期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2005
卷号：102
期号：48
页码：17332-17336
DOI：10.1073/pnas.0508809102
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：We describe conditions for rolling-circle amplification (RCA) of individual DNA molecules 5-7 kb in size by >109-fold, using {phi}29 DNA polymerase. The principal difficulty with amplification of small amounts of template by RCA using {phi}29 DNA polymerase is "background" DNA synthesis that usually occurs when template is omitted, or at low template concentrations. Reducing the reaction volume while keeping the amount of template fixed increases the template concentration, resulting in a suppression of background synthesis. Cell-free cloning of single circular molecules by using {phi}29 DNA polymerase was achieved by carrying out the amplification reactions in very small volumes, typically 600 nl. This procedure allows cell-free cloning of individual synthetic DNA molecules that cannot be cloned in Escherichia coli, for example synthetic phage genomes carrying lethal mutations. It also allows cell-free cloning of genomic DNA isolated from bacteria. This DNA can be sequenced directly from the {phi}29 DNA polymerase reaction without further amplification. In contrast to PCR amplification, RCA using {phi}29 DNA polymerase does not produce mutant jackpots, and the high processivity of the enzyme eliminates stuttering at homopolymer tracts. Cell-free cloning has many potential applications to both natural and synthetic DNA. These include environmental DNA samples that have proven difficult to clone and synthetic genes encoding toxic products. The method may also speed genome sequencing by eliminating the need for biological cloning.
关键词：DNA sequencing ; rolling-circle amplification ; synthetic DNA
