Axelrad Mary Derasmo, Budagov Temuri, Atzmon Gil
Department of Medicine, Albert Einstein College of Medicine, USA.
J Vis Exp. 2013 May 22(75):e50246. doi: 10.3791/50246.
Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.
端粒是位于染色体末端的重复DNA序列,其长度各异,在人类中可达15,000个碱基对。端粒作为一种生物保护机制,可防止每次细胞分裂时染色体磨损。当端粒长度达到一定程度时,会变得过短而无法进行复制,这一过程可能导致染色体不稳定或细胞死亡。端粒长度受两种相反机制调控:磨损和延长。磨损发生在每次细胞分裂时。相反,延长则部分受端粒酶调节,端粒酶会在染色体末端添加重复序列。通过这种方式,端粒酶可能逆转衰老机制并恢复细胞活力。这些是维持细胞生命的关键要素,可用于评估细胞衰老。在本手稿中,我们将描述一种准确、简短、精密且廉价的方法,用于评估多种组织和物种中的端粒长度。该方法利用了两个关键要素,即端粒序列的串联重复以及qRT-PCR检测测试样品差异拷贝数的灵敏度。此外,我们将描述一种简单的检测方法,用于评估端粒酶活性,作为端粒长度的补充性主要检测。