The C-Circle Assay for alternative-lengthening-of-telomeres activity.
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.
Cancer Cell Immortality Group, Adult Cancer Program, Prince of Wales Clinical School, UNSW Australia, NSW, Australia. Electronic address: j.henson@unsw.edu.au.
Jeremy D
JD
Henson
Cancer Research Unit, Children's Medical Research Institute, University of Sydney, Westmead, NSW, Australia.
Loretta M
LM
Lau
Cancer Cell Immortality Group, Adult Cancer Program, Prince of Wales Clinical School, UNSW Australia, NSW, Australia.
Sylvia
S
Koch
Cancer Cell Immortality Group, Adult Cancer Program, Prince of Wales Clinical School, UNSW Australia, NSW, Australia.
Nancy
N
Martin La Rotta
Children's Cancer Research Unit, The Children's Hospital at Westmead, Westmead, NSW, Australia.
Rebecca A
RA
Dagg
Cancer Research Unit, Children's Medical Research Institute, University of Sydney, Westmead, NSW, Australia.
Roger R
RR
Reddel
D014408:Biomarkers, Tumor / Q000235:genetics*; D004270:DNA, Circular / Q000032:analysis*; D004273:DNA, Neoplasm / Q000235:genetics*; D006801:Humans; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics; D016615:Telomere
D014408:Biomarkers, Tumor; D004270:DNA, Circular; D004273:DNA, Neoplasm
ALT; C-Circle Assay; Exonuclease I; Native telomeric DNA dot blot; Quick C-Circle Preparation; Rolling circle amplification; Synthetic C-Circle C96; Telomere; Telomere maintenance mechanism; Telomeric qPCR; γ-Irradiation; λ exonuclease