WO1997015687A1 - Dosage de l'activite de la telomerase - Google Patents

Dosage de l'activite de la telomerase Download PDF

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Publication number
WO1997015687A1
WO1997015687A1 PCT/US1996/009669 US9609669W WO9715687A1 WO 1997015687 A1 WO1997015687 A1 WO 1997015687A1 US 9609669 W US9609669 W US 9609669W WO 9715687 A1 WO9715687 A1 WO 9715687A1
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telomerase
primer
substrate
extended
seq
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PCT/US1996/009669
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English (en)
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Calvin B. Harley
Nam Woo Kim
Scott L. Weinrich
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Geron Corporation
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Priority claimed from US08/482,132 external-priority patent/US5837453A/en
Priority claimed from US08/631,554 external-priority patent/US5863726A/en
Priority claimed from US08/632,662 external-priority patent/US5804380A/en
Application filed by Geron Corporation filed Critical Geron Corporation
Priority to JP9512229A priority Critical patent/JPH11507839A/ja
Priority to AU63808/96A priority patent/AU6380896A/en
Publication of WO1997015687A1 publication Critical patent/WO1997015687A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)

Definitions

  • the present invention relates to telomerase, a ribonucleoprotein enzyme involved in telomere DNA synthesis, and provides assays and materials for identifying and measuring telomerase activity.
  • the invention relates to the fields of molecular biology, chemistry, pharmacology, and medical diagnostic and prognostic technology. Background
  • telomeres are specialized structures at the ends of eukaryotic chromosomes that function in chromosome stabilization, positioning, and replication (Blackburn and Szostak, 1984, Ann- Rey. Biochem. 53: 163-194; Zakian, 1989, Ann. Rev. Genetics 23:579-604; Blackburn, 1991 Nature 35jQ:569-573). In all vertebrates, telomeres consist of hundreds to thousands of tandem repeats of 5'-TTAGGG-3' sequence and associated proteins (Blackburn, 1991 ; Moyzis et al., 1988, Proc. Natl. Acad. Sci. 85:6622-6626).
  • TRF chromosome terminal restriction fragments
  • telomeres This shortening of telomeres has been proposed to be the mitotic clock by which cells count their divisions (Harley, 1991 , Mut. Res. 25.6:271-282), and a sufficiently short telomere(s) may be the signal for replicative senescence in normal cells (Allsopp et aL , 1992; Vaziri et al., 1993; Hastie et al. , 1990, Nature 346:866-868; Lindsey et al. , 1991, Mut. Res. 256:45-8; Wright and Shay, 1992, Trends Genetics 8:193-197).
  • telomere length or sequence with cell divisions suggesting that maintenance of telomeres is required for cells to escape from replicative senescence and proliferate indefinitely (Counter ej al., 1992, EMBO 11: 1921-1929; Counter et aL, 1994, Proc. Natl. Acad. S_ti. USA 91:2900- 2940).
  • Telomerase a unique ribonucleoprotein DNA polymerase, is the only enzyme known to synthesize telomeric DNA at chromosomal ends using as a template a sequence contained within the RNA component of the enzyme (Greider and Blackburn, 1985, Ceil 43:405-413; Greider and Blackburn, 1989, Nature 337:331- 337; Yu et al. , 1990, Nature 344:126-132; Blackburn, 1992, Ann. Rgv. Biochem. 61: 113-129).
  • telomerase activity has been identified in immortal cell lines and in ovarian carcinoma but has not been detected at biologically significant levels (that required to maintain telomere length over many cell divisions) in mortal cell strains or in normal non-germline tissues (Counter et aj., 1992; Counter et aj. , 1994; Morin, 1989. Cell 59:521-529). Together with TRF analysis, these results suggest telomerase activity is directly involved in telomere maintenance, linking this enzyme to cell immortality.
  • telomere activity as well as for identifying compounds that regulate or affect telomerase activity, together with methods for therapy or diagnosis of cellular senescence and immortalization by controlling or measuring telomere length and telomerase activity, have also been described. See PCT patent publication No. 93/23572, published November 25, 1993, and 95/13382, published May 18,1995, inco ⁇ orated herein by reference.
  • the identification of compounds affecting telomerase activity provides important benefits to efforts at treating human disease.
  • Compounds that inhibit telomerase activity can be used to treat cancer, as cancer cells express and require telomerase activity for immortality, and most normal human somatic cells do not express telomerase activity.
  • Compounds that stimulate or activate telomerase activity can be used to treat age-related diseases and other conditions relating to cell senescence.
  • telomerase substrate a radioactive deoxyribonucleotide triphosphate (dNTP) for labelling
  • dNTP radioactive deoxyribonucleotide triphosphate
  • telomere The phase of the repeats depends on the 3'- end sequence of the substrate; telomerase recognizes where the end is in the repeat and synthesizes accordingly to yield contiguous repeat sequences.
  • telomeric sequence oligonucleotides are efficient in vitro substrates, telomerase will also synthesize repeats using substrates comprising non- telomeric DNA sequences.
  • telomerase activity assays with increased sensitivity, speed, and efficiency of detecting telomerase activity as compared to the conventional assay, and this invention meets that need.
  • the assays can be used as diagnostic or prognostic aids, or for the identification, screening, and development of molecules that act as telomerase inhibitors.
  • the present invention also provides reagents, kits, and related methods and materials useful in the practice of the invention.
  • a method which comprises the steps of:
  • the method involves the extraction of telomerase activity, if any, from a cell sample using, for example, a buffer that comprises a non-ionic and/or a zwitterionic detergent.
  • the extracted telomerase is used to mediate extension of a telomerase substrate in a telomerase substrate extension reaction.
  • the telomerase activity is detected in situ, where a telomerase substrate is internalized by the cells in a sample and then extended by the telomerase in situ.
  • the extended telomerase substrate can be detected by numerous methods.
  • the extended telomerase substrate is replicated prior to detection by specific hybridization and subsequent extension of an oligonucleotide "primer" complementary to a telomeric repeat sequence.
  • primer extension is mediated using a template-dependent DNA polymerase or ligase, and the primer is extended by addition of nucleotides to the primer by the DNA polymerase.
  • the DNA polymerase used in this step is preferably a thermostable DNA polymerase.
  • PCR Polymerase Chain Reaction
  • the telomerase substrate lacks telomeric repeats to minimize primer dimer formation.
  • the primer extension can be mediated by a template-dependent DNA ligase, so that the primer is extended by addition of an oligodeoxyribonucleotide to the primer by the DNA ligase.
  • the DNA ligase is a thermostable DNA ligase, and the primer extension step is conducted by (1) heating the reaction mixture to denature duplex DNA molecules; and (2) cooling the reaction mixture to a temperature at which complementary nucleic acids can hybridize and the ligase can extend the primer by ligation.
  • the DNA ligase is a thermostable DNA ligase
  • LCR Chain Reaction
  • oligonucleotides (“ligomers”) complementary to the extended primer in the reaction mixture and by performing the heating and cooling steps from 5, 10, 15, 20, 30, or more times.
  • the present invention also provides a number of reagents such as oligonucleotides, primers, and oligomers, useful in the practice of the present invention. For instance, when one is using PCR to amplify a nucleic acid, one needs to avoid non-specific product formation. Such products can form by a variety of methods, including via interaction of the primers used in the process to form "primer-dimers.”
  • the present invention provides primers and reaction conditions designed specifically to minimize the problem of primer-dimer formation.
  • the invention provides primers that limit the size of the largest primer extension product to no more than a defined number of telomeric repeats more than the largest products of telomerase-mediated extension of the telomerase substrate.
  • Control nucleic acids that can be used in a quantitative telomerase assay are also provided.
  • the method detects extended telomerase products by employing RNA polymerase to synthesize multiple RNA copies of the products.
  • telomerase extended products contain a promoter sequence that is recognized by the RNA polymerase.
  • RNA copies of the extended telomerase substrate can be replicated by using reverse transcriptase to make DNA copies of the RNA.
  • a method comprises the steps of: (a) incubating a cell sample or an extract derived therefrom in a reaction mixture comprising a telomerase substrate and a buffer in which telomerase can catalyze extension of the telomerase substrate by addition of telomeric repeat sequences;
  • signal amplification is achieved by using a branched DNA probe.
  • branched DNA probes can be used with or without target nucleic acid amplification.
  • the various reagents can be labelled to facilitate identification and quantitation of telomerase-extended telomerase substrate.
  • kits comprising those reagents to facilitate practice of the method.
  • a kit comprises a telomerase substrate with or without instructions.
  • a preferred kit comprises the following reagents: CHAPS lysis buffer (10 mM Tris-Cl, pH 7.5; 1 mM MgCl 2 ; 1 mM EGTA; 0.1 mM benzamidine (AEBSF, PMSF or similar reagents can be used in place of or in addition to benzamidine); 5 mM /S-mercaptoethanol; 0.5% CHAPS; 10% glycerol), 10X TRAP reaction buffer (200 mM Tris-Cl, pH 8.3; 15 mM MgCl 2 ; 630 mM KCl; 0.05% Tween 20; 10 mM EGTA; 1 mg/ml BSA), 50X dNTP mix
  • telomere a TRAP primer mix e.g., ACX 0.1 ⁇ g/ ⁇ l; NT 0.1 ⁇ g/ ⁇ l; TSNT 0.01 amol/ ⁇ l
  • a quantitation standard e.g., TSR8; 0.1 amol/ ⁇ l
  • an assay system and apparatus which allow for high throughput detection of telomerase activity.
  • the assay system and apparatus provide for simultaneous separation of telomerase products from primers and nucleotides in multiple samples and allows for quantitative detection of the isolated telomerase products.
  • telomere activity is especially useful and applicable to the detection of telomerase activity in samples of biological material obtained from humans.
  • samples will contain cells or cellular materials and will typically be obtained from humans for the pu ⁇ ose of detecting a diseased state or other medical condition of interest, such as, cancer.
  • telomerase is not expressed by most normal post-natal human somatic cells, although low levels of telomerase activity can be detected in certain stem cells, activated cells of the hematopoietic system, and fetal tissues, so the presence of telomerase activity in a sample of human somatic tissue or cells indicates that cells of extended proliferative capacity, such as immortal cells, fetal cells, or hematopoietic cells, are present in the tissue. While not all cancer cells express telomerase activity, telomerase expression is required for cells to become immortal. Consequently, the presence of cells with telomerase activity is associated with many forms of cancer and can also serve to indicate that a particularly invasive or metastatic form of cancer is present.
  • the invention provides a method for diagnosis of a condition in a patient associated with an elevated (or reduced) level of telomerase activity within a cell.
  • the method involves determining the presence or amount of telomerase activity within the cells of the patient, and the method is therefore applicable to the detection of elevated (or reduced) levels of telomerase activity associated with, for example, prostate cancer, breast cancer, colon cancer, renal cancer, skin cancer, liver cancer, ovarian cancer, cervical cancer, lung cancer, urogenitary cancer, and leukemia, or, in the case of reduced levels, infertility.
  • the method can also be used for testing other diseased states and medical conditions, e.g., fetal cell testing to detect fetal cells in maternal blood as an indication of pregnancy or to detect telomerase as a marker for bone marrow proliferative capacity. Whether for diagnosis or other pu ⁇ oses, the method involves determining the presence or amount of telomerase activity within the cells by a telomerase substrate extension reaction, and replicating the extended telomerase substrate, for example, by primer extension, such as in the PCR.
  • FIG. 1 A is a schematic perspective of the Multiplex Electrophoretic Separator (MES) apparatus of the invention.
  • the parallel arrows depict the direction of the electrical field when the electrodes are attached to the electrical source.
  • FIG. IB is a cross sectional view of the MES apparatus.
  • telomere activity synthesizes telomeric DNA at the ends of chromosomes and is believed to be necessary for indefinite proliferation of immortal cells.
  • Analysis of chromosome terminal restriction fragments (TRF) in a wide variety of human cell types has shown that telomere length and sequence are stably maintained in immortal cell lines but not in dividing cultures of normal somatic cells. Telomere and telomerase biology are clearly important in the maintenance of the immortal cell state and other biological states.
  • telomerase activity in the cell sample with presence of the extended telomerase substrate correlating presence of telomerase activity in the cell sample with presence of the extended telomerase substrate.
  • the method essentially involves two reactions: (1) telomerase-mediated extension of a telomerase substrate; and (2) replication of telomerase-extended substrates.
  • telomerase-mediated extension of a telomerase substrate and (2) replication of telomerase-extended substrates.
  • Samples of particular interest include cell samples, which can be tissue or tumor samples, obtained for pu ⁇ oses of diagnostic analysis.
  • cell samples which can be tissue or tumor samples, obtained for pu ⁇ oses of diagnostic analysis.
  • the expression of telomerase activity in a variety of cells has been studied and discussed in the scientific literature.
  • Telomerase is expressed not only by certain pathogens (i.e., malaria, fungi, yeast, and ciliates) but also by immortal human cells, including certain types of tumor and cancer cells, but is not expressed by cells of normal somatic (as opposed to germline or embryonic) tissue, although low levels of telomerase activity can be detected in stem cells, fetal cells, and in certain activated cells of the hematopoietic system. Consequently, samples can be obtained for the purpose of determining whether a telomerase-expressing pathogen or cancer or tumor cell is present.
  • samples tested by the present method can be obtained from agriculturally important mammals, such as cattle, horses, sheep, or any other animal of veterinary interest, such as cats and dogs, and from the environment, i.e., for environmental testing for the presence of pathogens.
  • telomerase activity is assayed in vitro, requiring the preparation of a cell extract.
  • Methods for the preparation of cell extracts are known in the art (for example, see Scopes, 1987, Protein Purification:
  • the detergent lysis method is used which provides more uniform extraction of telomerase even at low cell numbers.
  • the method involves the steps of: (1) collecting a sample of cells; (2) lysing the sample in a lysis buffer comprising 0.01 to 5 % of a non-ionic and/or a zwitterionic detergent; (3) removing cellular debris by centrifugation; and (4) collecting supernatant separated from the cellular debris.
  • a cell extract can also be prepared merely by lysing a cell sample to release telomerase without further sample preparation.
  • non-ionic and/or zwitterionic detergents can be employed in the method.
  • Preferred non-ionic detergents include Tween 20, Triton X-100, Triton X-114, Thesit, NP-40, n-octylglucoside, n-dodecylglucoside, n-dodecyl- beta-D-maltoside, octanoyl-N-methylglucamide (MEGA-8), decanoyl-N- methylglucamide (MEGA- 10), and isotridecyl-poly(ethyleneglycolether) n
  • preferred zwitterionic detergents include CHAPS (3- ⁇ (3- cholamidopropyl)dimethylammonio ⁇ -l-propane-sulfonate), CHAPSO (3- ⁇ (3- cholamidopropyl)dimethyl-ammonio ⁇ -2-hydroxy- 1 -propane-sulfonate
  • telomerase activity assay of the invention is far superior to the conventional assay in detecting telomerase activity in such circumstances, as well as being faster to complete and more efficient.
  • the telomerase activity assay is applied to intact cells.
  • Intemalization of the substrates can be achieved using methods known in the art, for example, by passive intemalization of substrate oligonucleotides or other nucleic acid added to the media surrounding the cell sample (typicaUy at a concentration of 10-100 ⁇ M), by microporation using a detergent or Staphylococcus alpha toxins, by employing liposomes (e.g., LipofectAmineTM, LipofectinTM, LipofectAceTM available from
  • the sample is incubated to allow any active telomerase present in the cell to extend the substrate by de novo synthesis of telomere repeats. After incubation, the sample is fixed and permeabilized, for example, by treatment with a protease such as proteinase K, pronase, trypsin, pepsin, or the like.
  • a protease such as proteinase K, pronase, trypsin, pepsin, or the like.
  • telomerase-extended substrate is then amplified in situ by any of various methods known in the art, for example, using a primer extension reaction (e.g., employing established PCR or LCR protocols or primed-m situ labelling (PRINS; Koch, J., in "Nonradioactive in situ Hybridization Application Manual” (1992), Boehringer Mannheim, 31-33) or by other methods as described below.
  • a primer extension reaction e.g., employing established PCR or LCR protocols or primed-m situ labelling (PRINS; Koch, J., in "Nonradioactive in situ Hybridization Application Manual” (1992), Boehringer Mannheim, 31-33
  • PRINS primed-m situ labelling
  • the sample (or an aliquot thereof) is assayed in a reaction mixture comprising a telomerase substrate.
  • a telomerase substrate chosen in each case may vary depending on the type or origin of the telomerase activity for which one is testing, or the type of amplification or detection method employed.
  • the telomerase activity expressed by one organism may differ with respect to substrate specificity from that expressed by another organism. Consequently, if one is using the present method to determine whether a cancer cell of human origin is present in the sample, one employs a telomerase substrate recognized by human telomerase.
  • telomerase substrates are known for the telomerases of Tetrahymena, other fungi, mammahan, and human cells and can readily be identified for other types of cells.
  • the present method requires that the telomerase substrate not comprise a complete telomeric repeat sequence to minimize primer-dimer formation.
  • telomerase activity will depend upon the origin of the telomerase. For instance, Tetrahymena telomerase adds repeats of sequence 5'-TTGGGG-3' to the ends of telomerase substrates, while human telomerase adds repeats of sequence 5'- TTAGGG-3'.
  • the telomerase substrate should be a human telomerase substrate lacking the complete repeat sequence 5'-TTAGGG-3'.
  • a human telomerase substrate lack a telomeric repeat sequence from an organism that has a telomerase that adds a different repeat, so long as the presence of that different repeat sequence does not produce undesired results, such as excessive primer-dimer formation, as discussed further below.
  • the substrate can be a circular plasmid DNA that undergoes linearization at a specific site, either inducibly or spontaneously. Such a plasmid substrate is particularly useful for in situ applications.
  • An illustrative plasmid telomerase substrate is a vector that contains an insert with a unique restriction site (e.g., Isce I) located 3' to the telomerase substrate sequence.
  • unique means that the restriction site is not present in the genome of the cell under analysis.
  • the vector is a selectable, multi-copy vector with a mammalian origin of replication.
  • the method can further include a second expression plasmid that contains a gene coding for a restriction enzyme specific for the unique site, under the control of an inducible promoter. The two plasmids are co-infected into the target cell by methods known in the art, and are replicated.
  • the restriction enzyme cleaves the DNA of the telomerase substrate plasmid at the unique restriction site resulting in a linearized substrate plasmid, the ends of which are recognized as a telomerase substrate and can be elongated with TTAGGG repeats by telomerase.
  • the telomerase substrate to lack telomeric repeat sequences in some instances, in particular where the replication step of the present method involves the hybridization of a primer or probe to extended telomerase substrates.
  • the non-telomerase-mediated primer extension reaction involves hybridization of an oligonucleotide primer that hybridizes only to extended telomerase substrates. This addition is made under conditions such that, if extended telomerase substrates are present, the primer binds to the extended substrates and is then extended by enzymatic action. Because telomerase can extend the telomerase substrate only by the addition of telomeric repeats, the primer will necessarily comprise a sequence complementary to a telomeric repeat.
  • telomerase substrate employed in the telomerase extension reaction comprised a complete telomeric repeat
  • primer employed in the primer extension reaction could hybridize readily to unextended telomerase substrate, with potentially negative consequences.
  • the telomerase substrate can, however, comprise sequences highly related to a telomeric repeat sequence without compromising the validity of the results obtained.
  • an especially preferred human telomerase substrate of the invention is oligonucleotide M2, also known as TS, which contains a sequence at its 3 '-end that is identical to five of the six bases of the human telomeric repeat but otherwise contains no complete telomeric repeat sequences.
  • telomerase substrate be free of telomeric repeat sequences where the replication or detection method is not compromised by the presence of such repeats in the substrate, i.e. , where primer extension is mediated by a ligase activity, or replication is achieved by means in which specific hybridization of a probe or primer to a telomeric repeat sequence is not problematic.
  • the primer extension reaction conducted subsequent to the telomerase substrate extension serves to amplify the signal produced by the presence of telomerase activity in a sample (extended telomerase substrates) by producing a second signal (extended primers).
  • the primers can be extended by any means that requires the presence of extended telomerase substrates for primer extension to occur; preferred means are mediated by a template-dependent DNA or RNA polymerase, a template-dependent DNA ligase, or a combination of the two. With these means, if telomerase activity is present in the sample, an extended telomerase substrate is formed and then hybridizes to a primer, providing a substrate for either DNA or RNA polymerase or DNA hgase to produce a primer extension product.
  • primer extension product Once a primer extension product has formed, one can disassociate (typically by heating, but one could also use an enzyme or chemical process, such as treatment with helicase) the extended primer from the extended substrate. If additional primer and primer extension reagent is present in the sample, then a new primer/extended telomerase substrate complex can form, leading to the production of another extended primer.
  • primer extension and denaturation of extended primer/extended telomerase substrate complexes will be performed at least 5, 10, 15, 20 to 30 or more times.
  • telomerase substrate still present in the reaction mixture during the primer extension step can function as such a second primer.
  • the primer extension reagent is a DNA polymerase
  • a second primer is present, one has the requisite components for a polymerase chain reaction, more fully described in U.S. Patent Nos. 4,683,195; 4,683,202; and 4,965,188, provided the appropriate buffer and nucleoside triphosphates are present in the reaction mixture.
  • PCR amplification is a preferred mode for conducting the primer extension reaction step of the present invention and dramatically increases sensitivity, speed, and efficiency of detecting telomerase activity as compared to the conventional assay.
  • the protocol is termed "TRAP” for Telomeric Repeat Amplification Protocol and is illustrated, inter alia, in Example 2.
  • the telomerase substrate can also conveniently serve as a PCR primer (termed the "upstream primer”), although many other primer sequences can also be used.
  • the sequence of the other primer is chosen to avoid annealing of the telomerase substrate and the primer, because even minor levels of primer/telomerase substrate annealing can yield early cycle PCR products identical to telomerase products (e.g., TS plus (5'-AG[GGTTAG] n -3'), where n equals 1, 2, 5, 10, or more. In subsequent cycles, these products could serve as template for the production of PCR products, potentially resulting in a false positive result.
  • the present invention provides a variety of oligonucleotide primers and telomerase substrates for use in the PCR-based embodiment of the present invention.
  • One such primer (termed the “downstream primer”) is designated “CX" and is composed of sequences complementary to three imperfect telomeric repeats and one perfect repeat, 5'-(CCCTTA) 3 CCCTAA-3' (SEQ ID NO:l).
  • the single nucleotide difference in three of the repeats compromises the capacity of CX to anneal to the telomerase substrate TS (which, as noted above, contains 5 of 6 nucleotides of a telomeric repeat) by creating a 3' mismatch in the TS/CX duplex, thereby minimizing the formation of non-specific PCR products, such as primer-dimer.
  • TS telomerase substrate
  • Any possible alignment between these primers (CX and TS) nucleated by the telomeric sequence complementarity leads to a duplex in which the recessed 3' nucleotide is mismatched and so is not efficiently extended by polymerase.
  • a primer with sequences "complementary to a telomeric repeat” includes a primer that may contain one or more mismatched bases with respect to the telomerase substrate extension product to which the primer is intended to hybridize.
  • the number of mismatches that can be tolerated within this definition can vary depending upon the length and sequence composition of the primer, the temperature and reaction conditions employed during the PCR step, the purpose for which the assay is conducted, and the results desired.
  • the present invention provides several modifications of a basic PCR that, while not necessary to obtain the benefits of the present method, can greatly enhance the specificity, sensitivity, and efficiency of the present method for some applications and in certain embodiments.
  • the buffer provides a buffer in which both telomerase activity and DNA polymerase activity can be observed.
  • the use of such a buffer allows the artisan to conduct both the telomerase substrate extension reaction and a DNA polymerase-mediated primer extension reaction in the same reaction vessel, for example, in a tube (see Example 2).
  • Another modification relates to the use of relatively short oligonucleotides that are complementary to either the telomerase substrate or the return primer in the reaction mixture.
  • These short oligonucleotides are designed to have a melting temperature (with respect to the primer or telomerase substrate to which the short oligonucleotides hybridize) about 10°C lower than the annealing temperature of the primers used in the primer extension step and to prevent primer-dimer formation and/or non-specific primer extension, particularly at low temperatures.
  • the short oligonucleotides melt away from their complementary oligonucleotides at temperatures just below the ideal annealing temperatures for the primer extension step, preventing inappropriate primer extension at lower, non-specific temperatures.
  • the 3 '-end of the short oligonucleotide can be blocked to prevent addition of nucleotides to the short oligonucleotide. If the short oligonucleotide is designed to hybridize to the primer, then the 3 '-end of the short oligonucleotide should be blocked (e.g., with biotin, phosphate, digoxigenin, a fluorescein or an amino group) to prevent the short oligonucleotide from serving as a telomerase substrate.
  • a variety of other reagents and formats can be employed to ensure a high degree of specificity, including: (1) the use of T4 gene 32 protein (available, e.g., from Boehringer Mannheim); (2) the use of TaqStartTM antibody (available, e.g., from Clontech); and (3) the separation of the primer from the other reaction components by a wax barrier (e.g., AmpliwaxTM, available from Perkin Elmer) that melts only after the reaction mixture is heated at the end of the telomerase- mediated extension reaction. The pu ⁇ ose of the wax barrier is to separate the telomerase extension reaction from the amplification reaction.
  • a wax barrier e.g., AmpliwaxTM, available from Perkin Elmer
  • the DNA polymerase can be sealed under a wax layer thus separating it from other reaction components.
  • the primer can be sealed under a wax layer or barrier at the bottom of a tube with the other reaction components positioned on top of the barrier.
  • reaction components can be attached to solid matrices, such as polystyrene beads of about 1 micron to about 5 millimeters in diameter; plates or the wells of a microtiter plate such as those made from polystyrene or polyvinylchloride; glass beads; magnetic particles; polysaccharides; or other surfaces that can be coated, e.g. , with primer.
  • the primer is typically affixed to the solid matrix by adso ⁇ tion from an aqueous medium although other modes of affixation, as is well known to those of ordinary skill in the art, can also be used.
  • the solid surface is contacted with a solution of the primer and dried until the surface is coated with an appropriate amount of primer.
  • the amount of primer coated on the solid surface can be varied by adjusting the concentration of primer in the contacting solution.
  • the primer-coated solid matrix is then covered with hot molten wax that is left to solidify.
  • the wax barrier thus isolates the primer from the other reaction components (e.g., the telomerase substrate, dNTPs, buffer, DNA polymerase or ligase) until a temperature is reached at which the wax melts.
  • kits of the invention comprises a reaction tube or other solid support having a primer or polymerase separated from the telomerase reaction buffer and/or other reagents by a wax barrier.
  • the primer extension methods of the present invention have been used to test for telomerase activity in human cell lines and normal somatic cells as illustrated in Examples 2. Telomerase-positive extracts from human 293 kidney cells were produced routinely from IO 5 cells, as assessed by TRAP assay, with a lower limit for the conditions employed in this particular example of 10 2 cell equivalents for detection of telomerase activity.
  • Example 3 The telomerase activity assay method of Example 2 has been used to test for telomerase activity in various immortal cell lines and normal somatic cell cultures from different tissues and individuals, as illustrated by Example 3 below. As shown in Example 3, the difference in telomerase activity between immortal and normal somatic cells was estimated to be at least 1000-fold, supporting a direct role for telomerase in telomere dynamics in human cells. Those of skill in the art will recognize the detection limits noted above are valid only if one employs merely routine procedures.
  • the present method can be used to detect telomerase activity in a single cell, provided one is willing to use effort somewhat greater than what is typically considered routine, primarily for the single cell isolation step.
  • Example 11 illustrates this aspect of the invention.
  • the PCR-based embodiment of the present invention offers significant improvements over currently available methods for measuring telomerase activity in a sample.
  • Other novel variations of the present method also offer significant advantages.
  • the present method can be used to quantitate the telomerase activity in a sample by providing the number of telomerase products generated.
  • telomerase products generated.
  • staggered binding refers to the binding of a primer to a sequence in an extended telomerase substrate in a manner that leaves the 3 '-end of the extended telomerase substrate recessed and therefore available for extension by DNA polymerase.
  • DNA polymerase can add nucleotides to the 3 '-end of the extended telomerase substrate, creating molecules longer than those produced in the telomerase-mediated extension step.
  • synthetic oligonucleotides representing discrete telomerase extension products e.g., TS+4 (TS plus four telomeric repeats), were used to develop specific amplification conditions.
  • telomere extension products produced using a primer such as CX are not directly reflective of the length distribution of telomerase products generated in the telomerase substrate extension step, due to the staggered binding of primers to templates during the primer extension reactions.
  • a primer such as CX
  • an anchor sequence is a 5 '-terminal sequence of a PCR primer that is a non-telomeric repeat sequence (a sequence neither complementary nor identical to a complete telomeric repeat sequence) and that prevents the PCR product from "growing" on itself, for example as observed when the primer pairs TS/(CTR) 4 or TS/CX are employed.
  • such an oligonucleotide can comprise a 6 nucleotide anchor sequence (although the length is not critical and can be 3 to 5 to 15 to 30 or more nucleotides) at its 5 '-end followed by three repeats of CTR (C-rich telomeric repeat; 5'-CTAACC-3') sequence (e.g., the ACT primer; 5'-GCGCGG[CTAACC] 3 -3'; SEQ ID NO:2 is an anchored primer of the invention).
  • CTR C-rich telomeric repeat
  • the anchor sequence is the sequence of the telomerase substrate used in the telomerase-mediated extension step of the method, providing a "TS -anchored" primer (note that, in this context "TS" represents any telomerase substrate lacking a complete telomeric repeat sequence).
  • the anchored primer would thus comprise, in the 5'-to-3' direction, a telomerase substrate sequence and two or more complementary copies of the telomeric repeat sequence.
  • telomerase substrate in the reaction mixture can prime the synthesis of both strands of the duplex formed as a result of the first round of primer extension.
  • the primers TS (5'-AATCCGTCGAGCAGAGTT-3'; SEQ ID NO:3) and ACT (or another anchored primer) in the TRAP assay
  • MPP Most Processive Product
  • the use of an anchored primer such as ACT prevents the growth of telomerase products into longer versions during PCR.
  • the slowest migrating band reflects directly the length of the MPP of the original telomerase products before the PCR.
  • primer extension products that comprise many more telomeric repeats than present in the telomerase-extended telomerase substrates originally present in the reaction mixture after the telomerase substrate extension reaction.
  • the ACT primer is particularly preferred for purposes of the present invention in that it is more resistant to the types of primer- dimer interactions observed between TS and primers such as CX or CTR 4 .
  • a hybrid oligonucleotide can be used as a retum primer.
  • the hybrid has an anchor sequence followed by a primer-based sequence that contains mismatches in the complementary telomeric repeats, for example, ACX 5'- GCGCGG[CTTACC] 3 CTAACC-3' (SEQ ID NO:4) that has mismatches in 3 of 4 complementary telomeric repeats.
  • a primer-based sequence that contains mismatches in the complementary telomeric repeats, for example, ACX 5'- GCGCGG[CTTACC] 3 CTAACC-3' (SEQ ID NO:4) that has mismatches in 3 of 4 complementary telomeric repeats.
  • the resulting ACX is more resistant to primer-dimer formation than either the ACT or CX primer.
  • the utilization of the ACX primer in the TRAP assay provides similar benefits as the wax-barrier methodology in preventing primer-dimer formation, but simplifies the analysis, manufacture, and performance of the TRAP assay.
  • the TRAP assay can be further improved by reducing non-specific, template- independent, PCR artifacts (e.g., primer-dimer) by means other than or in addition to oligonucleotide selection and the use of a hot-start wax barrier.
  • non-specific, template- independent, PCR artifacts e.g., primer-dimer
  • DMSO dimethyl sulfoxide
  • glycerol optionally with glycerol
  • telomere extension e.g., telomere styrene styrene styrene styrene styrene styrene styrene styrene styrene styrene styrene styrene styrene styrene styrene styrene RNA polymerase; Perkin Elmer
  • a primer such as CX or a CX-based primer, hybridizes to a template (e.g., TS)
  • a template e.g., TS
  • the present invention also provides a variety of means to quantitate the amount of telomerase in a sample, although for most purposes, a qualitative result (telomerase activity present or absent) is sufficient.
  • a control oligonucleotide template added to each reaction mixture in a known amount, as illustrated below in Example 4.
  • An illustrative competitive control oligonucleotide of the invention comprises, in 5'-to-3' order, a telomerase substrate sequence, a spacer sequence (preferably 3 nucleotides in length, but which can be any sequence of nucleotides or length; a length of 3 nucleotides ensures that the internal control is different from that from the extended substrates, and a telomeric repeat sequence (typically present in multiple, e.g., 2 to 50, copies).
  • a spacer sequence preferably 3 nucleotides in length, but which can be any sequence of nucleotides or length; a length of 3 nucleotides ensures that the internal control is different from that from the extended substrates, and a telomeric repeat sequence (typically present in multiple, e.g., 2 to 50, copies).
  • a telomerase substrate sequence preferably 3 nucleotides in length, but which can be any sequence of nucleotides or length; a length of 3 nucle
  • control nucleic acid of any sequence can be added to the reaction mixture in known amounts and amplify the control with primers different from those used to amplify the extended telomerase substrate (non-competitive internal control).
  • the control oligonucleotide and/or the primers used to amplify the control oligonucleotide can be labelled identically to or differently from the label used to label the telomerase extension products.
  • a preferred internal control oligonucleotide comprises a combination of the controls described above where, in the PCR embodiment of the invention, the internal control competes with the telomerase extension product for only one primer and is thus termed a semi-competitive control.
  • a control comprises, in 5'-to-3' order, a sequence that is not a substrate for telomerase or a telomeric repeat, or a complementary sequence thereof followed by a telomeric repeat sequence.
  • An alternative semi-competitive control comprises, in 5'-to-3' order, a telomerase substrate sequence followed by a sequence that is neither a substrate for telomerase nor a telomeric repeat or a complementary sequence thereof.
  • the control can also be designed to be separated easily from the TRAP products for quantitation.
  • An example of such a semi-competitive internal control is 5'- AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3' (SEQ ID NO: 5; TSNT) that can be amplified by the TS telomerase substrate primer and the NT primer 5'-ATCGCTTCTCGGCCTTTT-3' (SEQ ID NO: 6).
  • the NT primer is not a substrate for telomerase.
  • the TSNT oligonucleotide is employed in a TRAP assay with TS, ACX (or any other retum primer) and NT, and the resulting 36 bp control duplex DNA is easily distinguished from the telomerase extension products, the smallest of which is 50 bp.
  • the control oligonucleotide can also be conveniently packaged into a kit with other reaction components.
  • the TaqManTM (Perkin Elmer) detection system can be employed to detect the presence of a nucleic acid in a sample.
  • This system is suitable for use in the telomerase assays of the invention and provides a rapid detection method that is non-radioactive and readily modified to a multiwell system.
  • a target specific probe that possesses both a fluorescent reporter dye tag and a quencher dye tag is inco ⁇ orated into the primer extension reaction (e.g., PCR amplification).
  • the tags are preferably positioned 6-8 nucleotides apart.
  • the TaqManTM probe hybridizes to the target sequence at an internal site under primer extension conditions and, if the primer is extended by the action of a DNA polymerase having 5 '-3' exonuclease activity, the 5 '-3' exonuclease activity of the polymerase degrades the hybridized probe freeing the reporter dye/dNTP from the proximity of the quencher dye.
  • the increase in the free reporter dye/dNTP complex results in an increase in fluorescence that is proportional to the amount of amplified product (Livak et aL, January 1995, Research News, Perkin Elmer Co ⁇ oration, 1-5; Lee et a ⁇ ., 1993, Nucl. Acids Res. 21:3711-3766).
  • TaqManTM probe and the two primers is limited. These sites comprise the telomerase substrate sequence (e.g., TS; 5'-AATCCGTCGAGCAGAGTT-3'; SEQ ID NO:3), and the telomeric repeat sequences (e.g. , TTAGGG), or their complementary sequences, with or without mismatches.
  • a TaqManTM probe that comprises C-rich telomeric repeat (CTR; 5'-CCCTAA-3') sequences can be used (e.g. , a probe that comprises four repeats of CTR; this probe is termed CTR .
  • the CTR probes could compete with the ACT primer (or other retum primers) as they hybridize to the same sites, which can lead to a reduction in PCR efficiency.
  • the probe can anneal to the telomerase substrate, such as TS, the use of hot-start PCR methodology in the assay may be useful; this can readily be accomphshed by separating the probe and the retum primer with a wax barrier from the remaining reaction components.
  • a preferred TaqManTM probe consists of a sequence complementary to the telomerase substrate; therefore such a probe does not compete with either the forward or retum primers, and thus does not result in primer-dimer formation.
  • Such a probe can form a duplex with the forward primer (e.g. , TS), which can decrease PCR efficiency during exponential amplification.
  • TS forward primer
  • telomerase can recognize and extend double- stranded substrates (see Example 8), and the reaction can proceed in the presence of forward primer-probe duplexes.
  • a TaqManTM probe that consists of a sequence complementary to the 3' region of the forward primer, followed by a telomeric repeat sequence (e.g., CTR sequence), can be used.
  • a telomeric repeat sequence e.g., CTR sequence
  • Such a probe specifically hybridizes to the junction between the forward primer and the telomeric repeat sequence and reduces not only competitive effect with the retum primer but also forward primer-probe duplex formation. With such a probe, generation of primer-dimer artifacts can be avoided by using hot-start TRAP methodology.
  • a probe comprising the sequence 5'-AACCCTAACCCTAACTCTGCT-3' (SEQ ID NO: 7), corresponding to the junction sequence between the 3' end of TS and the telomeric repeats of the telomerase product, can be used. Altematively, mismatches can be introduced into such probes to minimize potential interference with the telomerase substrate extension reaction, i.e. , by minimizing probe binding to TS during the extension step.
  • probes having the sequence 5'- CCTAACCCTAACCCCACTATGCT-3' (SEQ ID NO: 8) or 5'- CCTAACCCTAACCCTGTATATGCT-3' (SEQ ID NO: 9) can be used in this embodiment. Conditions are selected to allow the probe to bind preferentially to the extended telomerase substrate during the PCR reaction.
  • a TaqManTM probe consisting of the telomerase substrate sequence or telomere repeat sequences (e.g., 5'-TTAGGG-3') can also be used in the TaqManTM detection system.
  • these probes may compete with the telomerase substrate for telomerase, although competition can be minimized using hot start methodology.
  • reporter and quencher dyes are present at the two ends of a retum primer with a 3' mismatch, as exemplified by the primer MACX, which has the sequence 5'-
  • GCGCGG[CTTACC] 3 CTAACCAAT-3' SEQ ID NO: 10
  • the mismatched nucleotide at the 3' end of the primer is cleaved by the exonuclease activity releasing the reporter dye from the quencher prior to extension.
  • any amplification using such a primer results in a fluorescent signal.
  • This embodiment of the invention is not limited to fluorescence quenching, as other proximity indicating signals can be used; for example, a probe can be labelled with a radioactive label and detected with a scintillant.
  • the method of the invention can involve the conelation of telomerase activity in a sample with the formation (presence in the reaction mixture) of duplex nucleic acids composed of extended telomerase substrates annealed to extended primers, one can infer the presence of such molecules by the presence of either (1) an extended telomerase substrate; (2) an extended retum primer; (3) a duplex nucleic acid comprising both (1) and (2); or (4) hybridization of a probe to any of the foregoing.
  • PCR-based embodiment of the present method has been described in detail above and is exemplified in the Examples below, the present method can be practiced using any method of primer extension to provide target amplification or with a method that provides for signal amplification or both, as described below.
  • PCR provides for exponential accumulation of primer extension products, even linear accumulation of primer extension products can provide useful results.
  • telomere extension products can be made using DNA ligase to ligate together two oligonucleotides hybridized to the extended telomerase substrate. If, as in PCR, the duplex nucleic acid is then denatured, then one can repeat the process of ligation and denaturation many times to accumulate many complementary copies of the original extended telomerase substrate.
  • LCR LTS (5'-CCCAATCCGTCGAGCAGAGTTAG-3') (SEQ ID NO: 11), CLT (5'-TAACTCTGCTCGACGGATTCCC-3') (SEQ ID NO: 12), LC (5'-GGGTAACCCTAACCCTAACCC-3') (SEQ ID NO: 13), and LG (5'-GGTTAGGGTTAGGGTTAAA-3') (SEQ ID NO: 14).
  • the LC and CLT ligomers will anneal to an extended telomerase substrate and then be ligated with DNA ligase to form a template for ligation of the LTS and LG ligomers.
  • These ligomers have been selected so that no two ligomers can anneal to form a duplex nucleic acid that can be joined to another duplex nucleic acid in the mixture by the blunt-end ligation activity of DNA hgase.
  • a wide variety of such ligomers can be used in the method to minimize template- independent product formation.
  • LCR amplification of telomerase extension products produces an amplified product of uniform size and so is conducive to quantitative analysis.
  • thermostable polymerase and a hgase with primers, such as, for example TS, LC, and CLT, can be used to amplify telomerase-extended substrates.
  • primers such as, for example TS, LC, and CLT
  • ohgonucleotides complementary to adjacent portions of the telomerase extension product are employed to detect the presence of telomerase extension product.
  • the ohgonucleotides are constmcted so that when annealed to the telomerase extension product, the 5 '-end of one oligonucleotide can be ligated to the 3 '-end of the other.
  • One of the oligonucleotides is labelled with a fluorescent "reporter" label and the other with a "quencher” label.
  • the components can both be fluorescence emitters with the absorbance spectmm of the first being selected to overlap with the emission spectmm of the second; an example of such a pair would be fluorescein and rhodamine.
  • the quenching effect resulting from the proximity of the reporter to the quencher can be detected as an indication of telomerase extension products.
  • Subsequent ligation of the annealed ohgonucleotides is required for further amplification by LCR, although ligation is not required for detection using the assay.
  • This detection system is not limited to fluorescence and quenching but can be used with probes comprising any two components that interact in a detectable manner through adso ⁇ tion, modulation, or emission of electromagnetic or nuclear radiation.
  • Detection can, for example, be mediated by chemical, enzymatic or radiation events.
  • a radioactive label and a scintillant, or an enzyme pair in which the product of one enzyme is a substrate for the second can be used to provide a proximity signal. Any proximity indicating pair resulting in a detectable signal can be used; detection can be fluorometric, radiometric, or colorimetric, for example, (iii) RNA Polymerase-Mediated Replication
  • telomerase extended products are replicated by means of the action of an RNA polymerase.
  • a variety of methods for replicating nucleic acids using an RNA polymerase are known, e.g., nucleic acid sequence- based amplification (Compton, 1991 , Nature 350:91-92), self-sustained sequence replication (Guatelli et aj., 1990, Proc. Natl- Acad. Sri. USA 87:1874-1878), and strand displacement amplification (Walker et aj., 1992, Proc. Natl. Acad. Sci. USA 89:392-396).
  • an RNA polymerase that utilizes a single stranded DNA template e.g., N4 RNA polymerase
  • a single stranded DNA template e.g., N4 RNA polymerase
  • the telomerase substrate can comprise a promoter sequence operably linked to its 5' end or the promoter can be ligated to the substrate after extension of the substrate and prior to replication with the RNA polymerase.
  • Many RNA transcripts can be generated from a single DNA template thereby increasing the number of the target molecules.
  • the method employs a non-telomeric telomerase substrate sequence (e.g., TS) that can be extended by telomerase and, a retum primer, e.g., TS-telomerase substrate sequence (e.g., TS) that can be extended by telomerase and, a retum primer, e.g., TS-telomerase substrate sequence (e.g., TS) that can be extended by telomerase and, a retum primer, e.g., a retum primer, e.g.,
  • CTR 3 (comprising 3 repeats of the CTR sequence) that is complementary to telomeric sequences.
  • a DNA polymerase e.g., Taq polymerase, Klenow fragment, Stoffel fragment of AmpliTaqTM polymerase
  • An RNA polymerase that recognises a double stranded substrate e.g., T7 polymerase, T3 polymerase,
  • SP6 polymerase is then used to synthesize multiple RNA copies of the duplex telomerase extension product as directed by a promoter sequence inco ⁇ orated into the 5 '-end of the telomerase substrate or retum primer.
  • a promoter sequence inco ⁇ orated into the 5 '-end of the telomerase substrate or retum primer.
  • Any of a variety of promoters e.g., T7, T3, SP6 promoters, can be used for this pu ⁇ ose.
  • the promoter sequence can be present during the telomerase extension step or hgated at a later stage to the extension products.
  • amplification can also be achieved by using nucleic acid sequence-based amplification (NASBA).
  • an RNA polymerase (such as T7 RNA polymerase) synthesizes RNA copies of the extended telomerase substrate essentially as described above.
  • a reverse transcriptase is used to synthesize DNA copies of the RNA, RNaseH degrades the RNA strand and the single stranded DNA acts as a template for RNA synthesis, thus providing cyclic amplification.
  • reverse transcriptase can be used to synthesize cDNAs, or RNA polymerase can be used to extend primers, and the products can be amplified by primer extension (e.g., with PCR).
  • telomere activity is conelated to the presence of RNA copies of the extended telomerase substrate.
  • primers can be designed (for example, to decrease background), as would be apparent to one of ordinary skill in the art.
  • telomerase extension products are either first rephcated and/or then detected using branched DNA (bDNA) signal amplification (Urdea, 12 Sep. 1994, Bio/Tech. 12:926-928; U.S. Patent No: 5,124,246) which involves amplification of the signal produced upon probe hybridization to a target nucleic acid.
  • bDNA branched DNA
  • the assay for detecting telomerase activity with bDNA can be carried out in a multi-well plate, a format that is particularly useful for screening, because the assay is simple to perform with multiple samples, and commercial hardware is available. Furthermore, conditions can be chosen to allow quantitative detection of telomerase activity. Any telomerase substrate (e.g.
  • TS oligonucleotide 5'-AATCCGTCGAGCAGAGTT-3'; SEQ ID NO:3) can be used, and the substrate is incubated with a cell extract, allowing any telomerase present to extend the substrate (optionally bound to a solid support to facihtate detection) with the addition of telomeric repeats.
  • the substrate is linked at its 5' end to a well of a multi- well plate (or other sohd surface) using conventional chemical techniques. Multi-well plates with linked ohgonucleotides are also available commercially (Chiron Co ⁇ .).
  • the telomerase substrate is bound by hybridization to a complementary oligonucleotide bound to the solid surface. The telomerase extension reaction can occur on bound ohgonucleotide substrate or on free oligonucleotide substrate that is bound at a later stage.
  • the bDNA probe is comprised of a hybridizing portion complementary to the telomeric repeats (e.g., 5'-(CCCTAA) n -3' or its permutations) and so hybridizes with extended telomerase substrates.
  • the probe further comprises a branched region that provides multiple secondary probe binding sites. After washing to remove unbound probe, a labelled secondary probe specific for the branches of the bDNA is hybridized to the bDNA and is detected via the label.
  • the telomerase substrate can be immobilized after extension by telomerase by capturing the telomerase extension products by hybridization to immobilized ohgonucleotides complementary to telomeric repeats and detecting the products by hybridization to a probe complementary to the telomerase substrate.
  • the signal increases in direct proportion to the secondary probe-accessible-sites on the bDNA molecule, thus a rare population of target nucleic acids can be detected by bDNA hybridization.
  • Sensitivity can be further enhanced by probing the telomeric-repeat-complementary-bDNA (or telomerase substrate-complementary- bDNA) with a secondary bDNA probe specific for the branches of the primary bDNA probe (and a tertiary probe specific for the secondary probe, etc.), thereby presenting more numerous hybridization sites for the labelled probe.
  • bDNA to probe for extended telomerase substrates is not limited to use with cell extracts but can also be apphed to the in situ methodology of the present invention.
  • Extended telomerase substrates in cells fixed onto a microscope slide can be probed with bDNA instead of conventional linear nucleic acid probes.
  • shorter branch lengths are prefened.
  • the branches are usually less than about 60 nucleotides in length, preferably less than about 40 nucleotides in length. The improved probe uptake overcomes any decrease in sensitivity of the assay that may occur resulting from shorter branch length.
  • a secondary bDNA probe specific for the branches of the primary bDNA probe provides additional sensitivity.
  • the bDNA methodology can also be apphed to the detection of primer extension products, as would be understood by one of ordinary skill in the art upon reading this disclosure.
  • telomerase substrate can be immobilized to a sohd surface, e.g., a well of a microtiter plate, a tube, or beads.
  • the telomerase assay is employed to extend the telomerase substrate with telomeric repeats (e.g., TTAGGG).
  • the reaction can be stopped, if desired, by washing the sohd surface to remove soluble reactants, by the addition of denaturing amounts of detergent or divalent cation chelators, by heating, or by other means.
  • Extended telomerase substrate is then probed with a labelled nucleic acid complementary to the telomeric repeats.
  • a nucleic acid probe DNA, RNA, or peptide nucleic acid (PNA) or other nucleic acid analogues
  • PNA peptide nucleic acid
  • a particularly prefened probe is a polybiotinylated nucleic acid that can be detected by tyramide signal amplification, as described in U.S. Patent No. 5,196,306.
  • HRP Horseradish peroxidase
  • streptavidin binds to the biotin, and the HRP catalyzes the deposition of activated tyramide molecules labelled with fluorophores, biotin, or HRP, for further amplification, and thus the covalent deposition of fluorophores, biotin or HRP to the solid surface.
  • the deposited labels can be detected by standard techniques. Reagents While the disclosure above and Examples below illustrate the invention with results obtained using oligodeoxyribonucleotide telomerase substrates, probes, controls, and primers or ligomers, the present invention is not so limited.
  • nucleotide monomers in a nucleic acid are linked by phosphodiester bonds or analogues thereof.
  • Analogues of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranihdate, phosphoramidate, peptide, and the like linkages.
  • PNA is an ohgonucleotide with peptide bonds instead of phosphodiester bonds in its backbone. Because a PNA has no charge, a PNA has a higher binding affinity than a deoxyribonucleic acid.
  • a phosphorothioate ohgonucleotide having a telomeric or non-telomeric sequence can be prepared by methods known in the art and used as a telomerase substrate during the telomerase reaction. Unlike DNA polymerase, telomerase can recognize and extend phosphorothioate ohgonucleotide substrates.
  • telomere reaction can then be detected by the methods described herein, except that amplification of an extended telomerase product using phosphodiester primers and a polymerase is asymmetrical unless another primer is added.
  • Polymerases cannot utilize phosphothioate oligonucleotides as primers, so primer- dimer artifact formation is decreased in such an assay, although, compared to exponential amplification of the telomerase products, sensitivity of the assay may be compromised unless another primer is added. Sensitivity can be improved in this embodiment by using PCR-independent methods of replication or detection, such as by the use of branched DNA probes, as described above.
  • telomere-extended telomerase substrates or nucleic acids replicated therefrom in a reaction mixture can be labelled to facilitate identification of telomerase-extended telomerase substrates or nucleic acids replicated therefrom in a reaction mixture.
  • the internal control if any, can also be labelled with the same or a different label.
  • Any of a wide variety of labels can be used for pu ⁇ oses of the present invention.
  • Such labels include fluorescent (e.g., fluorescein-5 -isothiocyanate (FITC) and rhodamine), phosphorescent, chemiluminescent, enzymatic, and radioactive labels, as well ws various chromophores.
  • a preferred fluorescent label is an intercalating dye.
  • a particularly preferred fluorescent dye is SYBR Green I that exhibits enhanced fluorescence when bound to double- stranded nucleic acids but only minimal fluorescence with single-stranded DNA or RNA.
  • the label can merely be an unlabelled "tag", which in turn is recognized by a labelled molecule that binds to the tag.
  • a labelled molecule that binds to the tag.
  • biotin as the tag, use avidinylated or streptavidinylated horseradish peroxidase (“HRP”) to bind to the tag, and then use a chromogenic substrate (e.g., tetramethylbenzamie, TMB) to detect the presence of the HRP.
  • HRP horseradish peroxidase
  • the tag can be an epitope or antigen (e.g., digoxigenin), and an enzymatically, a fluorescentiy, or a radioactively labelled antibody can be used to bind to the tag.
  • telomeric repeat binding proteins that are known in the art. Such proteins have been identified as binding either to double-stranded or single-stranded telomeric repeats and native or recombinant proteins can be used. Typically, such proteins would be purified for use and detected by virtue of a label attached to, or an antibody specific for, the particular protein. Detection of the label may involve additional steps, depending on the needs of the practitioner and the particular label or detection means employed.
  • the practitioner may first separate reaction products from one another using gel electrophoresis, as exemplified below.
  • Other separation methods i.e., chromatography, can also be employed, but for some pu ⁇ oses, no separation will be performed, and the detection of extended telomerase substrates and/or primers will be carried out without removing the reaction mixture from the vessel in which the reaction was performed, as in a homogeneous PCR where one measures intercalation of a fluorophore in duplex DNA during amplification.
  • One important advantage of the present invention is the adaptability of the method to any detection format of interest.
  • the Multiplex Electrophoretic Separator is useful in analysis of primer extension products or any other samples with a mixture of target nucleic acid and non-specific dNTPs and/or primers (See Example 11) and is particularly useful for high throughput diagnostic assays.
  • the apparatus comprises a housing (1) containing a receptacle (2) adapted to receive a multiwell plate (3), and two electrodes (4).
  • the electrodes are typically parallel to each other and preferably movably attached to the housing to allow insertion of a multiwell plate onto the receptacle (2) that retains the multiwell plate between the two electrodes, and have the same configuration as the surfaces of the multiwell plate (i.e.
  • the electrode covers the surface of all wells present in the microtiter plate in preferred embodiments).
  • the multiwell plate typicaUy has 24, 48, 96 or more wells per plate, in single (i.e., a strip of microtiter weUs) or multiple rows.
  • Each well (5) is open at its upper surface (6) and has an open but sealable lower surface (7), e.g., a Silent MonitorTM 96 well plate, PaU Co ⁇ . , with removable filter bottom, that can be resealed with adhesive tape.
  • the microtiter plate is placed between two electrodes and connected to a means for applying an electrical current, such as outlets for connection to a power supply.
  • the electrodes can be made of sheets of any electroconducting material, such as metaUic sheets or wire metal grids (Fig. 1).
  • This apparatus can be used to separate a mixture of compounds in multiple samples by preparing an electrophoretic matrix, such as a polyacrylamide or agarose gel, in a sealed well. The seal is removed, the plate is placed in the receptacle (2) above an electrode, and the electrophoretic matrix is immersed in electrophoretic buffer. Altematively, the buffer contacting the negative and positive electrodes can be provided in two chambers, where the only points of contact between the two chambers is through the electrophoretic matrix. The mixture to be separated is prepared for electrophoresis essentiaUy as for conventional electrophoresis and then apphed to the surface of the electrophoretic matrix.
  • an electrophoretic matrix such as a polyacrylamide or agarose gel
  • a second electrode is placed above the multiwell plate, and both electrodes are connected to a power supply, thus allowing separation of the components of the reaction mixture. Electrophoresis is continued until the non- incoiporated dNTPs and primers have eluted from the matrix, after which the gel is optionally rinsed or washed and the products inside the matrix detected by an appropriate means for the label used.
  • a combination of labels can be used in labelling primers so that different products (e.g., primer extension products of extended telomerase substrates and of a control nucleic acid) can be detected in a single well.
  • Streptavidin-coated microtiter well-plates can also be used for detection of amplified or non-amplified telomerase extension products.
  • One iUustrative method involves labelling the 5' end of a telomerase substrate with biotin, capturing the extended telomerase substrates in the streptavidin-coated microtiter weU-plates, and detecting the presence of extended telomerase products by a labelled probe complementary to telomeric repeats.
  • the label can be a radioisotope or a fluorescent, phosphorescent, chemiluminescent, or enzymatic molecule, or any of various chromophores, epitopes or antigens.
  • telomerase substrates are captured by a biotinylated retum primer (e.g., (CTR) 4 , CX, ACT, ACX, LC) and detected by a probe complementary to the extended telomerase substrate
  • CTR biotinylated retum primer
  • the extended products can also be captured by capture probes bound to the plates that are complementary to the telomerase substrate or to telomeric repeats.
  • Extended products can also be captured by inco ⁇ orating biotin-labeUed dNTP (e.g. , biotin-dUTP) during telomerase extension or product amplification and capturing the extended product in the streptavidin plate. The products are then detected by probe complementary to the telomerase substrate or to telomeric repeats.
  • biotin-labeUed dNTP e.g. , biotin-dUTP
  • telomerase assay of the present invention which include research, diagnostic and other applications. Because the assay is fast, simple, and amenable to single reaction vessel reactions, the assay can be used in research and clinical laboratory settings where there is a need to detect telomerase- positive ceUs or samples.
  • Such apphcations include, but are not limited to: (i) detection of immortal ceUs in tumor biopsies for the identification of potential cancer ceUs, before or after therapy; (ii) identification in a ceU-based or ceU-free screen of agents capable of activating, derepressing, inhibiting, or repressing telomerase, including immortalizing agents (e.g., oncogenes) or compounds that activate telomerase and extend telomeres and replicative lifespan of ceUs; (iii) identification in culture systems or in vivo of stem ceUs, early progenitor ceUs, or fetal ceUs that possess telomerase activity; (iv) examination of telomerase regulation during differentiation and development; (v) identification of telomerase- positive fractions generated during purification of telomerase; (vi) identification of protozoal or fungal infections; (vii) diagnosis of diseased states or medical conditions characterized by a different level of telomerase activity in a patient relative to an
  • the diagnostic methods of the present invention can be employed with any cell or tissue type of any origin and can be used to detect an immortal cell of any origin, provided the cell expresses telomerase activity.
  • the detection of immortal cells wiU typically be used to detect the presence of cancer ceUs of any of a wide variety of types, including without limitation, sohd tumors and leukemias including: apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal ceU, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel ceU, mucinous, non-small cell lung, oat cell, papUlary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell carcinomas), histiocytic disorders, leukemia (e.g., B-cell, mixed-cell, null-cell, T-cell
  • telomerase activity is determined from a body fluid (e.g. urine, phlegm, sputum, saliva, blood), a fine needle aspirate or biopsy to diagnose cancer, such as urogenitory cancer, bladder cancer, lung cancer, and leukemia.
  • a body fluid e.g. urine, phlegm, sputum, saliva, blood
  • a fine needle aspirate or biopsy to diagnose cancer, such as urogenitory cancer, bladder cancer, lung cancer, and leukemia.
  • cancer such as urogenitory cancer, bladder cancer, lung cancer, and leukemia.
  • the cell sample is obtained by using a non-invasive method to collect a fluid sample and then isolating cells therefrom, for example, by centrifugation, filtration or other physical means. This simple method of obtaining cells for screening for different cancers is particularly suited to the chnical setting.
  • Telomerase activity can then be assayed, allowing for diagnosis based on the presence of telomerase activity.
  • the assay will be conducted to determine whether an elevated level of telomerase is present.
  • elevated level means that the absolute level of telomerase activity in the particular ceU is elevated compared to normal somatic ceUs in that individual, or compared to normal somatic cells in other individuals not suffering from a disease condition. GeneraUy, any detectable level of telomerase activity is considered elevated in ceUs from normal, post-natal human somatic tissue.
  • telomerase activity is present in germline ceUs, and low levels of telomerase activity can be detected in stem ceUs and certain hematopoietic system ceUs, such cells do not present problems for the practitioner of the present method.
  • Germline ceUs can be readily distinguished and/or separated from human somatic tissue samples, and the telomerase activity present in stem ceUs and certain hematopoietic cells is present at such low levels that the few such ceUs present in somatic tissue samples will not create false positive signals from a telomerase activity assay or can be detected and distinguished using other means.
  • telomerase activity in somatic ceUs is indicative of the presence of immortal ceUs, such as certain types of cancer cells, and can be used to make that determination even when the cells would be classified as non-cancerous by pathology.
  • immortal ceUs such as certain types of cancer cells
  • the method of the present invention allows cancerous conditions to be detected with increased confidence before cells become visibly cancerous.
  • the diagnostic tests of the invention can also be carried out in conjunction with other diagnostic tests. In some instances, such combination tests can provide useful information regarding the progression of a disease, although the present method for testing for telomerase activity provides much useful information in that regard as well.
  • the presence of telomerase activity can be used to determine where a patient is at in the course of progression of the disease, whether a particular tumor is likely to invade adjoining tissue or metastasize to a distant location, and whether cancer is likely to recur or has recurred.
  • Tests that may provide additional information in conjunction with the present method include diagnostic tests for DNA ploidy, fraction of cells in S-phase, nodal status, Her-
  • the level of telomerase activity can also be used to monitor the effectiveness of chemotherapeutics during cancer treatment.
  • the level of telomerase can be a monitor of the effectiveness of a telomerase inhibitor or retinoid therapy, or any other cancer therapy, where telomerase activity is decreased through telomerase inhibition, cellular differentiation, or cell death, respectively.
  • the level of telomerase can monitor the effectiveness of any oncolytic or tumor-debulking procedure by providing an estimate of the number of immortal ceUs within the patient.
  • Telomerase activity can also be determined as a marker for fetal ceUs, e.g. , coUected from cord or maternal blood. Identification of fetal ceUs in maternal blood is useful in diagnosing pregnancy or in the genetic testing of fetal ceUs, i.e. , to identify the ceUs to be tested.
  • Telomerase activity can also be used as a marker for bone marrow proliferation.
  • a bone marrow transplant is typicaUy more successful when less differentiated bone marrow ceUs are employed. Because hematopoietic cells lose telomerase activity as they differentiate, the presence of telomerase activity can be used as a marker for hematopoietic cells useful for transplant purposes. Any of the current assays for telomerase activity, as well assays that may be developed in the future can be used.
  • the present invention also provides kits for performing the diagnostic method of the invention. Such kits can be prepared from readily available materials and reagents and can come in a variety of embodiments.
  • kits can comprise, in an amount sufficient for at least one assay, any one or more of the foUowing materials: ohgonucleotide telomerase substrates (e.g., TS), control reagents (e.g., control ohgonucleotides (e.g., TSNT, TSR8), positive control extracts or cell peUets), and oligonucleotide primers (e.g., CX, ACT, ACX, LTS, CLT, LC, LG, NT), optionally provided together with any of the following: reaction vessels, buffers (e.g., cell lysis buffer, end-labelling buffer, TRAP reaction buffer), water (preferably RNase, DNase and protease- free), nucleotides, labels or stains (e.g., SYBR Green, fluorescer, labeUed ohgonucleotide probes, such as bDNA probes ), enzymes (e.g., oh
  • instructions include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like, to allow the user to carry out any one of the assays described above.
  • the kit comprises a reaction tube in which is placed a telomerase substrate and a primer.
  • a prefened form of this kit comprises such a tube in which the primer is separated from other reaction components by a wax barrier.
  • the primer may be coated on or attached to a glass bead.
  • kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user.
  • Other formats utilize oligonucleotide primers, such as the ACX primer, that aUow wax-free assay conditions.
  • the reagents of any diagnostic assay described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form, either independently or in a mixture of components to improve ease of use.
  • an enzyme or other degradable reagent is provided, conditions are chosen so as to stabilize the reagents, e.g. , storage at lower temperature, addition of stabUizing agents (e.g., glycerol or a reducing agent).
  • Unstable reagents can be provided together with or separately from the more stable components of the kit.
  • a solid support such as a multiwell plate, glass beads, or tubes, and one or more buffers can also be included as separately packaged elements in a kit.
  • kits discussed herein in relation to diagnostic methods or research apphcations are similar in a general sense to those customarily utilized in diagnostic systems and so can include glass and plastic (e.g. , polyethylene, polypropylene and polycarbonate) bottles, vials, plastic and plastic-foil laminated envelopes, and the like.
  • glass and plastic e.g. , polyethylene, polypropylene and polycarbonate
  • Example 1 Preparation of CHAPS-extracted Telomerase
  • cell extracts prepared using the zwitterionic detergent-based extraction method of the invention were tested for telomerase activity using the conventional telomerase assay.
  • ceU extracts were prepared from immortal 293 cells, which are known to express telomerase activity and are derived from human embryonic kidney ceUs transformed with fragments of adenovirus type 5 DNA. The ceUs were grown in
  • Joklik's medium containing 5% to 10% fetal bovine semm and then coUected by centrifugation (unless otherwise noted, the procedure below assumes that about 1 x IO 6 cells were collected), washed once in PBS, pelleted at 10,000Xg for 1 minute at 4°C, and resuspended in 1 ml of ice-cold PBS. The cells were peUeted again and resuspended in ice-cold lysis buffer [10 mM Tris-HCl (pH 7.5), 1 mM
  • MgCl 2 MgCl 2 , 1 mM EGTA, 0.1 mM PMSF (benzamidine or AEBSF can also be used), 5 mM jS-mercaptoethanol, DEPC-treated water, 0.5 % CHAPS (from Pierce), 10% glycerol] at a concentration of 20 ⁇ l of lysis buffer per 10 0 6 cells (depending on the purpose of the experiment).
  • the suspension was incubated on ice for 30 minutes and then spun in a microultracentrifuge at 10,000Xg for 30 minutes at
  • telomere activity was stable to multiple freeze-thaws.
  • the procedure for and conditions of the conventional telomerase assay were as described by Counter ej aj. , 1992; Counter et aj., 1994, EMBO J. 11: 1921- 1929; and Counter et aj., 1994, J. Virol. 68:3410-3414, using ohgonucleotide substrates at a concentration of 1 ⁇ M. See also Morin, 1989, CeU 59:521-529. The products were separated on an 8 % polyacrylamide sequencing gel and exposed ovemight to a PhosphorimagerTM screen (Molecular Dynamics, Sunnyvale, CA).
  • telomerase substrates used in the conventional assay were 5'- GTTAGGGTTAGGGTTAGG-3' (abbreviated as "(GTTAGG) 3 "; SEQ ID NO: 15); 5'-TTAGGGTTAGGGTTAGGG-3' (abbreviated as "(TTAGGG) 3 "; SEQ ID NO: 16), and 5'-AATCCGTCGAGCAGAGTT-3' (abbreviated as "TS”; SEQ ID NO: 3).
  • telomerase activity functioned as predicted for human telomerase.
  • the detergent-extracted activity produces a six nucleotide ladder of extension products characteristic of telomerase activity.
  • a shift in product phase is observed dependent upon the 3 '-sequence of the oligonucleotide telomerase substrate, as is expected for telomerase-mediated extension, and the extracted telomerase can extend a non-telomeric ohgonucleotide previously shown to be a telomerase substrate (Morin, 1991, Nature 353:454-456) with 5'-TTAGGG-3' repeats (as confirmed using dideoxynucleotide chain termination sequencing).
  • the activity was abolished by RNase treatment, as would be expected for telomerase activity (Greider and Blackburn, 1985, Cell
  • Example 2 PCR Amplification of Telomerase Extension Products
  • the reaction components include the telomerase substrate TS (the sequence of which is provided in Example 1, above), which telomerase extends by synthesizing telomeric repeats and which also functions as the upstream primer in the PCR step, and the downstream primer CX, the stmcture of which is defined by its sequence 5'-(CCCTTA) 3 CCCTAA-3' (SEQ ID NO: l).
  • telomerase is known to extend ohgonucleotides of non- telomeric sequence, such as the TS ohgonucleotide (Morin, 1991, Nature 353:454-
  • oligonucleotide substrate TS was used to avoid non-specific amplification due to PCR primer complementarity.
  • mismatches (relative to TS) in the downstream primer CX, single stranded binding protein T4 gene 32 protein, hot start PCR, and an annealing temperature of 50 °C were used to conduct the telomerase activity assays described in this Example.
  • a trace amount of bromophenol blue was added to the CX primer suspension to monitor possible leakage through the wax barrier prior to thermal cycling. While the addition of dye for this purpose is in no way required for practice of the present invention, dye addition can be a convenient method for monitoring the integrity of a manufacturing process. Tubes were then heated at 70°C, and 7-10 ⁇ l of molten wax (AmpliwaxTM, Perkin-Elmer) was pipetted into the bottom of the tube. After the wax was allowed to solidify at room temperature, the tubes were stored at 4°C. Tubes were warmed to room temperature before use.
  • molten wax AmpliwaxTM, Perkin-Elmer
  • the reaction solution contained 20 mM Tris-HCl, pH 8.3, 1.5 mM MgCl 2 , 63 mM KCl, 0.005% Tween 20, 1 mM EGTA, 50 mM each dNTP, 0.1 ⁇ g of TS ohgonucleotide, 0.5 mM T4 gene 32 protein, 0.1 mg/ml BSA, 2 Units of Taq DNA polymerase (optionaUy use 2 Units of Taq treated with an equal volume of TaqStartTM antibody from Clontech to enforce hot start PCR), and 1-2 ⁇ l of a CHAPS ceU extract.
  • the telomerase extension reaction can be conducted at temperatures ranging from about 10°C to about 42 °C, depending upon the source of the telomerase.
  • the telomerase reaction time can vary widely, depending upon the number of primer extension steps employed, the amount of telomerase expected to be in the sample, and the time available to the practitioner. TypicaUy, the telomerase reaction time will be between 5 and 60 minutes, but the time could be up to several hours.
  • the PCR cycles can be composed of cycle times and temperatures that vary widely.
  • the simplest PCR cycle comprises a duplex nucleic acid denaturation step followed by a primer annealing and extension step.
  • the heating method itself can be conducted at a wide range of temperature for any amount of time sufficient to denature but not damage the DNA.
  • the time and temperature of the primer annealing step depends to a great extent on the reaction buffer and primer sequence, concentration, and composition, as weU as the specificity required by the practitioner, while the time and temperature of the primer extension step depends greatly upon the type of DNA polymerase employed.
  • reaction mixture For analysis of the samples, one half of the reaction mixture was analyzed by electrophoresis in 0.5 X TBE on 15 % polyacrylamide non-denaturing gels. Visualization of the products was by ethidium bromide staining, silver staining, SYBRTM Green staining (Molecular Probes) autoradiography, or PhosphorimagerTM analysis (Molecular Dynamics, Sunnyvale, CA) of the gels.
  • Control samples were assayed, using: a sample from which the TS oligonucleotide was omitted; a sample from which the cell extract was omitted; a TRAP assay sample of an immortal 293 cell extract; a sample of 293 extract pretreated by incubation for 10 minutes at 65 °C to heat-inactivate the telomerase; a sample of 293 extract pretreated by incubation for 10 minutes with 0.5 ⁇ g of RNase (DNase-free, Boehringer
  • RNA component of telomerase a sample of phenol-extracted 293 extract (by mixing in an equal volume of a 1: 1 phenol: chloroform mixture, vortexing for 30 seconds, centrifuging to separate the phases, and collecting the aqueous phase); a sample of 293 extract pretreated with protease by incubation of the extract (50 ⁇ l) with 5 ⁇ g of Bromelain protease
  • TRAP Telomerase Repeat Amplification Protocol
  • telomerase detection in 10 2 cells was confirmed by TRAP assays of serial dilutions of an extract from IO 6 293 cells. This limit is a function of the TRAP assay conditions employed and should be considered a practical limit under the given set of conditions rather than an absolute limit of the sensitivity of the current method.
  • Example 15 is an iUustrative example describing the detection of telomerase activity in a single cell. Those of skill in the art wiU recognize that other means of increasing the sensitivity of the assay are available.
  • telomeres (5'-CCCTAA-3'), (SEQ ID NO: 17)] or CTR4 [(5 ; - CCCTAA-3') 4 (SEQ ID NO: 18)] instead of CX further increases sensitivity, although these primers are more likely to interact with the unextended TS primer.
  • the limit of sensitivity was also analyzed by titration of the synthetic telomerase product TS+4 (which contains oligonucleotide TS followed by four telomeric repeats). DUutions of TS+4 oligonucleotide were mixed with heat-treated (telomerase inactivated) 293 extract and analyzed in TRAP assays.
  • telomerase activity from mouse tissue (telomerase activity is present in somatic cells of mice) and cell extracts was detected by TRAP assay even though the mouse telomerase by conventional assay was shown to be mostly non ⁇ processive (i.e. , adds only a single repeat; Prowse et aj. , 1993, Proc. Natl. Acad. Sci. USA 90: 1493-1497), indicating that the TRAP assay is detecting very low levels of processive mouse telomerase activity that cannot be visualized by the conventional assay or mouse telomerase is more processive under TRAP conditions.
  • Reaction tubes were 0.2 ml Strip-easeTM tubes from Robbins Scientific (Sunnyvale, CA) and were autoclaved before use.
  • AU oligodeoxyribonucleotides were Ultrapure grade (HPLC -purified) obtained from Keystone Laboratory (Menlo Park, CA) and were suspended in DEPC-treated H 2 O or TE buffer (10 mM Tris.Cl, pH 7.6; ImM EDTA, pH 8.0) at a concentration of 1 mg/ml.
  • Taq DNA polymerase, Tween 20, and T4 gene 32 protein were purchased from Boehringer Mannheim. Radioisotopes were purchased from NEN-Dupont.
  • the dNTPs were purchased from Pharmacia and were aliquoted, stored at -20°C, and thawed (no more than twice) before use. All other reaction components were molecular biology grade and purchased from Sigma, except when otherwise noted. Diethylpyrocarbonate (DEPC)-treated, de-ionized, sterile H 2 O was used routinely.
  • DEPC Diethylpyrocarbonate
  • telomerase assays Assay of Telomerase Activity in Normal Somatic and Immortal Cells
  • Adherent cell cultures such as BJ ceUs, a normal somatic cell culture of human skin fibroblasts, were grown to 80% confluency prior to extract preparation.
  • the assays (10 5 cell equivalents per reaction) were conducted as described in Examples 1 and 2, above, and the results of the assay are summarized in Table 1 , below. Assays were performed on the same 10 cell extracts, which were prepared using the CHAPS detergent lysis method (see Examples 1 and 2, above).
  • Control samples were assayed with extracts pretreated with RNase, which should eliminate any telomerase activity in the sample.
  • the breast carcinoma line MCF-7/ADR-RES, pancreatic carcinoma line AsPC-1, prostatic carcinoma line PC-3, melanoma line M14, normal foreskin fibroblast cell culture BJ, lung carcinoma line NCI-H23, normal stromal fibroblast cell culture 31YO, normal lung fibroblast ceU culture IMR-90, ovarian carcinoma hne OVCAR-3, colon carcinoma line COLO205, and immortal kidney cell line 293 were assayed.
  • IO 6 cell equivalents were used per reaction.
  • HSF normal synovial fibroblast
  • telomere activity was unexpected.
  • an investigation of telomere length in these lines showed that the telomeres were longer than those of the normal somatic cells from which the lines were derived, which may indicate that the cells experienced a transient burst of telomerase activity. If the telomerase activity is not reinitiated, then the cells will not replicate indefinitely.
  • An important factor in the set-up of the TRAP assay is the environment where the initial reaction mixtures are made prior to the PCR step.
  • the ideal environment is free of contaminating ribonucleases and PCR amplified DNA products, which can cause enoneous negative and positive results, respectively.
  • a major source of PCR product (and RNase) contamination can be the person performing the experiment, who should maintain high standards of personal hygiene and avoid generation of aerosols of PCR products when opening or pipetting PCR products or disposing of gel buffer after the electrophoresis of PCR products.
  • a positive air displacement hood which blows in filtered air over the sample toward the investigator, is ideal. Separate solutions, pipettes, tubes, and tips should always be used and kept inside the hood.
  • Work space should be wiped with 10% bleach prior to set-up of the reaction, and the hood should be routinely UV-irradiated when not in use. Also, barrels of pipettes should be periodicaUy soaked in 10% bleach, even when aerosol-resistant tips are used.
  • the investigator should wear gloves and a disposable lab coat with elastic wrist straps; the lab coat should be periodically changed.
  • a dedicated work area for setting up TRAP reaction can be prepared by placing an acryhc shield of 45.7 cm (L) X 30.5 cm (W) X 61 cm (H) size from VWR (cat. # 56615-848) on a standard cubby-hole type desk. The top of the desk is covered either by a board or heavy cloth, and the front is blocked by the shield. This arrangement creates dead-air space, where the contaminants are prevented from falling into the working area from outside and the samples are physicaUy blocked from the investigator.
  • telomerase ribonucleoprotein
  • Lysis Buffer 0.5 % CHAPS or CHAPSO
  • the micro-extraction procedure involves the following steps:
  • tissue sample is minced with a sterile blade untU a smooth consistency is reached.
  • the sample is transfened to a sterile 1.5 ml centrifuge tube and lysis buffer is added.
  • the sample is homogenized with a motorized pestle on ice (— 10 sec) until a uniform consistency is achieved.
  • Connective tissue is placed in a sterile mortar and frozen by the addition of liquid nitrogen.
  • the sample is then pulverized by grinding with a matching pestle.
  • the thawed sample is then transfened to a sterile 1.5 ml centrifuge tube and resuspended in lysis buffer.
  • a mechanical homogenizer e.g., PowerGenTM Model 35 Homogenizer, Fisher, cat# 15-338-35H
  • AATCCGTCGAGCAGAGTT-3' SEQ ID NO: 3; HPLC purified, 1 ⁇ g/ ⁇ l); ACX primer (5'-GCGCGG[CTTACC] 3 CTAACC-3'; SEQ ID NO:4; HPLC purified, 0.1 ⁇ g/ ⁇ l); NT primer (5'-ATCGCTTCTCGGCCTTTT-3'; SEQ ID NO:6; HPLC purified, 0.1 ⁇ g/ ⁇ l); TSNT internal control (5'- AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3'; SEQ ID NO:5;
  • HPLC purified 0.01 amol/ ⁇ l
  • TSR8 quantitation standard 5'- AATCCGTCGAGCAGAGTTAG[GGTTAG] 7 -3'; SEQ ID NO: 19; HPLC purified, 1 amol/ ⁇ l
  • 2.5 mM dNTPs Pharmacia
  • Taq DNA polymerase Perkin Elmer; AmpliTaqTM
  • 10X TRAP Buffer 10X TRAP Buffer.
  • IPX TRAP Buffer Components For 5 ml
  • the substrate/primer can be end-labelled with [ 32 P] ⁇ ATP using the reaction mixture described below, or with other reagents, such as 5 '-biotin, digoxigenin, fluorescein or another fluorophore, depending on the particular detection and quantitation system to be employed:
  • the end-labelling reagents are combined in a reaction vessel and incubated for 20 minutes at 37°C, foUowed by 5 minutes at 95 °C.
  • the end-labelling conditions can be varied depending on the particular application and needs of the practitioner. For example, increased sensitivity of the assay can be achieved by increasing the specific activity of the labelled primer, as described in Example 15.
  • the foUowing materials are mixed in a PCR reaction tube.
  • End-labelled primer (0.1 ⁇ g/ ⁇ l TS) 1 ⁇ l
  • TSNT internal control (0.01 amol/ ⁇ l) 1 ⁇ l Taq polymerase (AmpliTaqTM, Perkin Elmer) 0.4 ⁇ l (2 Units)
  • Optional components include 0.2 ⁇ l of T4 gene 32 protein (5 mg/ml, available from Boehringer Mannheim), and 0.4 ⁇ l of TaqStartTM antibody (avaUable from Clontech; the polymerase is mixed with the antibody prior to the assay).
  • the telomerase extract can be replaced with one of the following: a negative control RNase-inactivated or heat-inactivated extract
  • the reaction is carried out according to the following steps:
  • TSNT internal control results in a specific PCR amphfication product that appears as a band on a gel 14 bp below the first products of the TRAP assay, regardless of RNase treatment or no extract control.
  • the internal control band can be used to normalize the PCR amplifications from different samples, and to calculate the number of telomerase products generated when used in combination with end-labelled TS ohgonucleotide substrate/primer as described below.
  • CE Analysis i) Measure the signal of the region of the gel lane conesponding to the TRAP product ladder bands from all samples including non-heat-treated (x) and heat- treated sample extracts (xj, CHAPS lysis buffer only control (r 0 ), and TSR8 quantitation control (r); ii) Measure the signal from the TSNT internal standard in non-heat-treated samples (c) and TSR8 quantitation control (C R ). ih) Quantitate the amount of telomerase product using the following formula:
  • TPG (units) x 100 (if 1 ⁇ l (0.1 amol) of TSR8 is used) (r - r 0 )/c R
  • TPG Total Product Generated
  • TS primers in 1 x 10 '3 amole or 600 molecules
  • telomerase activity from approximately 10 to 10,000 control cells in a 10 minute incubation at 30°C.
  • telomere extension products This calculation is valid only if the TS substrate is end-labelled and does not apply to a TRAP protocol in which direct inco ⁇ oration of radioactive dNTPs or non-radioactive quantitation is used for detection, because the signal would depend on the length of the products in that case.
  • the primers should be present in excess over templates for the quantitative analysis to be accurate. Therefore, if a sample has very high levels of telomerase activity, one can dilute the extract so that the PCR primers are not limiting. Altematively, one can add a control nucleic acid of any sequence to the reaction mixture in known amounts and amplify the control with primers different from those used to amplify the extended telomerase substrate.
  • the control oligonucleotide and/or the primers used to amplify the control ohgonucleotide can be labelled identically to or differently from the label used to label the telomerase extension products.
  • telomerase assay method This example iUustrates the telomerase assay method of the present invention when apphed in situ.
  • the method involves intemalization of a telomerase substrate by cells and detection of the extended telomerase substrate if telomerase is present in the ceUs.
  • telomerase substrate There is no limitation as to which telomerase substrate is used other than that imparted by the particular detection method used. In one embodiment, a plasmid telomerase substrate is used.
  • a preferred plasmid telomerase substrate is a selectable, multi-copy vector having a mammahan origin of replication, which vector comprises a non-telomeric telomerase substrate sequence (e.g., TS sequence, 5'-AATCCGTCGAGCAGAGTT-3') (SEQ ID NO:3) adjacent to a restriction site (e.g., Isce I) that is not present in the mammahan genome.
  • a restriction site e.g., Isce I
  • the restriction endonuclease specific for the restriction site is supphed to provide a linearized telomerase substrate.
  • a second plasmid containing the gene coding for the specific restriction enzyme (e.g., Isce I), operably linked to an inducible promoter, is coinfected with the first plasmid into the target ceUs.
  • the plasmid encoding the unique restriction enzyme expresses the restriction enzyme that cleaves the specific restriction site present on the insert of the telomerase substrate plasmid. This results in a linearized telomerase substrate plasmid that can be elongated with TTAGGG repeats by telomerase.
  • Intemalization of DNA substrate Intemalization of the substrates can be achieved using passive intemalization
  • oligonucleotides or DNA added to the cell media at a concentration of 10-100 ⁇ M
  • microporation by a detergent or Staphylococcus alpha toxins BRL, following the manufacturer's conditions
  • Uposomes e.g., LipofectAmineTM, LipofectinTM, LipofectAceTM, from BRL, foUowing the manufacturer's conditions
  • the cells are incubated at 37°C for 1-6 hours.
  • frozen non-fixed tissue is cut into a thin section on a cryostat, placed on a clean sterile microscopic glass slide and incubated at 37°C with media containing DNA telomerase substrate, or DNA telomerase substrate inco ⁇ orated into liposomes, for 1-6 hours.
  • the tissue is gently washed with PBS, and fixed using the methods discussed below. If active telomerase is present, the DNA substrate wiU be extended with de novo synthesis of TTAGGG repeats.
  • the ceUs or sectioned tissues are treated to stop the telomerase reaction and any degradative process and permeabilized to aUow for detection.
  • the samples can be washed or rinsed twice with PBS and fixed by any of various methods known in the art, such as with MeOH: Acetic acid (3:1 ratio, incubated overnight at -20°C), with buffered 10% formalin (4-15 hr at room temperature), with 3 % paraformaldehyde (4-15 hr at room temperature), with 4% formaldehyde (4-15 hr at room temperature), or with a commercially available fixative such as Permeafix (ORTHO, 1-5 hr at room temperature) can be used.
  • Permeafix ORTHO, 1-5 hr at room temperature
  • the cells are then fixed onto a microscopic glass shde by CytospinTM (Shandon) and dried ovemight at room temperature.
  • the fixed samples are then permeabilized by a protease treatment (e.g. , proteinase K, pronase, trypsin, pepsin [2 mg/ml]) for 10-60 minutes at room temperature.
  • the samples can then be washed or rinsed with PBS at room temperature for 10 min, washed briefly with 100% EtOH, and air dried.
  • the extended telomerase substrate if present, can be detected by various means.
  • the extended telomerase substrate if present, can be detected by various means.
  • PCR detection is used.
  • Ulustrative TS and CTR5 primers [5'-CCCTAA-3'] 5 , SEQ ID NO:20) are described below.
  • Other primer pairs and reaction conditions can be utilized as is apparent to one of skiU in the art.
  • reaction mix (10 mM Tris-HCl, 50 mM KCl, pH 8.3; 2.5 mM MgCl 2 ; 200 ⁇ M dNTPs; 1 ⁇ M TS and CTR5 primers; 10 U AmpliTaqTM DNA polymerase) is added to the sample heated to 70°C, sealed with a sUicone gasket and chp (following the manufacturer's protocol, Perkin Elmer), and amplified for 30 cycles of 94°C/40 sec, 55°C/90 seconds.
  • tagged dUTP or primers tagged by fluorescent labels, radioisotope, biotin or digoxigenin, with a ratio of tagged dUTP to dTTP of 94 ⁇ M T-dUTP: 106 ⁇ M dTTP
  • wash buffer 4X SSC; 0.05 % Tween 20
  • samples can be pretreated with dNTPs and a DNA polymerase without the primers, optionally with a DNA hgase.
  • the DNA polymerase preferably Taq DNA polymerase, forms a complementary copy of any single-stranded regions in the cellular DNA and the ligase, preferably a thermostable ligase, eliminates any nicks in the resulting product.
  • the addition of DNA polymerase in the amphfication step is thus not required if the sample is pretreated with a thermostable DNA polymerase.
  • Fluorescent in situ hybridization can be employed to identify telomerase positive cells in a mixed population of cells or tissues.
  • the nucleic acids are denatured by immersing the slide in 70% deionized formamide/2X SSC solution pre-warmed to 70-74°C for 2-3 minutes.
  • the slide is transferred to ice-cold 70% EtOH, and then to 95 % EtOH, and then to 100% EtOH (4 minutes in each solution).
  • Labelled probe e.g., 100-200 ng of a plasmid insert containing about 500 bp of 5'-TTAGGG-3' repeat sequence is used per slide.
  • the probe can be labelled with biotin, digoxigenin, radioisotope, or a fluorescent tag.
  • the probe is dried, resuspended in 10 ⁇ l of 100% deionized formamide, denatured by incubation at 75 °C for 8 minutes, and immediately cooled on ice.
  • 10 ⁇ l of 2X hybridization buffer (4X SSC; 4X Denhardt's solution; 20 % dextran sulfate; 100 mM Tris, pH 7.5
  • the probe/hybridization mix (20 ⁇ l) is added to the fixed sample, overlayed with a coverslip, and the coverslip sealed with mbber cement or nail polish before incubating the sample at 37 °C for 8-48 hours.
  • the coverslip is then removed and the sample is washed twice with 2X SSC/50% deionized formamide at 37°C, and then twice with 2X SSC at 37°C (5 minutes per wash).
  • the sample can be mounted in antifade solution (VectaShieldTM, VectaLab), or developed using a photographic emulsion, and viewed under a microscope.
  • Digoxigenin-labelled probes are detected following the manufacturer's conditions using conjugated anti-digoxigenin antibodies (Boehringer Mannheim). If the probe is labelled with biotin, the slide can be blocked with 2X SSC/1 % BSA for 10 minutes at room temperature, and then incubated in fluorescent conjugated avidin/2X SSC/1 % BSA (final avidin concentration: 5 ⁇ g/ml) at room temperature for 1 hour.
  • the slide can then be washed 5 minutes at room temperature in the foUowing series: 4X SSC, 4X SSC/0.1 % Triton X-100, 4X SSC, PN buffer (0.1 M Na 2 HPO 4 ; 0.1M NaH 2 PO 4 and 0.1 % Nonidet P40).
  • the sample can be mounted in antifade and viewed under a microscope.
  • the slide is blocked in PNM buffer (PN buffer plus 5 % [w/v] non-fat dried milk) at room temperature for 10 minutes, and incubated in a solution of biotinylated anti-avidin antibody (5 ⁇ g/ml) in PNM buffer at room temperature for 20 minutes.
  • the slide is then washed in the four-step wash series described above, blocked again with PNM buffer, and incubated with fluorescentiy conjugated avidin (5 ⁇ g/ml) in PNM buffer at room temperature for 20 minutes.
  • the slide is washed again by the four-step wash series, mounted in antifade and the result is viewed under a microscope.
  • telomere repeats by PRINS involves using an oligonucleotide probe specific for telomere repeats and chain elongation inco ⁇ orating labeUed nucleotides.
  • PRINS mixture (10 ⁇ l) of 5% (v/v) glycerol; 10 mM Tris-HCl, pH 8.3; 100 mM KCl; 0.05 % (w/v) Tween 20; 0.75 mM EGTA; 2.5 mM MgCl 2 ; 0.4 ⁇ M retum primer [e.g., CTR,]; 200 ⁇ M dATP, dGTP, dCTP; 110 ⁇ M dTTP; and 90 ⁇ M labelled dUTP, is placed on the fixed, permeabilized sample, sealed with a coverslip, anchored with nail polish, overlayed with mineral oU, and incubated at 70 °C for 30 minutes to 3 hours. After completion of the PCR, the sample is washed 3 times in wash buffer (4X SSC; 0.05% Tween 20) heated to 70°C for 2 minutes and the signal can be observed as described above.
  • wash buffer (4X SSC; 0.05% Tween 20
  • PCR products smaller than 200 bp from the ceUular matrix can be prevented by inco ⁇ oration of "bulky" dNTPs (e.g., a biotin-labeUed dNTP, a fluorescent-labelled dNTP or a digoxigenin-labeUed dUTP) into the PCR product or by incorporation of a product extension primer into the in situ PCR protocol.
  • "bulky" dNTPs e.g., a biotin-labeUed dNTP, a fluorescent-labelled dNTP or a digoxigenin-labeUed dUTP
  • IUustrative primers for such a protocol are a primer that contains multiple copies (three to four) of a 6 bp repetitive incomplete (or mismatched) telomeric sequence (e.g., [5'-TTTCCC-3'] 3Jl ) (SEQ ID NO:21 and SEQ ID NO: 22) at its 5' end, followed by a sequence that is specific for the target, an appropriate retum primer, and a third primer that contains incomplete repetitive sequences (e.g., [5'-TTTCCC-3'] 4 ) (SEQ ID NO:22).
  • These primers can be used to amphfy the specific target by in situ PCR.
  • the presence of the third primer elongates the PCR product due to its staggered-binding to the 3 '-end of the target PCR product.
  • the elongation of the PCR products can be further induced by decreasing the annealing temperature during the initial PCR condition.
  • the annealing temperature of the first primer to the target sequence is 60 °C
  • the sample is initially amplified for 15-20 cycles of 94° C/45 seconds and 60°C/45 seconds, then amphfied for 15-20 cycles of 94°C/45 seconds and 50° C/45 seconds.
  • the lowered annealing temperature in the second PCR step favors staggered-binding of the third primer to the repetitive sequences and thus the generation of elongated PCR products.
  • the resulting elongated PCR products are less prone to leakage through the cellular matrix, thus resulting in improved signal retention in in situ PCR analysis.
  • Example 6 Preparation of TRAP Reaction Beads This Example iUustrates the use of a wax barrier to separate the PCR retum primer from other reagents to ensure that the retum primer is accessible to the
  • a primer solution e.g., ACT; 5-10 ng/ ⁇ g
  • a trace amount of bromophenol blue is mixed with clean sterile glass beads (e.g. , — 300 micron in diameter, acid-washed, commercially available from Sigma).
  • the bromophenol blue can be added to monitor possible leakage through the wax barrier prior to thermal cycling. While the addition of dye for this pu ⁇ ose is in no way required for practice of the present invention, dye addition can be a convenient method for monitoring the integrity of a manufacturing process.
  • the mix is dried untU the beads are coated with an appropriate amount of dried primers (See Example 2), and the primer-coated beads are then mixed with hot molten wax.
  • the bead/wax mixture is dispensed onto a clean surface by pipetting and then allowed to solidify.
  • One bead droplet is added to a PCR tube and used in a TRAP assay using the conditions described in Example 2.
  • the primer solution e.g., ACT; 5-10 ng/ ⁇ l
  • glass beads ⁇ 1000 micron in diameter
  • An aliquot of hot molten wax ( — 5 ⁇ l) is placed into a plug mold having a covered lower surface and is left to harden.
  • the bead is placed on top of the hardened wax layer and a second aliquot of hot molten wax ( — 5 ⁇ l) is placed into the mold over the bead, and aUowed to harden.
  • the cover of the lower surface is removed and the finished plug is pushed through the mold.
  • One plug per TRAP assay is used instead of the conventional reaction tube, and the TRAP assay is conducted as described above in Example 2.
  • Wax-free/hot-start-free TRAP assay There are various formats for reducing primer-dimer artifacts other than the use of a wax barrier Ulustrated in Example 6, such as choice of reaction conditions.
  • DMSO dimethylsulfoxide
  • glycerol in combination with DMSO compensates for the potential enzyme destabilization caused by DMSO.
  • addition of about 5 % glycerol to samples in TRAP buffer containing about 5 % DMSO can be beneficial where enzyme stabUity is critical.
  • Appropriate primer design can also be employed to reduce primer-dimer formation.
  • An illustrative primer is the ACX retum primer (5'- GCGCGG[CTTACC] 3 CTAACC-3', SEQ ID NO:4) which is a chimeric oligonucleotide that has the anchor sequence of the ACT primer (5'-GCGCGG-3') (SEQ ID NO:2) at its 5' end followed by a CX primer-based sequence that contains mismatches (5'-[CTTACC] 3 CTAACC-3 ⁇ SEQ ID NO:23) in 3 of 4 complementary telomeric repeats (5'-TAACCC-3').
  • ACX retum primer 5'- GCGCGG[CTTACC] 3 CTAACC-3', SEQ ID NO:4
  • SEQ ID NO:4 a chimeric oligonucleotide that has the anchor sequence of the ACT primer (5'-GCGCGG-3') (SEQ ID NO:2) at its 5' end followed by a CX primer-based sequence that contains mismatches (5'-[CTTACC]
  • TRAP assays were performed essentially as described in Example 2 using 293 extracts with either TS and ACX, or TS and ACT, in the presence of about 5 % DMSO, using cold start conditions (i.e., without a wax barrier) and the products were separated on a 15% polyacrylamide gel.
  • Control samples lacked input extract, and one set of control samples included a synthetic telomerase product (TSR8; 5'- AATCCGTCGAGCAGAGTTAG[CGGTTAG] 7 -3' SEQ ID NO: 19).
  • TSR8 synthetic telomerase product
  • Results with no input 293 extract or with synthetic product showed that the TS and ACT combination resulted in primer-dimer artifacts, whereas no primer-dimer artifacts were observed with TS and ACX primers.
  • TS primer To test the robustness of the ACX primer, an attempt was made to induce primer-dimer artifact formation with TS primer by incubating the TRAP assay mixture including TS and ACX on ice without a wax-barrier, and then initiating a PCR amplification in a non-preheated thermocycler block.
  • the combination of TS and ACX primers consistently showed no primer-dimer artifact formations and had no detrimental effect on the efficiency of the TRAP reaction.
  • TS and ACX primers were equally resistant to primer-dimer formation in the absence of DMSO.
  • the utilization of the TS and ACX primers in the TRAP assay can replace the wax-barrier methodology for the TRAP assay, thus making the analysis, manufacturing, and the performance of the TRAP assay components more reproducible, simple, and reliable.
  • such primers can be used in various embodiments, including the wax- barrier methodology.
  • Example 8 TRAP product detection by TaqManTM detection system The method involves detecting extended telomerase substrates using the non- radioactive TaqManTM detection system (Perkin Elmer) modified for use in the
  • a TaqManTM probe that possesses both a fluorescent reporter dye tag and a quencher dye tag is inco ⁇ orated into the primer extension reaction (e.g., PCR amplification).
  • primer extension reaction e.g., PCR amplification
  • An illustrative probe for use in this detection system comprises repetitive CTR (5'-CCCTAA-3') sequences.
  • the CTR probe and retum primer e.g., ACT
  • ACT retum primer
  • a second iUustrative probe that comprises a sequence complementary to the telomerase substrate does not compete with either the TS or ACT primers, and thus does not result in primer-dimer formation.
  • the artisan may be concemed that the formation of a duplex between such a probe with the TS primer can potentially decrease PCR efficiency, telomerase was demonstrated to recognize and extend such double-stranded substrates in an assay using PCR detection as described below.
  • GTSI-1 A duplex DNA (GTSI-1) was constmcted by hybridizing an equal amount of M2A telomerase substrate primer (modified TS: 5'- GCCCAATCCGTCGAGCAGAGTTAG-3'; SEQ ID NO: 25) with its complementary sequence CM2A (5'-CTAACTCTGCTCGACGGATTGGGC-3'; SEQ ID NO: 26).
  • GTSI-1 was used in a TRAP assay with the return primer, HKC
  • the TRAP product was observed with this primer combination and was demonstrated to be RNase sensitive thus demonstrating the utUity of probes that are complementary to the telomerase substrate in detecting telomerase activity.
  • CM2A can be used as a TaqManTM probe.
  • a probe can be used which contains a sequence complementary to the 3' region of the TS primer, foUowed by a CTR sequence (e.g., 5'-AACCCTAACCCTAACTCTGCT-3'; SEQ ID NO:7) with a reporter dye at is 5' end and a quencher dye internal to the 5' end, preferably about 7 nucleotides internal to the 5' end.
  • This primer specificaUy hybridizes to the junction between the TS primer and the telomeric repeat sequence thereby reducing any competitive effect that may occur with the ACT-probe and TS-probe duplex formation.
  • a probe containing the TS sequence and at least one TTAGGG repeat can also be used in the TaqManTM detection system, preferably using the hot start methodology of Example 2.
  • the Multiplex Electrophoretic Separator is an apparatus that allows for analysis of multiple samples simultaneously.
  • the apparatus comprises two electroconducting sheets (e.g., copper) with the same configuration as a multiweU plate.
  • the two sheets act as electrodes (Fig. 1).
  • a 15 % non-denaturing polyacrylamide gel was prepared in a bottomless multiwell plate temporarily sealed to allow polymerization of the gel. After removal of the seal, the multiplex gel unit was placed equidistant from the two electrodes, and the complete apparatus was submerged in electrophoresis buffer (e.g., 0.5X TBE; 0.045 M Tris-borate, 0.001 M EDTA).
  • electrophoresis buffer e.g., 0.5X TBE; 0.045 M Tris-borate, 0.001 M EDTA.
  • radioactively labeUed TS primer was used as a substrate in a TRAP assay using 10-fold serial dilutions of 293 cell extracts (from 10 5 - 10 1 cell equivalents) essentially as described in
  • Example 2 As controls, 5-fold serial dUutions of the synthetic product TSR8 from 10 fmol - 16 amol (see Example 7) were also included. Each assay /dhution was prepared in triplicate.
  • each sample was loaded onto the top of a weU of the MES and an electric field was apphed across the gel.
  • the gel was washed with water and the product inside the gel detected by means of a PHOSPHORIMAGERTM apparatus (Molecular Dynamics).
  • PHOSPHORIMAGERTM apparatus Molecular Dynamics
  • a commercially available microtiter plate was used. As the plate material had not been optimized for the assay, scattering of the radioactivity by the plastic walls of the MES gel unit caused a haziness in the signal.
  • this apparatus can be used to distinguish negative samples (RNase controls and no extract) from positive samples, and telomerase activity could be detected in samples dUuted 10 000-fold with linear quantitation over an extensive range, up to 1 000-fold under the selected reaction conditions.
  • negative samples RNase controls and no extract
  • telomerase activity could be detected in samples dUuted 10 000-fold with linear quantitation over an extensive range, up to 1 000-fold under the selected reaction conditions.
  • various materials can be used in the MES, and assay conditions can be varied in accordance with this specification.
  • Example 10 Detection of Telomerase Activity with TRAP-SYBR Green
  • the TRAP assay is carried out on telomerase samples essentially as described above in Example 4, typically in a total volume of 50 ⁇ l, but using unlabeUed primer.
  • PCR parameters for the TRAP-SYBR Green assay are 22-35 cycles of a two step program, i.e., 94°C/30 seconds and 60°C/30 seconds. After electrophoresis, the gel is stained with SYBR Green to visualize products.
  • the reaction mixture is treated with 25 ⁇ l of 0.3M acetate buffer, pH 4.5 containing 500ng RNase and 10 units of Sl nuclease and incubated at 37°C for 15 minutes to digest excess primers and RNA in the telomerase extract.
  • electrophoresis aliquots (50 ⁇ l) of a 1:2000 dUution of SYBR Green dye (Molecular Probes) can be added to wells of a microtiter plate, and 50 ⁇ l aliquots of the digested TRAP assay are mixed with the dye for measurement of telomerase activity.
  • the double-stranded DNA/dye complex is detected with a fluorometric plate reader using an excitation wavelength of 497 nm and an emission wavelength of 520 nm.
  • a fluorometric plate reader using an excitation wavelength of 497 nm and an emission wavelength of 520 nm.
  • Example 11 Single-cell Telomerase Repeat Amphfication Protocol (STRAP) To illustrate the sensitivity of the TRAP assay, this Example describes a method for detecting telomerase activity in a single ceU.
  • Single cells can be obtained by various means, such as by serial dilution or using a fluorescence activated cell sorter (FACS) sorting either whole cell lines or specifically marked populations of cells. The latter method is illustrated in the present Example.
  • FACS fluorescence activated cell sorter
  • the cells are marked with fluorescentiy labelled or labelable antibodies, which antibodies recognize a specific marker.
  • the marker can differentiate ceUs of different life spans, function or are differentially expressed.
  • Cells were sorted into 25 ⁇ l of preparatory buffer containing 20 mM Tris ⁇ HCl (pH 8.3), 1.5 mM MgCl 2 , 63 mM KCl, 1 mM EGTA, 1 mg/mL BSA, 0.5 % Tween 20, 50 mM dNTPs, 100 ng of TS oligonucleotide, and DEPC-treated water.
  • the buffer was placed in 8 -tube microtiter format PCR strips.
  • the concentration of Tween 20 was sufficient to lyse the cell membrane.
  • the reaction time used in this Example although not limiting, was 60 minutes at 30°C.
  • the incubation was performed in the block of a microtiter format Perkin Elmer 9600 thermal cycler. One can also use a heat block or water bath.
  • amphfication buffer contained the same ingredients noted above except the TS ohgonucleotide was replaced by 100 ng of gamma end- labelled retum primer, ACX.
  • the ACX ohgonucleotide was labeUed in a reaction containing T4 polynucleotide kinase (NEB), 10X PNK buffer (NEB), and a ratio of 100 ⁇ Ci of 3000 Ci/mmol 7- 32 P ATP per 1 ⁇ g of ACX.
  • T4 kinase was used at a ratio of 40 U per 1 ⁇ g of ACX and incubated at 37 °C for 30 minutes, foUowed by a heat treatment of 65 °C for 15 minutes to inactivate the kinase.
  • Taq DNA polymerase (2U; Boehringer Mannheim) was added to the amplification buffer and amphfication was performed in a Perkin Elmer 9600 thermal cycler for 32 cycles of 94°C, 30 seconds and 60°C, 30 seconds. Products were analyzed by electrophoresis in 0.6X TBE on 15 % polyacrylamide non-denaturing gels.
  • Example 12 Method of detecting telomerase activity from body fluids
  • This Example provides a non-invasive, simple method, particularly useful in a chnical setting, for coUecting ceUs from body fluids for use in telomerase activity assays.
  • the method is described for coUecting cells from the urogenitary tract; however, it should be noted that the method is not hmited to use with urine, but can be apphed to other body fluids, such as, saliva, phlegm, sputum, blood, fine needle aspirates of tumors (e.g., breast tumors, prostate tumors, or other tumor types where fine needle aspirates are used).
  • body fluids such as, saliva, phlegm, sputum, blood, fine needle aspirates of tumors (e.g., breast tumors, prostate tumors, or other tumor types where fine needle aspirates are used).
  • Method 1 1. Collect voided urine (30-40 ml) in a 50 ml centrifuge tube. 2. Centrifuge at 1000 g for 15 minutes.
  • Extracts (2-5 ⁇ l) are used in a standard TRAP assay with 30-35 PCR cycles as described in Example 2. Sensitivity can be increased by addition of one or more radioactively-labelled dNTPs, in addition to the end-labeUed primers. The presence of telomerase activity is correlated with the presence of cancer ceUs in the urine sample and a diagnostic for urogenitary cancer.
  • the method can be used for any body fluid or fine needle aspirate, thus allowing the presence of telomerase activity to be correlated with the presence of cancer cells in the fluid or aspirate and a diagnostic for the particular cancer, e.g., lung cancer when sputum or phlegm is the tested fluid, and breast cancer when a fine needle aspirate of breast or other (e.g., prostate) tissue is tested.
  • a diagnostic for the particular cancer e.g., lung cancer when sputum or phlegm is the tested fluid
  • breast cancer when a fine needle aspirate of breast or other tissue is tested.
  • Example 13 TRAP Assay Reagents and Kit Formats
  • a variety of reagents and kit formats are provided for the practitioner's convenience in carrying out the TRAP assay.
  • a CHAPS lysis buffer is described.
  • a TRAP intemal control is described.
  • various kit formats are described. It will be apparent that variation in the reagents and kits are possible depending on their intended use.
  • a prefened CHAPS lysis buffer has the composition: 10 mM Tris-Cl, pH 7.5; 1 mM MgCl 2 ; 1 mM EGTA; 0.1 mM benzamidine; 5 mM ⁇ - mercaptoethanol; 0.5 % CHAPS; 10% glycerol.
  • Addition of KCl to a concentration of 1 mM, and an increase in the concentration of CHAPS detergent to 3%, in the CHAPS lysis buffer increases the efficiency of the telomerase extraction 2-5 fold.
  • a modified CHAPS lysis buffer for use in the TRAP assay contains 10 mM Tris-Cl, pH 7.5; 1 mM MgCl 2 ; 1 mM EGTA; 0.1 mM 4- (2-aminoethyl)-benzenesulfonylfluoride (AEBSF) or benzamidine; 5 mM ⁇ - mercaptoethanol; 0.5-3% CHAPS; and 1 mM KCl.
  • AEBSF (2-aminoethyl)-benzenesulfonylfluoride
  • the intemal control TSNT having the sequence 5'- AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3' (SEQ ID NO:5), can be amphfied by the TS telomerase substrate primer and a retum primer, NT, having the sequence 5'-ATCGCTTCTCGGCCTTTT-3' (SEQ ID NO: 6).
  • the NT is not a substrate for telomerase.
  • This control can be incorporated into a TRAP assay kit, optionally included in the 10X TRAP buffer (at a concentration of —0.002 amol/ ⁇ l) or in the primer mixture (0.01 amol/ ⁇ l), and results in a TRAP product of 36 bp.
  • kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user. Illustrative kits for performing the TRAP assay are provided below. Such kits can be prepared from readily available materials and reagents and can be easily varied as is apparent to one of ordinary skill in the art.
  • a kit comprises a telomerase substrate with or without instmctions.
  • a kit comprises a telomerase substrate and a retum primer.
  • a kit comprises a telomerase substrate, a retum primer and one or more buffers.
  • the buffer can be, for example, a ceU lysis buffer, end-labelling buffer or TRAP reaction buffer.
  • the TRAP reaction buffer can optionally contain a control reagent, e.g., control ohgonucleotides such as TSNT or TSR8, although such control reagents can be provided separately within the kit.
  • the kit can further comprise positive cell extracts or cell peUets, negative cell extracts or peUets, reaction vessels, water, nucleotides, labels, or enzymes.
  • the positive cell pellet can be 293 cells, HeLa cells or any other telomerase positive cell pellet or a panel of multiple cell types with varying amounts of telomerase activity.
  • the kits can be provided with or without instmctions.
  • instmctions describe how to use the reagents in the assay method described in Example 4.
  • a prefened kit comprises the following reagents: :
  • CHAPS lysis buffer (10 mM Tris-Cl, pH 7.5; 1 mM MgCl 2 ; 1 mM EGTA; 0.1 mM Benzamidine; 5 mM jS-mercaptoethanol; 0.5 % CHAPS; 10% glycerol)
  • a particularly prefened kit format further comprises a TRAP primer mix (e.g., ACX 0.1 ⁇ g/ ⁇ l; NT 0.1 ⁇ g/ ⁇ l; TSNT 0.01 amol/ ⁇ l) optionally with a quantitation standard (e.g., TSR8; 0.1 amol/ ⁇ l).
  • a TRAP primer mix e.g., ACX 0.1 ⁇ g/ ⁇ l; NT 0.1 ⁇ g/ ⁇ l; TSNT 0.01 amol/ ⁇ l
  • a quantitation standard e.g., TSR8; 0.1 amol/ ⁇ l
  • the kit comprises CHAPS lysis buffer, 10 x TRAP reaction buffer containing TSNT (0.002 amol/ ⁇ l), 50X dNTP mix, TS primer (1 ⁇ g/ ⁇ l), water (PCR grade; protease, DNase and RNase-free), a positive control cell peUet (IO 6 cells) or a panel of multiple cell types with varying amount of telomerase activity, TRAP reaction tubes (0.1 ⁇ g ACT sealed with wax), Taq DNA polymerase (5U/ ⁇ l), optionally provided with 10X End-labelling buffer (100 mM Tris-OAc; 100 mM MgOAc; 500 mM KOAc), polynucleotide kinase (lU/ ⁇ l) and gel loading dye.
  • 10 TRAP reaction buffer containing TSNT (0.002 amol/ ⁇ l), 50X dNTP mix, TS primer (1 ⁇ g/ ⁇ l), water (PCR grade; protease, DNase and RNase-free),
  • Example 14 Telomerase Activity Detection by Branched DNA (bDNA) Probes This Example illustrates the use of bDNA probes for detecting telomerase activity in a microtiter plate format.
  • the use of bDNA probes in the methods of the invention are not limited to such a format, as the bDNA probes can be used in various ways, for example, in in situ detection of telomerase extension products.
  • reagents for bDNA are avaUable from Chiron Co ⁇ .
  • nucleic acids can be linked to a solid surface using conventional attachment chemistry techniques, for example, via amide or ester linkages.
  • multi-well plates are prepared with the telomerASE substrate oligonucleotide, TS, (5'-AATCCGTCGAGCAGAGTT-3'; SEQ ID NO:3) bound to the plate at its 5' end.
  • Cell extracts are prepared as described in Example 1.
  • Telomerase buffer 100 ⁇ l; 20 mM Tris-Cl pH 8.3, 1.5 mM MgCl 2 , 63 mM KCl,
  • telomere cell extract 0.05 % Tween 20, ImM EGTA, 0.1 mg/ml BSA, 50 ⁇ M dNTPs) and 5 ⁇ l of CHAPS telomerase cell extract are added to the TS -bound microtiter plates and incubated for 30 minutes to 2 hours at 30°C. The reaction mix is then removed from the well and the well washed twice with 200 ⁇ l of IX SSC (8.76 g/1 NaCl, 4.41 g/1 Na citrate, pH7) at room temperature. Fifty microliters of IX SSC containing — 15 pmol of bDNA probe specific for the telomeric repeats (i.e.
  • telomerase products are then detected by an appropriate means, for example, by employing a fluorescent plate reader when a fluorescent label is used.
  • a further Ulustrative format involves the immobilization of the telomerase substrate to a microtiter plate using complementary nucleic acids.
  • Telomerase buffer 100 ⁇ l; 20 mM Tris-Cl pH 8.3, 1.5 mM MgCl 2 , 63 mM KCl, 0.05% Tween 20, ImM EGTA, 0.1 mg/ml BSA, 50 ⁇ M dNTPs;) containing 15 pmol of TS primer is added to a sterile standard microtiter plate together with 5 ⁇ l of
  • CHAPS telomerase cell extract (the amount of extract can be as high as 50% of the total reaction volume).
  • the plate is incubated for 30 minutes to 2 hours at 30°C, after which the solution is transferred to a second microtiter plate having complementary TS primers bound by their 5' ends (e.g. , one or more repeats of 5'-AACTCTGCTCGACGGATT; SEQ ID NO:24).
  • the second plate is then incubated at — 55 °C for 30 minutes with gentle shaking before removing the solution from the well and washing the well twice with 200 ⁇ l of IX SSC at 37°C.
  • Fifty microliters of IX SSC containing - 15 pmol of bDNA probe specific for the telomeric repeats is then added to the well.
  • the plate is incubated at — 55 °C for 30 minutes with gentle shaking after which the solution is removed from the weU.
  • the well is then washed twice with 200 ⁇ l of 0.1X SSC at 37°C.
  • Fifty microliters of IX SSC containing —50 pmol of rhodamine-labeUed secondary probe specific for the branched arms of the bDNA is then added to the weU. After incubating the plate at — 55 °C for 30 minutes with gentle shaking, the solution is removed and the well is then washed twice with 0.1X SSC at 37°C.
  • the fluorescence can be conveniently detected using a fluorescent plate reader after addition of 200 ⁇ l of 0.1X SSC although other methods exist, such as direct visualization.
  • telomerase reaction described above in a complementary TS-bound microtiter plate, thus eliminating the transfer step, immobilizing after the telomerase extension reaction with nucleic acids complementary to telomerase repeats or immobilizing prior to the telomerase reaction using nucleic acids complementary to the telomerase substrate.
  • labels can be used other than a fluorescent label and the signal from such labels can be increased by probing the bDNA specific for the telomerase extension product with a second bDNA probe specific for the first bDNA branches, in which case the second bDNA probe is labelled (directly or indirectly with a further probe).
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:3:
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE synthetic
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

On peut mesurer l'activité de la télomérase dans un échantillon grâce à un protocole à deux réactions. La première réaction concerne la formation de produits de développement d'un substrat de télomérase à partir d'un substrat de télomérase. La deuxième réaction concerne la réplication desdits produits de développement d'un substrat de télomérase et/ou l'amplification d'un signal produit par une sonde liée. La présence d'une activité de télomérase dans un échantillon de tissu somatique humain ou de cellules humaines est liée de manière certaine à la présence d'un cancer et elle peut être utilisée pour diagnostiquer une maladie ou d'autres états intéressants pour la médecine ainsi que l'évolution de la maladie ou de la rémission chez un patient.
PCT/US1996/009669 1995-06-07 1996-06-07 Dosage de l'activite de la telomerase WO1997015687A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP9512229A JPH11507839A (ja) 1995-06-07 1996-06-07 テロメラーゼ活性検定
AU63808/96A AU6380896A (en) 1995-06-07 1996-06-10 Telomerase activity assays

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US08/482,132 US5837453A (en) 1992-05-13 1995-06-07 Telomerase activity assays
US08/482,132 1995-06-07
US08/631,554 1996-04-12
US08/631,554 US5863726A (en) 1993-11-12 1996-04-12 Telomerase activity assays
US08/632,662 US5804380A (en) 1993-11-12 1996-04-15 Telomerase activity assays
US08/632,662 1996-04-15

Publications (1)

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WO1997015687A1 true WO1997015687A1 (fr) 1997-05-01

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JP (1) JPH11507839A (fr)
AU (1) AU6380896A (fr)
WO (1) WO1997015687A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0811693A2 (fr) * 1996-06-03 1997-12-10 Kyowa Hakko Kogyo Co., Ltd. Procédé de détermination de l'activité intracellulaire de la télomérase
EP0882802A1 (fr) * 1996-02-02 1998-12-09 Chugai Seiyaku Kabushiki Kaisha Procede d'evaluation de l'activite de la telomerase
WO1999009212A1 (fr) * 1997-08-18 1999-02-25 Queen Mary & Westfield College Test de telomerase
WO2000046601A1 (fr) * 1999-02-02 2000-08-10 Frank Larsen Detection d'activite telomerase
EP1333094A2 (fr) 1996-10-01 2003-08-06 Geron Corporation Sous-unité catalytique de la télomérase humaine
US6664046B1 (en) 1999-12-16 2003-12-16 Roche Molecular Systems, Inc. Quantitation of hTERT mRNA expression
WO2006119569A1 (fr) * 2005-05-11 2006-11-16 Genetic Technologies Limtied Procedes d’enrichissement de cellules fœtales
US20120070842A1 (en) * 2009-06-01 2012-03-22 Harley Calvin B Assay for Telomerase Activity Using Microfluidic Device
US8374838B2 (en) 2007-10-22 2013-02-12 The Wistar Institute Method for identifying a compound that modulates telomerase activity
US9447467B2 (en) 2009-04-21 2016-09-20 Genetic Technologies Limited Methods for obtaining fetal genetic material

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008182920A (ja) * 2007-01-29 2008-08-14 Sony Corp 蛍光標識オリゴヌクレオチドと該オリゴヌクレオチドを利用する方法

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0882802A4 (fr) * 1996-02-02 2001-01-03 Chugai Pharmaceutical Co Ltd Procede d'evaluation de l'activite de la telomerase
EP0882802A1 (fr) * 1996-02-02 1998-12-09 Chugai Seiyaku Kabushiki Kaisha Procede d'evaluation de l'activite de la telomerase
EP0811693A2 (fr) * 1996-06-03 1997-12-10 Kyowa Hakko Kogyo Co., Ltd. Procédé de détermination de l'activité intracellulaire de la télomérase
EP0811693A3 (fr) * 1996-06-03 2001-03-21 Kyowa Hakko Kogyo Co., Ltd. Procédé de détermination de l'activité intracellulaire de la télomérase
EP1783139A2 (fr) 1996-10-01 2007-05-09 Geron Corporation Sous-unité catalytique de télomérase humaine
EP1333094A2 (fr) 1996-10-01 2003-08-06 Geron Corporation Sous-unité catalytique de la télomérase humaine
EP2213740A1 (fr) 1996-10-01 2010-08-04 The Regents of the University of Colorado Sous-unité catalytique de la télomérase humaine
WO1999009212A1 (fr) * 1997-08-18 1999-02-25 Queen Mary & Westfield College Test de telomerase
WO2000046601A1 (fr) * 1999-02-02 2000-08-10 Frank Larsen Detection d'activite telomerase
US6664046B1 (en) 1999-12-16 2003-12-16 Roche Molecular Systems, Inc. Quantitation of hTERT mRNA expression
WO2006119569A1 (fr) * 2005-05-11 2006-11-16 Genetic Technologies Limtied Procedes d’enrichissement de cellules fœtales
US8374838B2 (en) 2007-10-22 2013-02-12 The Wistar Institute Method for identifying a compound that modulates telomerase activity
US9447467B2 (en) 2009-04-21 2016-09-20 Genetic Technologies Limited Methods for obtaining fetal genetic material
US20120070842A1 (en) * 2009-06-01 2012-03-22 Harley Calvin B Assay for Telomerase Activity Using Microfluidic Device

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Publication number Publication date
AU6380896A (en) 1997-05-15
JPH11507839A (ja) 1999-07-13

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