The predominant method for
DNA cloning is by propagation in biological hosts, but this method has limitations because certain sequences are difficult to clone using any combination of available hosts or vectors. Recently, multiply-primed
rolling circle amplification (MPRCA) has been applied to overcome the problems of the
DNA cloning via
host cells. However, when MPRCA is used to amplify from minute quantities of
DNA template, the products are mostly by-product
DNA molecules generated by false priming and primer dimer formation. This study demonstrates that MPRCA using
random RNA primers[#x02014]instead of
DNA primers[#x02014]blocked the synthesis of by-products and succeeded in amplifying one copy of a circular
DNA molecule more than 1012-fold to give microgram quantities of amplification product without using submicroliter reaction volumes. Furthermore, a ligation strategy was elaborated to circularize only the desired
DNA sequence and eliminate undesired ligation-products. A combination of these methods was able to amplify and ligate a large construct without undesired
DNA sequences and at microgram quantities within one day. Therefore, these methods have the possibility to improve
DNA cloning techniques that have been restricted by the limitations of
PCR methods or by the
host cell. Address correspondence to Shigeru Sugiyama, Nano-Biotechnology Laboratory, Food Engineering Division, National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. e-mail: ssugi@affrc.go.jp.
