By Patrick Lo
Published: September 18, 2008

New York, NY, Sept. 18—Padlock probes combined with rolling circle amplification (RCA) is a sensitive method to detect short DNA sequences. The padlock probe is a circularizable oligonucleotide consisting of a linker region and 3′ and 5′ sequences complementary to a DNA sequence of interest. Once bound and ligated, the probe creates a single-stranded circular DNA molecule that can be amplified by RCA.

Comparison of rolling circle amplification loop-mediated isothermal amplification (RCA-LAMP) reaction to hyperbranching. Source: BioTechniques.

The sensitivity of ligase to mismatches ensures that only exact matches to the target sequence will be ligated and then amplified.

While this method is precise, it is limited by the fact that RCA is a linear amplification process that can take several hours to produce a detectable signal.

Researchers at the University of California, San Diego (La Jolla, CA) have combined the padlock probe/RCA method with loop-mediated isothermal amplification (LAMP) to achieve rapid signal development.

After ligation of the hybridized padlock probe, a primer similar to the inner primers of LAMP initiates RCA. Another primer then binds the resulting RCA concatemer at multiple spots to prime replication of additional strands, with each new strand displacing the one in front, and these then become substrates for LAMP.

The resulting isothermal RCA-LAMP reaction increases speed significantly over the comparable hyperbranching method and improves convenience for field applications where thermal cycling may be difficult.

The work is described in a paper published in the Aug. 2008 issue of BioTechniques.