Journal: Frontiers in Microbiology
Article Title: Rapid Identification of Emerging Human-Pathogenic Sporothrix Species with Rolling Circle Amplification
Figure Lengend Snippet: Schematic of RCA of circularized padlock probes . (A) S. brasiliensis (Sbra-RCA) padlock probe design. (B) CAL is amplified by PCR with primers CAL -Fw and CAL -Rv; PCR products are submitted to ligation. Circularization of padlock probes occurs only if both probe arms hybridize correctly to the target sequence. (C) Upon specific hybridization, the phosphorylated 5′ end and the free hydroxyl at the 3′ end of the probe are joined by Pfu DNA ligase. After ligation, non-circularized probes and single-stranded primers are removed with Exo I and Exo III (optional step). (D) Circularized padlock probes serve as DNA template and signal amplification occurs via RCA, using Bst DNA polymerase and the primers RCA1 and RCA2. DNA synthesis occurs continuously for 1 h under isothermal amplification (65°C). DNA products are detected by gel electrophoresis or directly with SYBR Green I.
Article Snippet: The exonucleolysis mix consisted of 0.5 μL Exo I (New England BioLabs, Ipswich, MA, USA), 1 μL 10 × Exo I Buffer (New England BioLabs), 0.2 μL Exo III (New England BioLabs), 1 μL 10 × Exo III Buffer (New England BioLabs), and 7.3 μL ultra-pure water.
Techniques: Amplification, Polymerase Chain Reaction, Ligation, Sequencing, Hybridization, DNA Synthesis, Nucleic Acid Electrophoresis, SYBR Green Assay