AMPLIFICATION AND CLONING OF SINGLE DNA MOLECULES USING ROLLING CIRCLE AMPLIFICATION

摘要

The present invention relates, e.g., to a method for amplifying a small number of copies (e.g. a single copy) of a single-stranded circular DNA molecule (e.g. having a size of about 5-6 kb) by an isothermal rolling circle mechanism, using random or partially random primers and a F29-type DNA polymerase. The method, which can also be used for amplifying DNAs by non-rolling types of multiple displacement amplification, comprises incubating the reaction components in a small volume, e.g. about 10µl or less, such as about 0.6µl or less. The degree of amplification can be about 109 fold, or higher. A method for cell-free cloning of DNA, using the rolling circle amplification method of the invention, is described.