Open AccessRapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification
Author(s) -
Frank B. Dean,
John Nelson,
Theresa L. Giesler,
Roger S. Lasken
Publication year - 2001
Publication title -
genome research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.556
H-Index - 297
eISSN - 1549-5469
pISSN - 1088-9051
DOI - 10.1101/gr.180501
Subject(s) - rolling circle replication , biology , multiple displacement amplification , applications of pcr , in vitro recombination , dna , dna polymerase , microbiology and biotechnology , polymerase chain reaction , plasmid , dna nanoball sequencing , sequencing by ligation , ancient dna , primer dimer , cloning (programming) , dna sequencing , primase , genomic library , molecular cloning , genetics , dna extraction , multiplex polymerase chain reaction , complementary dna , gene , base sequence , reverse transcriptase , computer science , programming language , population , demography , sociology
We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques. Using random primers and phi29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods and traditional DNA isolation methods. Reaction products can be used directly for DNA sequencing after phosphatase treatment to inactivate unincorporated nucleotides. Amplified products can also be used for in vitro cloning, library construction, and other molecular biology applications.
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