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Rolling Circle Amplification (RCA)

Rolling Circle Amplification (RCA) is the signal amplification engine behind both PRISM and SPRINTseq. It amplifies a circularized DNA probe into a long, single-stranded DNA concatemer (DNA nanoball or "rolony").

Mechanism

  1. Circularization: The padlock probe hybridizes to the target and is ligated into a circle.
  2. Polymerization: Phi29 DNA Polymerase (a highly processive strand-displacing polymerase) rounds the circle continuously.
  3. Result: A long strand containing thousands of tandem repeats of the probe sequence.

Protocol for Tissue

Reagents

  • Phi29 DNA Polymerase (e.g., NEB or similar)
  • dNTPs
  • BSA
  • Glycerol

Procedure

  1. Wash: Wash slides with PBS-T (0.05% Tween-20) 3 times.
  2. Pre-incubation: Add RCA pre-mix (buffer only) for 5 min.
  3. Amplification:
    • Add reaction mix with Phi29.
    • Seal chamber (optional, to prevent evaporation).
    • Incubate at 30°C.
    • Duration: Typically 2 hours to Overnight depending on desired rolony size.
  4. Stop: Wash with PBS-T to remove polymerase.

Troubleshooting

Problem Possible Cause Solution
No signals Ligation failed Check ligase activity and probe design.
High background Non-specific binding Increase wash stringency or add blocking agents (BSA/tRNA).
Smeary signals Over-amplification Reduce RCA time.