Journal
METHODS
Volume 114, Issue -, Pages 74-84Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2016.08.016
Keywords
C-Circle Assay; ALT; Telomere; Telomere maintenance mechanism; Quick C-Circle Preparation; Rolling circle amplification; Native telomeric DNA dot blot; Telomeric qPCR; lambda exonuclease; Exonuclease I; Synthetic C-Circle C96; gamma-Irradiation
Funding
- Cancer Institute NSW Career Development Fellowship
- Sony Foundation
- Cure Brain Cancer Foundation
- Cancer Council NSW
- Harry Banks Sutherland Rotary PhD Scholarship
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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 gamma-irradiation. (C) 2016 Elsevier Inc. All rights reserved.
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