Rolling circle amplification

Rolling circle amplification has a variety of potential uses, all of which rely upon the isothermal linear amplification of a single stranded circle of DNA. Padlock probes use rolling circle amplification to identify small sequences, polymorphisms, or mutations. A linear probe is designed to have target-complementary segments of 15-20 nucleotides at both ends, separated by a linker of approximately 50 nucleotides. The linker can include sequence elements that allow for amplification or identification of individual probes. Upon hybridization to a target DNA or RNA sequence, the two ends of the probe become juxtaposed and can be joined by a DNA ligase, ensuring a similar level of specificity to that of PCR. Circularized probes can then be detected in a number of ways. For example, the primer can be designed to hybridize to the padlock with which primer extension creates a linear tandem repeat of the circle that can be probed with labeled decorator oligos. Hyper-branched RCA occurs if a second primer is added which copies the RCA product, resulting in faster replication than normal RCA. Digestion with a restriction enzyme, known to cut the padlock only once, allows the resolution of the product on an agarose gel.52 It is also possible to use PCR to amplify a padlock probe if the primers used amplify across the ligation point.

This method can also be used for in situ allelic discrimination. For allelic discrimination, there are two probes which only have 20 nucleotide target specific sequences which differ at the 3' nucleotide. A single anchor probe is designed to hybridize adjacent to the rolling circle probes. When ligase is added, the anchor probe will ligate to the allele discriminating probe only if the 3' nucleotide matches the target sequence. Amplification can then proceed as described above. Genotype can then be determined by using different labels for each decorator probe.23

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