Mizuta Ryushin, Mizuta Midori, Kitamura Daisuke
Research Institute for Biological Sciences, Tokyo University of Science, Noda, Chiba, Japan.
Arch Histol Cytol. 2003 May;66(2):175-81. doi: 10.1679/aohc.66.175.
Rolling circle amplification (RCA) of plasmid DNA using random hexamers and bacteriophage phi29 DNA polymerase is an increasingly applied technique for amplifying template DNA for DNA sequencing. We analyzed this RCA reaction at a single-molecular level by atomic force microscopy (AFM) and found that multibranched amplified products containing tandem repeats of a circle unit are formed within 1 h. We also used the RCA product of a GFP expression vector for the protein expression in cells, and found that the crude RCA product from one bacterial colony is sufficient for the GFP expression. Thus, the RCA reaction is useful in amplifying DNA for both DNA sequencing and protein expression.
使用随机六聚体和噬菌体phi29 DNA聚合酶对质粒DNA进行滚环扩增（RCA）是一种越来越多地应用于扩增用于DNA测序的模板DNA的技术。我们通过原子力显微镜（AFM）在单分子水平上分析了这种RCA反应，发现含有环状单元串联重复序列的多分支扩增产物在1小时内形成。我们还将绿色荧光蛋白（GFP）表达载体的RCA产物用于细胞中的蛋白质表达，发现来自一个细菌菌落的粗RCA产物足以用于GFP表达。因此，RCA反应在扩增用于DNA测序和蛋白质表达的DNA方面都很有用。
