Detection Of Telomerase Activity In Cancer Cells

Human telomerase is a protein complex consisting of a human telomerase reverse transcriptase catalytic subunit that uses the human telomerase RNA component of the complex as a template for adding TTAGGG repeats to the end of chromosome.Telomerase activity can be detected in most primary human tumor specimens and tumor-derived cell lines.Most normal human cells lack telomerase activity and their telomeres shorten with each cell division,until they cannot be shortened and apoptosis,except for expression in a few proliferating cells,such as germ cells and stem cells.In most immortal and cancer cells,the ribonucleoprotein telomerase maintains telomere length by adding TTAGGG repeats to the ends of the chromosome,repairing DNA damage during cell division and allowing cells to multiply indefinitely or even live forever.Telomerase maintenance mechanisms are basis for unlimited replication potential and a biomarker of cancer.The effective detection of telomerase is of great significance for the biomedical research and cancer diagnosis.Therefore,the detection of telomerase activity is of great importance.In order to better understand the biological and clinical significance of telomerase in human malignant tumors,the accurate detection of telomerase activity is required.In this thesis,quencher-free molecular beacon(MB)-assisted quadratic signal amplification strategy is developed to sensitively detect telomerase.The presence of telomerase can catalyze the addition of telomere repeat unit(TTAGGG)_n to the 3'end of the telomerase substrate chain,generating telomerase extension products with telomere repeats.With the addition of DNA polymerase,primer extension along the template replaces the extension product of telomerase to form a double strand.At the same time,the 2-AP MB single strand in the double strand can be degraded by T7 exonuclease to release the free 2-AP molecule and primer extension strand,and the primer extension strand is retained due to phosphorylation modification at the 5'end of the primer.The free primer extension strand can be combined with another 2-AP MB to form a new double strand,generating an enhanced fluorescent signal.We combine fluorescent molecular probes with DNA to make their fluorescence highly quenched.The presence of telomerase can promote target-specific secondary signal amplification,producing an enhanced fluorescence signal.The experimental design and steps of this strategy are simple,and the operation is easy,enabling the sensitive detection of telomerase activity.