaction products are amplified by PCR using two primers, called TS and CX primers. Finally, the amplified products are electrophoresed to detect the telomerase activity by confirming ladders in autoradiography. This method has improved the detection sensitivity, enabling the detection of telomerase activity even in a small number of cells, such as 10 cells.
Among the above three steps, however, the detection system must involve electrophoresis, which requires complicated operations and a long period of time. For example, 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 detected is limited and, in the case of
32
P, its handling, such as treatment of gel or a large amount of waste liquid, is not easy. In addition, it will take a long time to conduct a series of operations, such as preparation of gel, electrophoretic separation (analysis) and exposure (detection), usually 2 to 48 hours.
These problems are very inconvenient, especially in the diagnosis of progression and prognosis of cancer which requires real time analysis, and it is also difficult to analyse a large amount of samples. Thus, there is a need for the development of another detection system which may replace the electrophoresis or autoradiography.
DISCLOSURE OF INVENTION
It is an object of the present invention to provide a method enabling rapid detection 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 with a high sensitivity by constructing a system comprising the DNA extension reaction with a teromerase in combination with the hybridization protection assay (HPA) developed by Gen-Probe, leading to the completion of the present invention.
That is to say, the present invention is a method for the detection of telomerase activity comprising amplifying an oligonucleotide sequence extended by a DNA extension reaction with a telomerase and hybridizing the resulting amplified product with a probe labelled with a non-radioactive labelling material to detect the telomerase activity. The non-radioactive labelling material includes, for example, acridinium esters, luminol, isoluminol, pyrogallol, protohemin, aminobutylethyl-n-isoluminol, aminohexylethyl-n-ethyl-isoluminol, and acridine derivatives.
The acridine derivatives may include, for example, those represented by the following formula I:
wherein X denotes a halogen, or a group represented by the following formula II:
in which X
1
is a nitrogen, phosphorus, boron or arsenic atom, R
1
is an alkoxy or aryloxy group, or a substituted or unsubstituted alkyl, alkenyl or aryl group, and R
2
is a hydrogen atom, or, an alkoxy or aryloxy group, or a substituted or unsubstituted alkyl, alkenyl or aryl group, or the following formula III:
—X
2
—R
2
(III)
in which X
2
is an oxygen or sulfur atom and R
2
is as defined above, Y denotes an oxygen or sulfur atom, or NH, R
3
denotes a hydrogen atom, an amino, hydroxy, thiol, carboxylic acid, halogen, nitro, alkoxy or aryloxy group, or a substituted or unsubstituted acetyl, alkyl, alkenyl or aryl group, R
4
denotes a substituted or unsubstituted alkyl, alkenyl or aryl group, and at least one of R
1
, R
2
, R
3
and R
4
comprises a reactive site capable of chemical bonding. The reactive site capable of chemical bonding herein means a site which binds to a probe. For example, when the bonding is effected through a linker and an amino linker having an amino group at the end thereof, said reactive site is a site which binds to the amino group. The substance capable of binding to this site includes, for example, a carboxylic acid derivative, preferably an acid halide or ester.
However, the present invention is not limited to the non-radioactive labelling materials described above. In addition to the acridine derivatives represented by the above formula I, those represented by the chemical formula I as shown in Japanese Patent No. 2602315 are also encompassed.
The oligonucleotide sequence extended through DNA extension reaction by a telomerase is amplified by a polymerase chain reaction using an oligonucleotide primer comprising at least a base sequence represented by “AGNGTT” wherein N is A, T, G or C at the 3′ side and/or an oligonucleotide primer comprising the base sequence represented by SEQ ID NO: 21. This amplification may also be effected by RNA synthetic reaction using an oligonucleotide primer comprising at least a base sequence represented by “AGNGTT” wherein N is A, T, G or C at the 3′ side and/or an oligonucleotide primer comprising the base sequence represented by SEQ ID NO: 21. A promoter sequence is added to at least one of oligonucleotide primers used in the RNA synthetic reaction.
Further, the amplification may also be carried out by polymerase chain reaction or RNA synthetic reaction using an oligonucleotide primer having any sequence which does not hybridize with the sequence represented by “TTAGGG” and has been added to the 5′ side of either an oligonucleotide primer comprising the base sequence represented by SEQ ID NO: 21 or an oligonucleotide primer comprising a base sequence having at least one nucleotide deleted, substituted or added in the base sequence of said primer.
In the present invention, the conditions under which no hybridization occurs may include, for example, 30 to 120° C., preferably 37 to 90° C.
The oligonucleotide primer comprising at least a base sequence represented by “AGNGTT” wherein N is A, T, G or C at the 3′ side includes that represented by
Hashimoto Junko
Hirose Minoru
Yoshimura Tadashi
Chugai Seiyaku Kabushiki Kaisha
Davidson Davidson & Kappel LLC
Tung Joyce
Zitomer Stephanie
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