In situations where certain DNA sequences cannot be cloned by the traditional method of insertion into a vector and growth in a biological host (for example, due to their length or repetitive nature), cell-free cloning is an attractive alternative. A recent and innovative technique for cell-free cloning uses multiply primed rolling circle amplification (MPRCA), which can amplify DNA molecules in submicroliter reaction volumes and is flexible in terms of the length or sequence to be amplified.
MPRCA is based on rolling circle amplification primed by random DNA primers, which afford the technique its flexibility, but also contribute to its main disadvantage. When the quantity of DNA template is very low, the majority of the amplification product arises from undesired DNA synthesis derived solely from the primers, producing amplicons that are found in control reactions lacking template DNA.
H. Takahashi, S. Sugiyama, and their colleagues at the National Agriculture and Food Research Organization (Ibaraki, Japan) investigated whether random RNA primers could effectively promote MPRCA and block synthesis of the unwanted byproducts observed with the random DNA primers.
Their hypothesis was based on the fact that the bacteriophage φ29 DNA polymerase used in RCA can use RNA as a primer for DNA synthesis but not as a template. They found that MPRCA using thiophosphate-linked RNA primers can efficiently amplify circular DNA molecules without creating byproducts or requiring submicroliter reaction volumes, allowing for the 1012-fold amplification of a single copy of a plasmid. The researchers then demonstrated that MPRCA using RNA primers is suitable for some types of cell-free cloning if a specific dephosphorylation/ligation strategy is first employed. Under these conditions, unwanted ligation products would be linear and easily eliminated by exonuclease treatment. The desired circular products would then be amplified, yielding sufficient product even if the ligation efficiency was low.
The complete paper was published in the July 2009 issue of BioTechniques.