Phi29 DNA Polymerase-Based DNA Amplification

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By Abhay Patki, Shinjae Rhee, J. Anthony Mamone, and John R. Nelson

The first phase of the international effort to determine the entire sequence of the human chromosome set is now complete. Scientists involved in the Human Genome Project plan to finish the gaps in the human genome sequence soon, along with completing the database of the most common sequence variations that distinguish one person from another. This knowledge base, freely available to any interested person over the Internet, will revolutionize the fields of biology and medicine. This will also lead to functional genomics research that will continue to produce valuable data relating genes to diseases.

As a result of these new research areas, as well as additional genomes that need to be sequenced, researchers are performing similar numbers of sequencing reactions compared to one to two years ago. With the ongoing need for DNA sequencing, researchers are looking for alternatives to drive down sequencing costs without negative effect on sequence quality. More efficient and reliable methods are desired to prepare DNA templates to get longer readlengths, avoid failed reactions, and reduce reagent and labour costs.

Traditional methods to prepare DNA templates for sequencing require overnight growth of cultures followed by multiple steps aimed at isolating and purifying plasmid or phage DNA for use in sequencing reactions. These protocols are often laborious and expensive.

The TempliPhi™ technology uses Phi29 — the DNA polymerase from Bacillus subtilis — and rolling circle amplification (RCA) technology (1,2) to amplify circular DNA templates for sequencing (Fig. 1) as an alternative to more classical plasmid preparations. The protocol is simple, requiring less than 20 minutes of hands-on time to amplify 96 samples from bacterial colonies (Table 1A, 1B). The kit potentially eliminates the need for liquid cultures and subsequent template-specific preparation protocols. By reducing the time, labour and reagents commonly used in template preparation, the kit delivers a cost-effective method for preparing DNA of consistent quality and quantity. Use of the kit can also result in higher success rates and longer readlengths of sequencing reactions.

Rolling Circle Amplification by Phi29 DNA Polymerase

Phi29 DNA polymerase, first described by M. Salas and co-workers (3-5), is a highly processive enzyme that incorporates at least 70,000 nucleotides per binding event (3). The enzyme does not pause when encountering a double-stranded template, performing strand displacement synthesis in the absence of added helicase or DNA binding proteins. Its rate of nucleotide (nt) incorporation is 25-50 nt/second, and we have observed linear kinetics for two days using this enzyme (data not shown). The polymerase has an associated 3’-5’ exonuclease “proofreading” activity (4), and has a reported error rate of 5 x 10-6 (5), about 100-fold lower than that of Taq DNA polymerase (6). Results using TempliPhi kit-amplified DNA confirmed the low error rate for Phi29 DNA polymerase (7). In addition, sequence analysis has been performed on numerous known plasmids and bacteriophages that have been amplified using the kit (>20,000 nt of sequence, >10,000-fold amplification) with no amplification errors detected.

Amplification Kinetics

Amplification using TempliPhi kit starts with nanogram amounts of double-stranded DNA. RCA is initiated when random hexamers bind at multiple locations on the circular template and are extended by Phi 29 DNA polymerase. The extending fragments from hexamers are eventually displaced at their 5’-ends, and as polymerization and strand displacement continue, single-stranded products of complementary and tandemly repeated copies are generated. New random hexamers bind to this single-stranded displaced product, again priming DNA synthesis at multiple sites. Continued elongation and strand displacement result in branching and exposure of new binding sites for the hexamers. This exponential amplification, involving ever-increasing and self-propagating strand displacement and DNA fragment accumulation, generates up to 107 copies of each circular template. A demonstration of the degree of amplification possible is shown in Figure 2. There is typically a brief lag followed by rapid synthesis of product until the supply of nucleotides is exhausted.

Although the standard protocol uses 1 ng of purified DNA as the input template for optimal kinetics, we have found that as little as 0.1 picogram of pUC18 DNA can be amplified efficiently (data not shown). As long as the amount of target template is > 0.01 pg, the target will out-compete non-specific amplification by the random hexamers. The result of non-specific amplification can be seen in lanes containing no input DNA.

Starting Material for DNA Amplification

The plasmid DNA present in bacterial colonies can be used directly as a template for TempliPhi amplifications. This is of great convenience as clones can be screened at the colony stage without having to wait for liquid cultures to grow and without the need for DNA plasmid preparation. Purified circular DNA can also be used. We have also tested amplification of lower copy number plasmids including pBR322, pBeloBAC11 and pBACe3.6. In general, 1 to 100% of the contents of a colony should be used for amplifications of these low-copy plasmids and performed for eight to 16 hours at 30ºC to achieve maximal yields. The TempliPhi reaction is performed at 30ºC, consequently, relatively inexpensive incubators or heating blocks can be used (although reactions can be conveniently performed in a thermal cycler). The success rate and efficiency of amplification among various DNA templates are similar, even for GC- and AT-rich templates. Microlitre volumes of saturated broth cultures or colonies/plaques picked from agar plates can be added directly to the reaction. While the kinetics of amplification may vary depending on the source of starting material, no differences were observed in DNA quality, and sequencing results are similar regardless of whether broth or plate cultures are used. Because some components of used culture media (found in saturated liquid cultures) and large amounts of agar can inhibit the amplification reaction, small volumes of liquid culture (0.25 to 2 µL) and a medium such as 1X LB with an appropriate antibiotic are recommended (7). The amount of agar carried over during colony or plaque picking should be minimized.

Liquid cultures provide good templates for TempliPhi reactions. This is particularly useful to researchers who prefer to grow small overnight cultures for archival storage. There is no significant difference in amplification yield when using 0.001 to 1 µL of a saturated overnight E. coli culture as initial template for amplifications. We have obtained good sequencing data by amplification of a culture dilution that contains between one and 10 E. coli cells (7). For low copy number plasmids, as little as 0.03 µL of overnight shaking culture contains sufficient template for amplification (data not shown). Again, as described for colonies, longer incubation at 30ºC for these types of vectors is beneficial.

Among other possible starting materials, even a small portion of glycerol stock can be amplified, as well as plasmid DNA stored on FTA™ paper (data not shown). This DNA can be stored at room temperature. M13 plaques are also excellent starting materials for amplification. Interestingly, since the products from the amplification of M13 single-stranded DNA circles are double-stranded, both strands of M13 clones can be sequenced.

Typical Sequencing Results

An additional benefit of the above protocol is the ability to use the amplified product directly in sequencing reactions without requiring template purification. A typical sequencing result on MegaBACE™ obtained without post-amplification purification is shown in Figure 3. The amplified products gave excellent results with BigDye™ sequencing kits and on any ABI PRISM ABI DNA sequencing instruments.

One of the major reasons for sequencing failure is DNA concentration variability. Perhaps one of the most beneficial observations when using the kit method is the normalization of DNA concentrations. Since amplification reactions continue until the nucleotide pool is depleted, each reaction contains a similar final concentration of sequencing template (100 to 250 ng/µL). There is typically a significant increase in success rates as a result of this normalization.

Other Applications

DNA amplified using the TempliPhi kit can be used for restriction digestion, ligation and cloning. Small amounts of vector DNA can be amplified and after standard restriction digestion and ligation, can be used for transformation, yielding similar transformation efficiencies to that of traditionally purified plasmid transformations (data not shown).

Bacterial artificial chromosome (BAC) DNAs are difficult to purify, amplify and sequence. This DNA can be amplified using the kit either from small crude DNA preparations obtained from typical alkaline lysis preparations or directly from lysate of colony, and can be successfully sequenced (Fig. 4).

References
1) Lizardi P.M. et al. 1998. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat. Genet. 19:225-232.

2) Dean F.B. et al. 2001. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11:1095-1099

3) Blanco L. et al. 1989. Highly efficient DNA synthesis by the phage Phi 29 DNA polymerase. Symmetrical mode of DNA replication. J. Biol. Chem. 264:8935-8940.

4) Blanco L. and M. Salas. 1996. Relating structure to function in Phi29 DNA polymerase. J. Biol. Chem. 271:8509-8512.

5) Esteban J.A. et al. 1993. Fidelity of Phi 29 DNA polymerase. Comparison between protein-primed initiation and DNA polymerization. J. Biol. Chem. 268:2719-2726.

6) Dunning A.M. et al. 1988. Errors in the polymerase chain reaction. Nucleic Acids Res. 16:10393.

7) Nelson J. R. et al. 2002. TempliPhi, Phi29 DNA polymerase based rolling circle amplification of templates for DNA Sequencing. Biotechniques, SNP supplement. June 2002: pg 44-47.

Acknowledgement
We would like to thank Fuller C., Gielser T., Farchaus J. W., Cai Y. C., Sundaram S. T., Ortiz-Rivera M., Hosta L. P., Reddy P., Hewitt P., and C. Palaniappan for help with this manuscript.