Abhay Patki shows how a novel amplification strategy provides a rapid, simple and versatile method for preparing DNA templates that can be used directly in cycle sequencing reactions with no need for purification. Its simple format eliminates the multiple steps of commonly used template preparation schemes and allows DNA template preparation and sequencing to be completed in a single day.
Traditional methods for preparing 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. They are also less than ideal for use in high-throughput sequencing laboratories where rapid, simple, low-cost procedures that can be automated are needed.
Preparing templates
TempliPhi Amplification Kits address these issues, preparing templates in foursix hours that can be directly used in sequencing the reactions using any chemistry and on any platform.
The TempliPhi technology uses rolling circle amplification (RCA) (Fig. 1) and Phi29 DNA polymerase licensed exclusively to Amersham Biosciences to amplify circular DNA for sequencing.
The protocol is simple, requiring less than 20 minutes of hands-on time to amplify 96 samples from bacterial colonies, while also eliminating the need for overnight cultures and template-specific preparation protocols.
By reducing the time, labour, and reagents commonly used in template preparation, the kits deliver a cost-effective method for preparing DNA of consistent quality and quantity.
RCA by Phi29 DNA polymerase
TempliPhi Amplification Kits have been developed specifically to prepare templates for DNA sequencing.
The method utilises bacteriophage Phi29 DNA polymerase to exponentially amplify circular DNA templates by RCA. Under isothermal conditions, the kits can produce microgram quantities of DNA from picograms of starting material in a few hours, eliminating the need for cell cultures and conventional plasmid or M13 preparations.
A number of characteristics make Phi29 DNA polymerase it an ideal choice for use in the TempliPhi protocol. Phi29 DNA polymerase is active at 30°C; consequently, amplification is performed in an incubator or heat block at this temperature, eliminating the need for a thermal cycler (although reactions can be conveniently performed in a cycler). Because of the enzyme's extraordinarily high processivity, a single binding event is capable of replicating more than
70 000 bases.
In four-six hours the enzyme can generate
35 µg of amplified DNA from as little as 1 pg of template DNA. Amplification errors are minimal as a result of the enzyme's high-fidelity and proofreading activity.
Phi29 DNA polymerase exponentially amplifies circular DNA templates under isothermal conditions by RCA. In the TempliPhi protocol, RCA (Fig. 1) is initiated when random hexamers bind at multiple locations on the template, thus priming DNA polymerisation by Phi29 DNA polymerase.
Synthesis begins around the circular template, with multiple priming occurring on new strands, as well as on the original template. The extending hexamers are eventually displaced at their 5'-ends upon completion of one revolution of the circularised template, and as polymerisation and displacement continue, a single-stranded product of complementary concatamers is generated.
Random hexamers bind to this product, again priming DNA synthesis at each site. Continued elongation and strand-displacement results in branching, and exposure of new recognition sites for the hexamers.
Eventually, strand-displacement causes the release of double-stranded DNA fragments from the parent template, and these fragments (1x, 2x, 3x to nx circle unit length) accumulate exponentially.
This geometric amplification, involving ever-increasing and self-propagating strand-displacement and DNA fragment accumulation, is called hyperbranching and generates up to 109 copies of each circular template. A demonstration of the degree of amplification possible is shown in
Fig. 2.
Starting material for TempliPhi
Suitable starting material for TempliPhi includes purified circular DNA or host cells containing the plasmid to be sequenced. Plasmids, M13 phage, or any circular DNA can be amplified; linearised and nicked DNA, however, should not be used with the kit.
The amplification 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 TempliPhi reaction.
While the kinetics of amplification may vary depending on the source of starting material, no differences are 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 (saturated cultures) and large amounts of agar can inhibit the TempliPhi reaction, small volumes of culture (0.252 µl) and a medium such as 1x LB with an appropriate antibiotic are recommended. The amount of agar carried over during colony or plaque picking should be minimised (Fig. 3).
The products of the TempliPhi reaction are high-molecular-weight, linear, double-stranded, tandem-repeated copies of the input circular template. When starting with M13 clones, the TempliPhi product is double-stranded DNA and can be sequenced with forward and reverse primers.
The amplified DNA is compatible with both DYEnamic ET terminator and ABI PRISM BigDye sequencing chemistries and can be used directly for cycle sequencing without any purification on all MegaBACE and ABI PRISM DNA analysis platforms. For templates requiring the use of more than 500 ng in the sequencing reaction, a precipitation may be helpful.
While amplification of the control DNA is complete in four to six hours, incubation can be continued for up to 18 hours without compromising yield or quality of amplified DNA (Fig. 4). The reaction simply ceases to generate more product when the nucleotides are consumed.
Simple protocol
The simple TempliPhi protocol makes it extremely easy to use and requires minimum hands-on time. Because DNA is amplified from colonies and plaques, overnight cultures are not required, saving costs in time, labour, and reagents. Throughput is greatly increased since templates can be prepared and sequenced in one day.
In addition, the TempliPhi protocol is not vector-discriminating. Amplification yields from plasmids and phage are equal, so individual template-specific protocols are not required. Plasmid copy number does not matter, and even single- or low-copy vectors can be used.
Yields from the reaction are independent of the amount of starting material. In fact, if the reaction is allowed to go to completion, experiments have shown that the final yields of amplified DNA are the same even with 10-fold differences in the amount of starting material (Fig. 5D).
By using random hexamers for priming in the amplification, this protocol eliminates the requirement for custom primers, thus providing versatility and broad applicability for any circular template.
Amplified DNA generated by TempliPhi is used directly for sequencing with no need for purification or quantitation. Because amplifications are uniform, sequencing failures are avoided, and sequencing pass rates and read-lengths are improved compared with samples prepared by traditional methods.
Amplified DNA can be archived, so space and long-term storage costs affiliated with storage of glycerol stock cultures are eliminated.
Typical results
The TempliPhi reaction is highly efficient in amplifying DNA, even from picogram quantities of starting material. Fig. 5C, lane a1 pg' shows that a significant amount of amplified product is generated from as little as 1 pg of pUC18 when the reaction is allowed to go to completion.
Some amplified product can be observed after only four hours with input amounts of 415 pg (Fig. 5B, lanes 4 pg and 15 pg). When amplification yield was quantified, reactions using only 62 pg of template yielded 3.2 µg of amplified product after 12 hours (Fig. 5C, lane 62 pg).
Uneven amounts of starting material appear to have little effect on yields, especially if the amplification is allowed to go to completion.
In the experiment shown in Fig. 5D, TempliPhi reactions were prepared using either an entire bacterial colony or only one-tenth of a colony. Even with a 10-fold difference in the amount of starting material used, amplifications are uniform within four hours and essentially complete since no significant differences were observed between four and twelve hour incubations.
The pattern in lane 0 for the 12 hour incubations is common for negative control reactions (no input DNA). This is expected in highly-sensitive, exponential amplification reactions where even primers can serve as amplification substrates. These non-specific amplicons are not made in the presence of input circular templates.
One of the greatest benefits of the TempliPhi protocol is the direct use of amplified product in sequencing reactions with no need for template purification. u
Abhay Patki is Marketing Manager, Genetic Variation, with Amersham Biosciences, Piscataway, New Jersey, USA. Thanks are due to J Nelson, T Giesler, YC Cai, and C Palaniappan for providing data for this article, which is adapted from Life Science News issue 9 and used with kind permission of Amersham Biosciences Corp (C) 2001 .
Order numbers for the kits are as follows: TempliPhi 100 Amplification Kit (100 reactions), 25-6400-10; TempliPhi 500 Amplification Kit (500 reactions),
25-6400-50; TempliPhi DNA Sequencing Template Amplification Kit (10 000 reactions),25-6400-01.
