Fig. 5
From: Molecular techniques for pathogen identification and fungus detection in the environment
Schematic representation of the steps in padlock probe technology coupled with hyperbranched rolling circle amplification (H-RCA) for SNPs detection. 1. The hybridization of padlock probes (containing the complementary sequences at the 5′ and 3′ ends) to the target templates. 2. During a perfect match, the probe forms a circular molecular with the aid of DNA ligase; while in the case of mismatch, no circular molecules formed. 3. Non-hybridized template will be removed during the exonucleolysis reactions (digestion by exonucleases I and III). 4. H-RCA is performed using two pre-designed primers and Bst polymerase, but no amplification will take place in the absence of a circular molecular. 5. The accumulation of dsDNA products during isothermal rolling circle amplification of DNA minicircles is monitored in a real time PCR thermocycler with the addition of SYBR green.