| Research Abstract |
Telomeres are specific structures found at the end of chromosomes in eukaryotes. In human chromosomes, telomeres consist of thousands of copies of 6 base repeats (TTAGGG). Although human somatic cells induce cell-death by reduction of telomeric repeats with cell division, cancer cells induce extension of telomeric repeats by telomerase. Telomerase is a ribonucleoprotein that synthesizes and directs the telomeric repeats onto the 3’ end of existing telomeres using its RNA component as a template. Therefore, telomerase participates in malignant transformation or immortalization of a cell, and attracts attention as anticancer drug screening and diagnostic tumor marker. Recently, telomeric repeat amplification protocol (TRAP) is used as universal method of telomerase assay. However, these approaches generally employ acrylamide gel electrophoresis after amplifying telomeric repeat by polymerase chain reaction (PCR); as a result, the TRAP method requires considerable time and skill for us. I
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n this study, for rapid and high sensitive detection of telomerase activity, we developed novel telomerase assay using bioluminescent detection method; that is, pyrophosphates produced by telomerase reaction and PCR are converted ATP by pyruvate phosphate dikinase (PPDK), and ATP is detected by firefly luciferin-luciferase reaction.As a result, the detection limit of pyrophosphate was 1.0×10-15mol/assay. For optimal bioluminescent assay of telomerase activity, we designed the specific primers to the telomeric repeat and selected the efficient Ta q polymerase for PCR. Sequences of the sense and antisense primers for PCR amplification of telomerase reaction product were 5’-AATCCGTCGAGCAGAGTT-3’ and 5’-CTAACCCTAACCCTAACC-3’, respectively. In study of Taq polymerase, efficient PCR amplification could be obtained by use of TITANIUM Taq DNA polymerase. After the telomerase reaction and subsequent PCR, the released pyrophosphate was detected by the proposed bioluminescent assay. As a result, positive cell (500 cells) and inactive cell (prepared by heating at 85℃ for 10 min) could be clearly identified. Then, time course of bioluminescent intensity are examined. The maximum bioluminescence intensity was maintained for about two minutes. The detection limit of cells with telomerase was examined with 500, 250, 100, 50, 10, 5, 1 cell. 1 cell/assay was detectable by telomerase reaction for 30 minutes and PCR consisting of 33 cycles. PCR cycle number also was examined, 25 cycles was detectable. Presently, we are examining simpler and rapid bioluminescent detecting method, and we are developing its applying to clinical chemistry of cancer and to basic research such as regenerative medicine. Less
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