Atomic force microscopy analysis of rolling circle amplification of plasmid DNA.
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.
Mizuta
Ryushin
R
Research Institute for Biological Sciences, Tokyo University of Science, Noda, Chiba, Japan. mizuta@rs.noda.tus.ac.jp
Mizuta
Midori
M
Kitamura
Daisuke
D
D000818:Animals; D017103:Bacillus Phages / Q000201:enzymology / Q000235:genetics*; D019556:COS Cells; D002522:Chlorocebus aethiops; D004270:DNA, Circular / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010957:Plasmids / Q000235:genetics*; D017422:Sequence Analysis, DNA; D020080:Tandem Repeat Sequences; D013698:Templates, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000378:metabolism
D004270:DNA, Circular; D004279:DNA, Viral; D008164:Luminescent Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D004259:DNA-Directed DNA Polymerase