Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification

Abstract

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.

Figure 1

Scheme for multiply-primed rolling circle amplification. Oligonucleotide primers complementary to the amplification target circle are hybridized to the circle. The 3′ ends of the DNA strands are indicated by arrowheads to show the polarity of polymerization. Thickened lines indicate the location of the original primer sequences within the product strands. The addition of DNA polymerase and deoxynucleoside triphosphates (dNTPs) to the primed circle results in the extension of each primer, and displacement of each newly synthesized strand results from elongation of the primer behind it. Secondary priming events can subsequently occur on the displaced product strands of the initial rolling circle amplification step. It is possible that the last intermediate shown in the figure makes only a small contribution to the overall yield of amplified DNA.

Figure 2

Comparison of amplification efficiency between singly- and randomly-primed M13 single-strand circular DNA. Rolling circle reactions were performed as described and contained either 1 ng of singly-primed single-strand M13 DNA or 1 ng of random hexamer-primed single-strand M13 DNA. Reactions were incubated at 34°C for 24 h and aliquots were taken to measure DNA synthesis as indicated.

Figure 3

Amplification of pUC19 from colonies and M13mp19 from plaques. Amplification reactions were performed as indicated directly on material picked from a colony or a plaque. Half of the radioactively labeled reaction products were cleaved with EcoRI and both cleaved and uncleaved samples were analyzed by agarose gel electrophoresis. The positions of linear, duplex M13, and pUC19 DNA are indicated.

Figure 4

Effect of exonuclease-resistant random primers on amplification. Annealing reactions (20 μL) were performed as described except that the template DNA was M13 double-strand RF DNA and the primers were either exo-resistant or exo-sensitive hexamers. Reactions contained 1 ng of M13 DNA with exo-resistant or exo-sensitive primers and φ29 DNA polymerase as indicated. Reactions were incubated at 34°C for 13 h. DNA synthesis was quantitated by the incorporation of radioactive nucleotide.

Figure 5

Sequencing of DNA amplified from a saturated bacterial culture. A saturated culture of XL1-blue (Stratagene) containing a random library plasmid (2- to 3-kb inserts in pUC18, 2 μL) was amplified as described for 12 h (see Methods). The amplified DNA was treated with calf intestine phosphatase, heated to 95° for 3 min, and sequenced by using the DYEnamic ET terminator sequencing kit and run on a MegaBACE 1000 DNA sequencer.