Cell Biology > Apoptosis

User Ratings

Your rating: None
Your rating: None
Your rating: None
 Add your comments

Analysis of Telomeres and Telomerase

Brittney‐Shea Herbert1,  Jerry W. Shay1,  Woodring E. Wright1

1The University of Texas Southwestern Medical Center, Dallas, Texas

Publication Name: 
Current Protocols in Cell Biology
Unit Number: 
UNIT 18.6
DOI: 
10.1002/0471143030.cb1806s20
Online Posting Date: 
November, 2003
GO TO THE FULL TEXT:
PDF or HTML at Wiley Interscience
Are you the author of this protocol? Login or register and return to this page.

Abstract

This unit describes techniques to analyze telomeric length and telomerase activity in human cells. Telomere length can be determined by a modification of Southern blotting in which the analysis of chromosome terminal restriction fragments (TRFs) provides the average lengths of all telomeres in a cell population. Telomerase activity can be measured in vitro by a sensitive and efficient polymerase chain reaction (PCR)-based detection method, also known as telomeric repeat amplification protocol (TRAP). These assays can be used to study the in vitro cellular effects of aging and cancer treatments on telomere biology and telomerase activity.

Keywords: telomeres; terminal restriction fragment (TRF); telomerase; TS primer; PCR

     
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Interscience

Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Terminal Restriction Fragment Size Determination to Measure Average Telomere Lengths
  • Support Protocol 1: Making A Labeled Molecular Weight Marker for Running on an Agarose Gel
  • Support Protocol 2: Making Kinased Radiolabeled Telomeric Repeat Probe
  • Support Protocol 3: Synthesis of A High–Specific Activity Telomeric Repeat Probe
  • Basic Protocol 2: Measurement of Telomerase Activity by the Telomeric Repeat Amplification Protocol
  • Alternate Protocol: Measurement of Telomerase Activity by the Telomeric Repeat Amplification Protocol Using Fluorescent Primers
  • Support Protocol 4: Lysis of Tissue Samples for the Telomeric Repeat Amplification Protocol
  • Support Protocol 5: Making Radiolabeled TS Primer for Telomeric Repeat Amplification Protocol
  • Support Protocol 6: Making the Primer Mix for Telomeric Repeat Amplification Protocol
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Interscience

Materials

Basic Protocol 1: Terminal Restriction Fragment Size Determination to Measure Average Telomere Lengths

 Materials
  • Cell pellet from ³300,000 cells and appropriate medium
  • Quick-prep lysis buffer (see recipe)
  • Triton X-100
  • 20 mg/ml proteinase K
  • TE buffer, pH 7.5 to 8.0 (appendix 2A)
  • Restriction enzyme mix: equal volumes of HinfI, RsaI, MspI, CfoI, HaeIII, and AluI restriction enzymes (Boehringer Mannheim; 10 U/µl each before mixing, final 1.67 U/µl each)
  • 10× TAE buffer (appendix 2A)
  • 10× TBE buffer (appendix 2A)
  • 10,000 cpm radiolabeled molecular weight markers mixed with ³500 ng unlabeled, StyI-digested DNA (or other appropriate unlabeled, digested DNA; see Support Protocol 1)
  • Denaturing solution: 0.5 M NaOH/1.5 M NaCl
  • Neutralizing solution: 1.5 M NaCl/0.5 M Tris×Cl, pH 8.0 (see appendix 2A for Tris×Cl)
  • Hybridization solution (see recipe)
  • Radiolabeled telomeric repeat probe (see Support Protocols 2 and 3)
  • 20× SSC (see recipe)
  • 0.1× SSC/0.1% (w/v) SDS
  • 2-ml polypropylene screw-cap tubes (Sarstedt) with an optional extra set of screw caps to be cut vertically with jigsaw to form open screw-cap ring (Fig. 18.6.1A)
  • 37°, 55°, and 70°C water baths heating blocks, or PCR machine
  • Dialysis membrane sheets or tubing (e.g., Spectra/Por; Spectrum), molecular weight cutoff (MWCO) ³6000 to 8000
  • Wide-bore pipet tips
  • Whatman 3MM filter paper
  • Hybridization oven, 42°C, and appropriate hybridization containers, or equivalent
  • PhosphorImager, including phosphor screens and ImageQuant software (Molecular Dynamics)
  • Additional reagents and equipment for agarose gel electrophoresis (appendix 3A)

Support Protocol 1: Making A Labeled Molecular Weight Marker for Running on an Agarose Gel

 Materials
  • ~250 ng/µl DNA molecular weight marker (e.g., DNA digested with StyI)
  • 10× React 2 buffer (Roche)
  • 100 µM dAGT mix: 100 µM each dATP, dGTP, dTTP in H2O, stored up to 1 year at –20°C
  • [-32P]dCTP (3000 Ci/mmol)
  • 2 U/µl Klenow fragment of E. coli DNA polymerase I
  • QIAquick nucleotide removal kit (Qiagen)

Support Protocol 2: Making Kinased Radiolabeled Telomeric Repeat Probe

 Materials
  • (T2AG3)4 or (C3TA2)4 oligonucleotide (20 pmol/µl or 0.16 µg/µl)
  • [-32P]ATP (3000 Ci/mmol)
  • 5× forward or exchange reaction buffer (GIBCO/BRL)
  • 10 U/µl T4 polynucleotide kinase (GIBCO/BRL)
  • QIAquick nucleotide removal kit (Qiagen)

Support Protocol 3: Synthesis of A High–Specific Activity Telomeric Repeat Probe

 Materials
  • 10 pmol/µl GTU4 oligonucleotide dissolved in TE buffer: 5¢-GGGUUAGGGUUAGGGUUAGGGAAA-3¢
  • 100 pmol/µl T3C3+9 oligonucleotide dissolved in TE buffer: 5¢-TTTCCCTAACCCTAA-3¢
  • 1 M NaCl
  • 10× buffer M: 10 mM Tris·Cl (pH 7.5)/10 mM MgCl2/50 mM NaCl/1 mM dithioerythritol (DTE; Roche)
  • 2 M Tris·Cl, pH 7.4 to 7.6 (appendix 2A)
  • 10 mg/ml BSA (e.g., Ambion)
  • 1.25 mM dAdT: 1.25 mM each of dATP and dTTP
  • [-32P]dCTP (3000 Ci/mmol)
  • 5 U/µl Klenow large fragment of E. coli DNA polymerase I
  • 1 U/µl uracil deglycosylase (UDG)
  • 37°, 95°, and 99°C water baths heating blocks, or PCR machines
  • PhosphorImager, including phosphor screens (Molecular Dynamics)
  • Additional reagents and equipment for DNA acrylamide gel electrophoresis (appendix 3A)

Basic Protocol 2: Measurement of Telomerase Activity by the Telomeric Repeat Amplification Protocol

 Materials
  • 100,000 cells grown in culture or tissue lysate containing 6 µg protein (see Support Protocol 4)
  • Liquid nitrogen
  • NP-40 lysis buffer (see recipe), ice cold
  • RNase
  • 50× dNTP mix: 2.5 mM each dATP, dTTP, dGTP, and dCTP in RNase-free water
  • 10× TRAP buffer (see recipe)
  • Radiolabeled TS primer (see Support Protocol 5)
  • Primer mix (see Support Protocol 6)
  • RNase-free H2O (DEPC-treated)
  • 50 mg/ml BSA, ultrapure (Ambion)
  • 5 U/µl Taq DNA polymerase
  • Loading dye (see recipe)
  • 0.5 M NaCl/50% (v/v) ethanol/40 mM sodium acetate (pH 4.2), optional
  • DNase-, RNase-free 0.5-ml microcentrifuge and PCR tubes
  • Tabletop centrifuge (e.g., 5415D; Eppendorf), room temperature and, optionally, 4°C
  • 37°C water bath or 85°C heating block
  • Thermal cycler
  • PhosphorImager with phosphor screens and ImageQuant software (Molecular Dynamics)
  • Additional reagents and equipment for nondenaturing acrylamide gel electrophoresis (unit 6.5)

NOTE: Most of the reagents are included in the TRAPeze kit (Intergen).

Alternate Protocol: Measurement of Telomerase Activity by the Telomeric Repeat Amplification Protocol Using Fluorescent Primers

 Additional Materials (also see Basic Protocol 2)
  • 100 ng/µl fluorescently labeled TS primer: 5¢-Cy5-AATCCGTCGAGCAGAGTT (Integrated DNA Technologies), HPLC or PAGE purified

Support Protocol 4: Lysis of Tissue Samples for the Telomeric Repeat Amplification Protocol

 Additional Materials (also see Basic Protocol 2)
  • 50 to 100 mg tissue sample, frozen at –80°C
  • Washing buffer (see recipe), ice cold
  • BCA protein assay kit (Pierce)
  • Kontes tubes and disposable pestles (VWR)
  • Hand-powered drill

Support Protocol 5: Making Radiolabeled TS Primer for Telomeric Repeat Amplification Protocol

 Materials
  • 100 ng/µl TS primer (5¢-AATCCGTCGAGCAGAGTT-3¢)
  • [-32P]ATP (3000 Ci/mmol), sterile
  • 10 U/µl T4 polynucleotide kinase and 5× forward kinase buffer (GIBCO/BRL)
  • Sterile, RNase-free H2O
  • RNase-free 1.5-ml microcentrifuge tubes
  • 37° and 85°C water baths or heating block, or PCR machine

Support Protocol 6: Making the Primer Mix for Telomeric Repeat Amplification Protocol

 Materials
  • TSNT oligonucleotide: 5¢-AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3¢
  • RNase-free H2O
  • 1 µg/µl ACX primer: 5¢-GCGCGGCTTACCCTTACCCTTACCCTAACC-3¢
  • 1 µg/µl NT primer: 5¢-ATCGCTTCTCGGCCTTTT-3¢
  • 10% (v/v) bleach
  • RNase-free 0.5-ml PCR tubes, including some that are siliconized
Looking for materials?  Suppliers Guide
     
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Interscience

Figures

  • Figure 18.6.1
    Construction of the dialysis tube for the terminal restriction fragment (TRF) protocol (see Basic Protocol 1). Large numbers of these open-ended caps can easily be prepared as follows. (A) Screw the caps onto a tube so that the tube functions as a handle and cut off the end of the cap using a jigsaw. (B) The threads of the cap are attached as a ring on the tube (arrow). These rings are reusable as tube closures.

    View Image
  • Figure 18.6.2
    Assembly of the dialysis tube for the terminal restriction fragment (TRF) protocol (see Basic Protocol 1). (A) A wet 1- to 2-cm2 piece of dialysis membrane is placed on the tube containing the sample (save the original screw cap). (B) An open-end screw cap (threaded rings) is carefully threaded on the tube over the membrane without tearing. (C) The tube (arrow) is inverted and placed in a floating rack in 1× TE buffer. There should be no droplets remaining on the walls of the tube and no bubbles on the membrane when inverted, as this will prevent dialysis.

    View Image
  • Figure 18.6.3
    Annealed priming and template oligonucleotides for synthesis of a high–specific activity telomeric repeat probe. The template contains a small mismatch at its 3¢ end (GGGAAA rather than GGGTTA) so the priming oligonucleotide will anneal in the correct spot.

    View Image
  • Figure 18.6.4
    Telomere elongation by telomerase. Telomerase acts on the ends of chromosomes to add telomeric repeat sequences. Telomerase (hTERT) adds hexameric TTAGGG repeats by using its own RNA template (hTR) as a primer to add nucleotides. Telomerase then repositions its template RNA downstream for the addition of more telomeric repeats.

    View Image
  • Figure 18.6.5
    The telomeric repeat amplification protocol (TRAP; see Basic Protocol 2). In the first step, telomerase-mediated extension occurs on a nontelomeric oligonucleotide that serves as a substrate for telomerase (TS). In the second step, the TS-telomerase products are specifically amplified by the polymerase chain reaction (PCR) using the TS as an upstream primer and ACX as the reverse, downstream primer. The addition of telomeric repeats in the first step results in a TRAP ladder of 6-bp increments as seen on a 10% (w/v) acrylamide gel. Adapted from the manual for the TRAPeze Telomerase Detection Kit (Intergen).

    View Image
  • Figure 18.6.6
    Determination of telomere length using the terminal restriction fragment (TRF) protocol in sequentially passaged (indicated by population doubling level, or the number of doublings the population has accrued, after infection) H1299 lung carcinoma cells infected with a dominant-negative mutant of human telomerase to inhibit telomerase and induce telomere shortening. Telomere length was determined by digesting genomic DNA with a mixture of six restriction enzymes having four-base recognition sites (see Basic Protocol 1), and then analyzing the DNA on a 0.7% (w/v) agarose gel. DNA cut with StyI was used as a molecular weight marker.

    View Image
  • Figure 18.6.7
    Analysis of telomerase activity using the telomeric repeat amplification protocol (TRAP; see Basic Protocol 2). Human cancer cell extracts (500 cell equivalents of H1299 lung carcinoma cells) are positive for telomerase activity as evidenced by the 6-bp incremental TRAP ladder. Treating the extract with heat inactivates the telomerase activity as evidenced by no TRAP ladder. Lysis buffer only serves as a negative control. Each TRAP reaction includes a 36-bp internal standard control. Ten-fold increases of H1299 cell equivalents (10 to 1000 cell equivalents) results in increased intensity of the TRAP ladder. The 1000 cell sample in this gel cannot be readily quantitated because of the faint signal in the internal standard (due to competition by excess telomerase products).

    View Image

Literature Cited

 Literature Cited
    Allsopp, R.C., Vaziri, H., Patterson, C., Goldstein, S., Younglai, E.V., Futcher, A.B., Greider, C.W., and Harley, C.B. 1992. Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 89:10114-10118.
    Collins, K. 2000. Mammalian telomeres and telomerase. Curr. Opin. Cell Biol. 12:378-383.
    Cong, Y.-S., Wright, W.E., and Shay, J.W. 2002. Human telomerase and its regulation. Microbiol. Mol. Bio. Rev. 66:407-425.
    Counter, C.M., Avilion, A.A., LeFeuvre, C.E., Stewart, N.G., Greider, C.W., Harley, C.B., and Bacchetti, S. 1992. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J. 11:1921-1929.
    Gollahon, L.S. and Holt, S.E. 2000. Alternative methods of extracting telomerase activity from human tumor samples. Cancer Lett. 159:141-149.
    Greider, C.W. and Blackburn, E.H. 1985. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43:405-413.
    Harley, C.B., Futcher, A.B., and Greider, C.W. 1990. Telomeres shorten during ageing of human fibroblasts. Nature 345:458-460.
    Holt, S.E., Norton, J.C., Wright, W.E., and Shay, J.W. 1996. Comparison of the telomeric repeat amplification protocol (TRAP) to the new TRAP-eze telomerase detection kit. Methods Cell Sci. 18:237-248.
    Kim, N.-W., Piatyszek, M.A., Prowse, K.R., Harley, C.B., West, M.D., Ho, P.L.C., Coviello, G.M., Wright, W.E., Weinrich, S.L., and Shay, J.W. 1994. Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011-2015.
    Norton, J.C., Holt, S.E., Wright, W.E., and Shay, J.W. 1998. Enhanced detection of human telomerase activity. DNA Cell Biol. 17:217-219.
    Ouellette, M.M., Liao, M., Herbert, B.-S., Johnson, M., Holt, S.E., Liss, H.S., Shay, J.W., and Wright, W.E. 2000. Subsenescent telomere lengths in fibroblasts immortalized by limiting amounts of telomerase. J. Biol. Chem. 275:10072-10076.
    Piatyszek, M.A., Kim, N.W., Weinrich, S.L., Hiyama, K., Hiyama, E., Wright, W.E., and Shay, J.W. 1995. Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP). Methods Cell Sci. 17:1-15.
    Wright, W.E., Shay, J.W., and Piatyszek, M.A. 1995. Modifications of a telomeric repeat amplification protocol (TRAP) result in increased reliability, linearity and sensitivity. Nucl. Acids Res. 23:3794-3795.
    Wright, W.E., Brasiskyte, D., Piatyszek, M.A., and Shay, J.W. 1996. Experimental elongation of telomeres in immortal human cells extends the lifespan of immortal × normal cell hybrids. EMBO J. 15:1734-1741.
 Internet Resource
    http://www.swmed.edu/home_pages/cellbio/shay-wright/

Includes TELORUN for calculating mean TRF.

     
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Interscience
Looking for Answers?
Do you have tips, tricks, or improvements to share?

Join the Conversation

Post new comment

The content of this field is kept private and will not be shown publicly.
CAPTCHA
This question is for testing whether you are a human visitor and to prevent automated spam submissions.