Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification.
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.
Dean
F B
FB
Molecular Staging, Inc., New Haven, Connecticut 06511, USA.
Nelson
J R
JR
Giesler
T L
TL
Lasken
R S
RS
D017103:Bacillus Phages / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics* / Q000378:metabolism; D004270:DNA, Circular / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010957:Plasmids / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic; D014764:Viral Proteins / Q000378:metabolism
D017931:DNA Primers; D004270:DNA, Circular; D004279:DNA, Viral; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases