Amplification and Quantitation of Telomeric Extrachromosomal Circles.
Amplification and Quantitation of Telomeric Extrachromosomal Circles.
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DOI：
10.21769/bioprotoc.4627
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发表时间：
2023-03-05
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影响因子：
0.8
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中科院分区：
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端粒是覆盖线性染色体末端的结构，在维持基因组完整性和建立细胞复制寿命方面发挥关键作用。在干细胞和癌细胞中，端粒通过端粒酶或端粒替代延长（ALT）途径被主动延长。该途径的特征在于几个标志性特征，包括可以探测以评估ALT活性的染色体外富含C的环状DNA。这些所谓的C环是ALT相关的DNA损伤修复过程的产物，同时作为迭代端粒延伸的潜在模板。这种双功能性质使得C环具有高度敏感性和特异性的ALT标志物。在这里，我们描述了一个C-环测定，改编自以前的报告，使C-环丰度在哺乳动物细胞进行了广泛的实验扰动的定量。该方案将快速C环制备（QCP）方法与基于荧光的DNA定量、滚环扩增（RCA）和使用定量PCR检测C环的DNA分离相结合。此外，包含具有良好表征的端粒维持机制（TMM）的内部标准允许对具有未知TMM状态的细胞进行可靠的基准测试。总的来说，我们的工作建立在现有的协议，以创建一个通用的工作流程，在体外C-圈定量和确认TMM身份。
Telomeres are structures that cap the ends of linear chromosomes and play critical roles in maintaining genome integrity and establishing the replicative lifespan of cells. In stem and cancer cells, telomeres are actively elongated by either telomerase or the alternative lengthening of telomeres (ALT) pathway. This pathway is characterized by several hallmark features, including extrachromosomal C-rich circular DNAs that can be probed to assess ALT activity. These so-called C-circles are the product of ALT-associated DNA damage repair processes and simultaneously serve as potential templates for iterative telomere extension. This bifunctional nature makes C-circles highly sensitive and specific markers of ALT. Here, we describe a C-circle assay, adapted from previous reports, that enables the quantitation of C-circle abundance in mammalian cells subjected to a wide range of experimental perturbations. This protocol combines the Quick C-circle Preparation (QCP) method for DNA isolation with fluorometry-based DNA quantification, rolling circle amplification (RCA), and detection of C-circles using quantitative PCR. Moreover, the inclusion of internal standards with well-characterized telomere maintenance mechanisms (TMMs) allows for the reliable benchmarking of cells with unknown TMM status. Overall, our work builds upon existing protocols to create a generalizable workflow for in vitro C-circle quantitation and ascertainment of TMM identity.