ROLLING CIRCLE AMPLIFICATION
Templates are effectively prepared by rolling circle amplification, or RCA (Fig 2). The starting material for amplification can be a small amount of bacterial cells containing a plasmid, an isolated plasmid, an M13 phage, or any circular DNA sample. A small portion of bacterial colonies can be picked from agar plates and added directly to the TempliPhi amplification reaction. Alternately, microliter quantities of a saturated culture can serve as starting material.With TempliPhi 100/500 Kits, amplification is completed in 4-6 h at 30 °C without the need for thermal cycling (Fig 3). The TempliPhi amplification reaction produces high molecular weight, double-stranded concatemers of circular template. Note that when starting with M13 clones, the TempliPhi amplification product is double-stranded DNA and can be directly sequenced with forward and reverse primers. Amplified DNA can be used in a cycle sequencing reaction without any purification.
Click here to see an animation explaining rolling circle amplification
Fig 2. A schematic of the TempliPhi amplification process.
Random hexamer primers anneal to the circular template DNA at multiple sites. Phi29 DNA polymerase extends each of these primers. When the DNA polymerase reaches a downstream-extended primer, strand displacement synthesis occurs. The displaced strand is rendered single-stranded and available to be primed by more hexamer primer. The process continues, resulting in exponential, isothermal amplification.
Fig 3. Kinetics of DNA amplification using TempliPhi 100 Amplification Kit.
Amplification of 1ng pUC19 DNA over 24 hrs. The amount of DNA was quantitated using PicoGreen™ dsDNA Quantitation Reagents at the given times. Data is representative of triplicate experiment.
Previous Next |
