Rapid Amplification Of Plasmid And Phage DNA Using Phi29 DNA ...

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RESULTS

Increased Yield in RCA Using Random Hexamer Primers

In multiply-primed RCA, the use of multiple primers annealed to a circular template DNA generates multiple replication forks (Fig.1). RCA proceeds by displacing the nontemplate strand. In this way, product strands are “rolled off” of the template as tandem copies of the circle. Random priming allows synthesis of both strands, resulting in double-stranded product. A cascade of priming events results in exponential (or hyperbranched) amplification.

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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.

φ29 DNA polymerase was chosen because of its capacity to perform strand displacement DNA synthesis for more than 70,000 nt without dissociating from the template (Blanco et al. 1989) and its stability, which allows efficient DNA synthesis to continue for many hours. Random priming of M13 single-stranded DNA gave significantly more DNA synthesis compared with a single specific primer (Fig.2). Pyrophosphatase was added to the reaction to eliminate the inhibitory accumulation of pyrophosphate. The M13 DNA was amplified 375-fold in 24 h by using random hexamers.

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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.

Amplification of Plasmid and Bacteriophage DNA from Colonies or Plaques

A small amount of material from bacterial colonies or plaques was picked and heated in the presence of random hexamer primers, as described. The heating step inactivates nucleases, releases the plasmid or phage DNA from cells or phage particles, and denatures the DNA, allowing primer annealing. Amplification was performed in the presence of radioactively labeled deoxycytidine triphosphate (dCTP) to quantify DNA synthesis and to visualize the reaction products after gel electrophoresis. Cleavage of the amplification products by restriction endonuclease EcoRI gave linear 7.2-kb (M13) and 2.7-kb double-stranded (pUC19) DNA fragments, demonstrating that the amplification product was indeed tandem repeats of the target (Fig.3). No DNA products were observed by using colonies that did not contain plasmid DNA (not shown).

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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.

Approximately 80%of the multiply-primed RCA products on the gel were converted to the linear form by EcoRI digestion. This high yield of specific product occurred in spite of the presence of an excess of bacterial genomic DNA over plasmid DNA. For 200 copies of pUC19, genomic DNA would be in an eightfold excess in the cell. Therefore, in addition to being efficient, amplification of the circular vector DNA was highly selective. The short heating step may not release the bacterial DNA from its association with the bacterial membrane (Kornberg and Baker 1992), leaving it less accessible for amplification than the more easily released pUC19 DNA. In addition, a plasmid DNA would be copied with orders of magnitude greater frequency than the bacterial chromosome during the linear RCA phase of the amplification. Finally, the large chromosomal DNA would have a greater likelihood of acquiring RCA-terminating nicks than would a small vector DNA.

Exonuclease-Resistant Random Primers Increase Amplification by φ29 DNA Polymerase

The degradation of primers by the proofreading, 3′-5′ exonuclease activity of φ29 DNA polymerase reduces yields. Primers resistant to degradation were used to prolong the reaction and allow the use of higher concentrations of DNA polymerase. Exonuclease-resistant (exo-resistant) random-hexamer primers were made by using thiophosphate linkages for the two 3′ terminal nucleotides (5′-NpNpNpNpsNpsN-3′). With exo-resistant primers, RCA yield increased linearly when using up to five units of φ29 DNA polymerase (Fig. 4). Up to a 10,000-fold amplification was achieved starting with 1 ng of M13 template. This compared favorably with the 375-fold amplification observed by using exo-sensitive primers (Fig. 2). It corresponded to an amplification rate of ∼800 copies per hour, a 40-fold improvement over the rate of 20 copies per hour achieved with linear RCA. No amplification was seen in the absence of added primer. Analysis by agarose gel electrophoresis (Fig. 4, inset) confirmed the improvement in yield using exo-resistant primers and showed the average product length to be greater than 40 kb. These results are consistent with the observation that, in assays containing more than 0.3 units of φ29 DNA polymerase per nanogram of DNA using unmodified primers, the amplification yield was decreased (Fig. 4) and primers are degraded (data not shown).

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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.

DNA Sequencing by Using Template Amplified by RCA with Random Hexamer Primers

DNA from a saturated culture of XL1-blue transformed with a plasmid from a DNA library was amplified directly by using thiophosphate-modified random hexamers and φ29 DNA polymerase. Amplification products were treated with calf intestinal alkaline phosphatase to dephosphorylate remaining dNTPs. The sample was heated to inactivate the enzymes and used directly as a template for DNA sequencing (Fig. 5). The random hexamer used in the amplification does not need to be removed before sequencing, presumably because it does not anneal at the elevated temperatures used in cycle-sequencing reactions. The quality and read length of the sequence was indistinguishable from that obtained with ∼100 ng DNA template purified by standard methods. Similar results were obtained by using bacterial colonies in place of saturated cultures (data not shown).

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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.