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General description
Telomeres are specific structures found at the end of chromosomes in eukaryotes. In human chromosomes, the telomeres consist of thousands of copies of 6 base repeats (TTAGGG)(1-3). It has been suggested that telomeres protect chromosome ends since damaged chromosomes lacking telomeres undergo fusion, rearrangement and translocation (2). In somatic cells, telomere length is progressively shortened with each cell division both in vivo and in vitro (4-7) due to the inability of the DNA polymerase complex to synthesize the very 5′ end of the lagging strand (8,9).
Telomerase is a ribonucleoprotein that synthesizes and directs the telomeric repeats onto the 3′ end of existing telomeres using its RNA component as a template (10-14). Telomerase activity has been shown to be specifically expressed in immortal cells, cancer and germ cells (15,16) where it compensates for telomere shortening during DNA replication and thus stabilizes telomere length (7,17). These observations have led to a hypothesis that telomere length may function as a "mitotic clock" to sense the number of cell divisions and eventually signal replicative senescence or programmed cell death when a critical telomere length is achieved. Therefore, expression of telomerase activity in cancer cells may be a necessary and essential step for tumor development and progression (16,18-20). The causal relationship between expression of telomerase and telomere length stabilization and the extension of the life span of the human cell has recently been reported (21).
Telomerase is a ribonucleoprotein that synthesizes and directs the telomeric repeats onto the 3′ end of existing telomeres using its RNA component as a template (10-14). Telomerase activity has been shown to be specifically expressed in immortal cells, cancer and germ cells (15,16) where it compensates for telomere shortening during DNA replication and thus stabilizes telomere length (7,17). These observations have led to a hypothesis that telomere length may function as a "mitotic clock" to sense the number of cell divisions and eventually signal replicative senescence or programmed cell death when a critical telomere length is achieved. Therefore, expression of telomerase activity in cancer cells may be a necessary and essential step for tumor development and progression (16,18-20). The causal relationship between expression of telomerase and telomere length stabilization and the extension of the life span of the human cell has recently been reported (21).
The TRAPeze XL Telomerase Detection Kit is a homogenous fluorescence assay for rapid and sensitive telomerase activity measurement in cell and tissue extracts. The TRAPeze XL Kit incorporates the use of the novel Amplifluor fluorescence energy transfer-labeled primers into the original TRAPeze Telomerase Detection Kit reaction so that quantitative measurements are obtained without radioactivity. Additionally, the unique design of Amplifluor primers enables homogeneous signal amplification and quantitation directly in the unopened PCR vessel. The closed-tube assay format eliminates the risk of contaminating unprocessed samples with PCR amplicon. The TRAPeze XL Telomerase Detection Kit procedure does not require post-PCR manipulations such as gels or enzyme-linked immunosorbant assays (ELISAs).
Recommended Taq polymerases: must be non-proofreading, having no exonuclease activity and capable of "hot-start." Titanium Taq, Platinum Taq are suggested.
Recommended Taq polymerases: must be non-proofreading, having no exonuclease activity and capable of "hot-start." Titanium Taq, Platinum Taq are suggested.
The development of a sensitive and efficient PCR-based telomerase activity detection method, TRAP (Telomeric Repeat Amplification Protocol)(15, 22), has made possible large scale surveys of telomerase activity in human cells and tissues (15, 23-29). To date, telomerase activity has been detected in over 85% of all tumors tested spanning more than 20 different types of cancers (30-31).
The TM=["TRAPEZE"] XL Kit is a highly sensitive in vitro assay for the fluorometric detection of telomerase activity. It incorporates refinements to the original TRAP assay that were first introduced in the gel-based TM=["TRAPEZE"] Telomerase Detection Kit (Cat #S7700). As in the original TM=["TRAPEZE"] Kit, primer sequence modifications that reduce amplification artifacts and an internal PCR control are included. In addition, the TM=["TRAPEZE"] XL Kit uses fluorescence energy transfer (ET) primers to generate fluorescently labeled TRAP products which permit nonisotopic, quantitative analysis of telomerase activity.
The unique design of these ET primers (TM=["Amplifluor"] primers) allows detection and quantification of telomerase activity by directly measuring fluorescence emission in the reaction vessels. Since TM=["Amplifluor"] primers will fluoresce only upon incorporation into the TRAP products or the internal control, post-PCR sample manipulations such as electrophoretic gel or ELISA analyses are eliminated, thereby reducing the the risk of carry-over contamination. Quantitative analysis is not compromised when detection is performed in a high-throughput 96-well format unlike platforms utilizing a qualitative ELISA. In addition, inclusion of an internal control labeled with a second fluorophore serves to both monitor PCR amplification and aid in the quantitation of telomerase activity.
The TM=["TRAPEZE"] XL Kit is a highly sensitive in vitro assay for the fluorometric detection of telomerase activity. It incorporates refinements to the original TRAP assay that were first introduced in the gel-based TM=["TRAPEZE"] Telomerase Detection Kit (Cat #S7700). As in the original TM=["TRAPEZE"] Kit, primer sequence modifications that reduce amplification artifacts and an internal PCR control are included. In addition, the TM=["TRAPEZE"] XL Kit uses fluorescence energy transfer (ET) primers to generate fluorescently labeled TRAP products which permit nonisotopic, quantitative analysis of telomerase activity.
The unique design of these ET primers (TM=["Amplifluor"] primers) allows detection and quantification of telomerase activity by directly measuring fluorescence emission in the reaction vessels. Since TM=["Amplifluor"] primers will fluoresce only upon incorporation into the TRAP products or the internal control, post-PCR sample manipulations such as electrophoretic gel or ELISA analyses are eliminated, thereby reducing the the risk of carry-over contamination. Quantitative analysis is not compromised when detection is performed in a high-throughput 96-well format unlike platforms utilizing a qualitative ELISA. In addition, inclusion of an internal control labeled with a second fluorophore serves to both monitor PCR amplification and aid in the quantitation of telomerase activity.
Components
CHAPS Lysis Buffer (13.5 mL) 5X TRAPEZE™ XL Reaction Mix (1.12 mL) TS primer RP Amplifluor® primer K2 Amplifluor® primer TSK2 template dA, dC, dG and dTTP diluted in : 100 mM Tris-HCl, pH 8.3 7.5 mM MgCl2 315 mM KCl 0.25% TWEEN® 20 5 mM EGTA 0.5 mg/mL BSA PCR - Grade Water (8.2 mL) protease, DNase, and RNase-free; deionized TSR8* (control template) (45 µL) 0.2 amole/µL TSR8 template Control Cell Pellet Telomerase positive cells (106 cells) * Caution - refer to Sec. II. Kit Components, Precautions.
Storage and Stability
Precautions 1. Because the TRAPEZE™ XL Telomerase Detection Kit detects the activity of telomerase, a RNase sensitive ribonucleoprotein, and not merely the presence of the RNA or protein components of telomerase, the assay requires enzymatically active cell or tissue samples. Furthermore, due to the sensitivity of the TRAPEZE™ XL Kit assay, which can detect telomerase activity in a very small number of cells, a special laboratory setup and significant precautions are required to prevent PCR carry-over contamination and RNase contamination. These precautions are discussed in detail in Sec. V. Appendix, Laboratory Setup and Precautions and TRAPEZE™ XL Telomerase Detection Kit Station Setup (Area 1). 2. For Research Use Only. Not for use in diagnostic procedures.
Legal Information
Amplifluor is a registered trademark of Merck KGaA, Darmstadt, Germany
TRAPEZE is a trademark of Merck KGaA, Darmstadt, Germany
TWEEN is a registered trademark of Croda International PLC
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
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Warning
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Hazard Classifications
Met. Corr. 1
Storage Class
8A - Combustible corrosive hazardous materials
wgk_germany
WGK 3
flash_point_f
Not applicable
flash_point_c
Not applicable
Certificates of Analysis (COA)
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