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Methods in Cell Science

, Volume 17, Issue 1, pp 1–15 | Cite as

Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP)

  • Mieczyslaw A. Piatyszek
  • Nam W. Kim
  • Scott L. Weinrich
  • Keiko Hiyama
  • Eiso Hiyama
  • Woodring E. Wright
  • Jerry W. Shay
Genetics — Cellular Immortality

Abstract

The association of human telomerase activity with an indefinite replicative capacity of cells in vitro and advanced tumors in vivo is gaining wide support. The increasing interest in studying various aspects of telomerase expression in cancer required the development of a sensitive and reliable protocol for the extraction and detection of telomerase activity in cell culture material, and from small tissue samples obtained from biopsy, surgical reaction of tumors, and autopsy. Recently a novel procedure for the extraction and detection of telomerase activity was developed (Science 1994; 266: 2011–2015) which resulted in an estimated 104 fold improvement in detectability compared with previous methods. The described procedures not only dramatically increased sensitivity but also allowed fast and efficient detection of telomerase activity in a large number of samples. A number of technical aspects which are of eritical importance for reproducibility and reliability of this assay using clinical material are addressed in this report. In addition, new methods to perform telomerase assays without the use of radioisotopes are described.

Key words

Cancer Cellular immortality Telomerase Telomeres 

Abbreviations

AEBSF

4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochlorine

BCA

bieinchoninic acid

CCD camera

charged coupled device camera

CHAPS

3-[(3-cholamidopropyl)-dimethyl-ammonio]-l-propanesulfonate

DEPC

diethyl pyrocarbonate

DPBS

Dulbecco's phosphate buffered saline

EGTA

ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid

HPLC

high pressure liquid chromatography

HPV

human papillomavirus

PDA

piperazine diacrylamide

PCR

polymerase chain reaction

T4g32 protein

T4 gene 32 protein

TRAP

telomeric repeat amplification protocol

TRF

terminal restriction fragment

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Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • Mieczyslaw A. Piatyszek
    • 1
  • Nam W. Kim
    • 2
  • Scott L. Weinrich
    • 2
  • Keiko Hiyama
    • 3
  • Eiso Hiyama
    • 4
  • Woodring E. Wright
    • 1
  • Jerry W. Shay
    • 1
  1. 1.Department of Cell Biology and NeurosciencesUniversity of Texas Southwestern Medieal Center at DallasDallasUSA
  2. 2.Geron CorporationMenlo ParkUSA
  3. 3.Sacond Department of Internal MedicineHiroshima University School of MedicineHiroshimaJapan
  4. 4.Department of General MedicineHiroshima University School of MedicineHiroshimaJapan

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