Improvement & Application Of Telomeric Repeat Amplification Protocol(TRAP)and Research On Telomerase Characteristic

Telomerase is a ribonucleoprotein complex that plays a critical role in telomere maintenance and cellular immortality. It adds short GGTTAG repeats to the 5'end of eukaryotic chromosomes, using its RNA component as template. Telomerase is a potentially useful tumor marker. The Telomeric Repeat Amplification Protocol (TRAP) is a sensitive procedure to measure telomerase activity in small samples of cell or tissue extracts. in the original protocol, the reverse primer can bind anywhere along the telomeric repeats during the amplification process. Thus, TRAP products may be both shorter and longer than the ofig4nal telomerase products due to staggered annealing of the primers. A new TRAP assay was developed with modified primers and amplification system, which does not change the distribution of the starting telomerase products during amplification. Thus it is suitable for studying the mechanism of telomerase- catalyzed reactions and the effects of various factors on processivity of the enzyme. There were two key improvements in the modified method. First, return primer (P2) is a 3Omer oligonucleotide containing two kinds of sequences. The 3?part isl2 nt sequences that hybridize to telomeric repeats. The 5?part is 18 nt sequence made of three kinds of dNTPs except dGTP and containing random sequences. Second, During telomerase-mediated extension reaction, the reaction system was absent of dCTP. Once the original telomerase products were genetated, P2 primer would ,~nneal to their 3?end and yield a 13 nt long non- telomeric overhang which act as primer binding site in subsequent PCR amplification. Thus the PCR products directly reflect the characterization of original telomerase products. To prove this assumption three chemically synthesized telomerase products comprising P1 primer extended with 2, 5, 8 telomeric repeats (R2, R5 and R8) were amplified with P2 reverse primers. Only one band was generated 91 in each reaction, The sizes of the products are in agreement with the proposed amplification mechanism (59, 77 and 95 fit). The telomerase avtivity was also detected using modified method. 293 cell extracts produced the expected DNA ladder with 6 nt increments. The shortest product has the same electrophoretic mobility of R2 band. The products generated from dilution of 293 cell extracts are nearly linear from --30 to 1000 cells. The reaction parameters such as pH, 2~ concerntration and PCR annealing temperature were optimized. Tumor specimens were detected using modified TRAP. Differences on telomerase activity, Among the 40 specimens, 37 were telomerase positive. There was no telomeerase activity in the nomal human tissues. There was telomerase activity in placenta as well as immortal cell linessuch as 293, hela,SIVIMC, Hep2 and MCf7. The processivity and band distribution were found among the specimens. The expression of hTERT gene of the specimens was detected by RT-PCR. There was no parallel between telomerase activity and hTERT expression level. The relationship between activity and disease degree was also investigated. The dNTP concerntration dependance on telomerase activity was studied by this method. Telomerase activity and processivity were both affected by dNTP concerntration. The minimum concemtration of various dNTPs, which support processive synthesist was different. The nuclease activity of telomerase was also shown in 293 cells. Var...