Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells
Figure Lengend Snippet: Flow diagram of RCA in situ . Pretreatment prior to in situ RCA detection of the Tp53 gene. Restriction enzymes were used to cut ≈20 base pairs either 3′ or 5′ of the probe binding site. Afl III was used for digestion 5′ of the binding site, and Bbs I was used for digestion 3′ of the target. Cells were then treated with exonuclease III, which digests DNA 3′ → 5′ starting with 3′ hydroxyl left by the endonuclease, resulting in staggered single-stranded DNA. The DNA strand remaining following Afl III digestion in this case is the complement to the sense probe sequence. DNA remaining following Bbs I digestion is the complement to the antisense probe sequence. Bbs I digestion constitutes a negative control for the RCA process using the sense probe, and Afl III constitutes a negative control for the antisense probe. The two ends of the sense probe create an incomplete circle as they anneal to the complementary site on the DNA digested with Bbs I. The DNA strand digested with Afl III is complementary to the sense probe and allows it to anneal and ligate, completing the circle and locking the probe onto the target. Targets other than Tp53 may require different endonucleases.
Article Snippet: Either Afl III or Bbs I (0.1 unit/μl, New England Biolabs) was applied for 12 h at 37°C.
Techniques: Flow Cytometry, In Situ, Binding Assay, Sequencing, Negative Control