用于端粒替代延长活性的C环检测法。

The C-Circle Assay for alternative-lengthening-of-telomeres activity.

作者信息

Henson Jeremy D, Lau Loretta M, Koch Sylvia, Martin La Rotta Nancy, Dagg Rebecca A, Reddel Roger R

机构信息

Cancer Cell Immortality Group, Adult Cancer Program, Prince of Wales Clinical School, UNSW Australia, NSW, Australia.

Cancer Research Unit, Children's Medical Research Institute, University of Sydney, Westmead, NSW, Australia.

出版信息

Methods. 2017 Feb 1;114:74-84. doi: 10.1016/j.ymeth.2016.08.016. Epub 2016 Sep 3.

DOI:10.1016/j.ymeth.2016.08.016

PMID:27595911

Abstract

The C-Circle Assay has satisfied the need for a rapid, robust and quantitative ALT assay that responds quickly to changes in ALT activity. The C-Circle Assay involves (i) extraction or simple preparation (Quick C-Circle Preparation) of the cell's DNA, which includes C-Circles (ii) amplification of the self-primed C-Circles with a rolling circle amplification reaction and (iii) sequence specific detection of the amplification products by native telomeric DNA dot blot or telomeric qPCR. Here we detail the protocols and considerations required to perform the C-Circle Assay and its controls, which include exonuclease removal of linear telomeric DNA, production of the synthetic C-Circle C96 and modulation of ALT activity by γ-irradiation.

摘要

C 环分析满足了对一种快速、稳健且定量的谷丙转氨酶（ALT）分析方法的需求，该方法能对 ALT 活性的变化迅速做出反应。C 环分析包括以下步骤：（i）提取或简单制备（快速 C 环制备）细胞 DNA，其中包括 C 环；（ii）通过滚环扩增反应对自身引物 C 环进行扩增；（iii）通过天然端粒 DNA 斑点杂交或端粒定量聚合酶链反应（qPCR）对扩增产物进行序列特异性检测。在此，我们详细介绍进行 C 环分析及其对照所需的方案和注意事项，包括用核酸外切酶去除线性端粒 DNA、合成 C 环 C96 的制备以及通过γ射线照射调节 ALT 活性。