Notes

1. Although Amplifluor primers are light-sensitive and long exposure to light should be avoided, the author found little decrease in fluorescence signal after 6 mo of storage at -80°C.

2. The T42 and TSR8 are oligonucleotide targets utilized for the PCR amplification. The working area can easily be contaminated from handling the highly concentrated oligonucleotide preparations. These oligomers should be synthesized

Fig. 2. Standard curve.(Please see Companion CD for a color version of this figure.)

by a DNA synthesizer different from those used for synthesis of other primers. In addition, the oligomer solutions should be prepared using specifically designated pipets in a physically separated work area.

3. All immortalized cultured cells express telomerase. Although the author observed several-fold variation of telomerase activity among different cell lines, any cultured cell lines can be used for this purpose. The volume of CHAPS lysis buffer used is adjusted for the number of cells to be extracted. To determine the volume of CHAPS lysis buffer for each sample, establish cell number by counting or extrapolation from tissue weight.

4. When preparing extracts from tumor samples, add RNase inhibitor to CHAPS lysis buffer prior to the extraction for a final concentration of 100-200 U/mL.

5. The problem most commonly associated with the TRAP assay is the use of sample extracts with high protein concentration. Many samples isolated from human tissues and fluids contain Taq polymerase inhibitor(s). In addition, presence of large quantities of proteins in the reaction mixture can occasionally cause amplification of nonspecific PCR products. Therefore, it is essential to control the amount of input proteins in the assay. Diluting the samples below the recommended maximum protein concentration minimizes most of these problems. See Subheading 3.3. for the effect of sample dilution on accurate analysis of clinical samples.

6. The extracts for the TRAP assay should be quick-frozen on dry ice after each use. Aliquots should not be freeze-thawed more than five times to avoid loss of telomerase activity. In addition, aliquoting reduces the risk of contamination.

7. You may alter the order of step b and steps c, I, and e. add telomerase extracts or controls at the bottom of the PCR reaction tube first, and then carefully add 48 | L of the Master Mix.

0 2 4 6 3 10 12 14 16 IS 20 22 24 26 2S 50 32 34 56 38 40

Cycl*

Cycl*

Fig. 3. Amplification plots. (A) TSR8-FAM window. (B) TSR8-ROX window. (C) Experimental samples (approx 1-5) -ROX window. (Please see Companion CD for a color version of this figure.)

References

1 Blackburn, E. H. (1991) Structure and function of telomeres. Nature 350, 569563.

2 Zakitan, V. A. (1989) Structure and function of telomeres. Ann. Rev. Genet. 23, 579-604.

3 Watson, J. D. (1972) Origin of concatemeric T7 DNA. Nature New Biol. 239, 197-201.

4 Olovnikov, A. M. (1973) A theory of marginotomy: the incomplete copying template margin in enzymic synthesis of pronucleotides and biological significance of the phenomenon. J. Theor. Biol. 41, 181-190.

5 Greider, C. W. and Blackburn, E. H. (1989) A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeats synthesis. Nature 337, 331337.

6 Morin, G. B. (1989) The human telomere terminal transferase enzyme is a ribo-nucleoprotein that synthesizes TTAGGG repeats. Cell 59, 521-529.

7 Kim, N. W., Piatyszek, M. A., Prowse, K. R., et al. (1994) Specific association of human telomerase activity with immortal cells and cancer. Science 266, 20112014.

8 Shay, J. W. and Bacchetti, S. (1997) A survey of telomerase activity in human cancer. Eur. J. Cancer 33, 787-791.

9 Bodnar, A. G., Ouellette, M., Frolkis, M., et al. (1998) Extension of life-span by introduction of telomerase into normal human cell. Science 279, 349-352.

10 Bacchetti, S. and Counter, C. M. (1995) Telomeres and telomerase in human cancer. Int. J. Oncology 7, 423-432

11 Counter, C. M., Avilion, A. A., LeFeuvre, C. E., et al. (1992) Telomerase shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J. 11, 1921-1929.

12 Nazarenko, I. A., Bhatnagar, S., and Hohman, R. J. (1997) A closed tube format for amplification and detection of DNA based energy transfer. Nucleic Acids Res. 25,2516-2521.

13 Uehara, H., Nardone, G., Nazarenko, I. A., and Hohman, R. J. (1999) Detection of telomerase activity utilizing energy transfer primer: comparison with gel- and ELISA-based detection. Biotechniques 26, 552-558.

14 Myakishev, M., Khripin, Y., Hu, S., and Hamer, D. (2001) High throughput SNP genotyping by allele-specific PCR with universal energy transfer-labeled primers. Genome Res. 1, 163-169.

15 Stryer, L. (1978) Fluorescence energy transfer as a spectroscopic ruler. Ann. Rev. Biochem. 47, 819-846.

16 Wu, P. and Brand, L. (1994) Resonance energy transfer: methods and applications. Anal. Biochem. 218, 1-13.

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