Telomere measurement by quantitative PCR

Department of Human Genetics, University of Utah, 15 N 2030 E, Room 2100, Salt Lake City, UT 84112, USA

^{Tel: +1 801 585 5520; Fax: +1 801 581 7796; Email: rcawthon@genetics.utah.edu}

Received 2002 Jan 4; Revised 2002 Mar 23; Accepted 2002 Mar 23.

Abstract

It has long been presumed impossible to measure telomeres in vertebrate DNA by PCR amplification with oligonucleotide primers designed to hybridize to the TTAGGG and CCCTAA repeats, because only primer dimer-derived products are expected. Here we present a primer pair that eliminates this problem, allowing simple and rapid measurement of telomeres in a closed tube, fluorescence-based assay. This assay will facilitate investigations of the biology of telomeres and the roles they play in the molecular pathophysiology of diseases and aging.

Abstract

ACKNOWLEDGEMENTS

I thank Mark Leppert for access to the Utah CEPH DNA samples, Richard Kerber and Ken Smith for guidance with statistical analysis and Robert Weiss, Jeff Stevens, Shannon Odelberg, Hilary Coon and Elizabeth O’Brien for helpful discussions. This work was supported by the Allied Signal Award for Research on Aging and by NIH grants K01 AG00767, R29CA69421 and AG13478.

ACKNOWLEDGEMENTS

REFERENCES

References

1. Allshire R.C., Dempster,M. and Hastie,N.D. (1989) Human telomeres contain at least three types of G-rich repeat distributed non-randomly. Nucleic Acids Res., 17, 4611–4627.
2. Lansdorp P.M., Verwoerd,N.P., van de Rijke,F.M., Dragowska,V., Little,M.T., Dirks,R.W., Raap,A.K. and Tanke,H.J. (1996) Heterogeneity in telomere length of human chromosomes. Hum. Mol. Genet., 5, 685–691. [[PubMed]
3. Bryant J.E., Hutchings,K.G., Moyzis,R.K. and Griffith,J.K. (1997) Measurement of telomeric DNA content in human tissues. Biotechniques, 23, 476–478, 480, 482, passim. [[PubMed]
4. Norwood D. and Dimitrov,D.S. (1998) Sensitive method for measuring telomere lengths by quantifying telomeric DNA content of whole cells. Biotechniques, 25, 1040–1045. [[PubMed]
5. Nakamura Y., Hirose,M., Matsuo,H., Tsuyama,N., Kamisango,K. and Ide,T. (1999) Simple, rapid, quantitative and sensitive detection of telomere repeats in cell lysate by a hybridization protection assay. Clin. Chem., 45, 1718–1724. [[PubMed]
6. White R., Leppert,M., Bishop,D.T., Barker,D., Berkowitz,J., Brown,C., Callahan,P., Holm,T. and Jerominski,L. (1985) Construction of linkage maps with DNA markers for human chromosomes. Nature, 313, 101–105. [[PubMed]
7. Higuchi R., Fockler,C., Dollinger,G. and Watson,R. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology, 11, 1026–1030. [[PubMed]
8. Boulay J.L., Reuter,J., Ritschard,R., Terracciano,L., Herrmann,R. and Rochlitz,C. (1999) Gene dosage by quantitative real-time PCR. Biotechniques, 27, 228–230, 232. [[PubMed]
9. Slagboom P.E., Droog,S. and Boomsma,D.I. (1994) Genetic determination of telomere size in humans: a twin study of three age groups. Am. J. Hum. Genet., 55, 876–882.
10. Harley C.B., Futcher,A.B. and Greider,C.W. (1990) Telomeres shorten during ageing of human fibroblasts. Nature, 345, 458–460. [[PubMed]
11. Levy M.Z., Allsopp,R.C., Futcher,A.B., Greider,C.W. and Harley,C.B. (1992) Telomere end-replication problem and cell aging. J. Mol. Biol., 225, 951–960. [[PubMed]
12. Hultdin M., Gronlund,E., Norrback,K., Eriksson-Lindstrom,E., Just,T. and Roos,G. (1998) Telomere analysis by fluorescence in situ hybridization and flow cytometry. Nucleic Acids Res., 26, 3651–3656.