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Preparing plasmid templates for DNA sequencing is the most time-consuming step in the sequencing process. Current template preparation methods rely on a labor-intensive, multistep procedure that takes up to 24 h and produces templates of varying quality and quantity. The TempliPhi™ DNA Sequencing Template Amplification Kit eliminates the requirement for extended bacterial growth prior to sequencing and saves laboratory personnel hands-on time by eliminating the centrifugation and transfer steps currently required by older preparatory methods. In addition, costly purification filters and columns are not necessary, as amplified product can be added directly to a sequencing reaction. Starting material can be any circular template from a colony, culture, glycerol stock, or plaque. Based on rolling circle amplification and employing bacteriophage Phi29 DNA polymerase, the method can produce 3–5 μg of template directly from a single bacterial colony in as little as 4 h. Implementation of these procedures in a laboratory or core sequencing facility can decrease cost on tips, plates, and other plasticware, while at the same time increase throughput.
Core sequencing facilities and genome centers devote considerable time and expense to prepare DNA templates for sequencing. Currently, the most common methods of plasmid DNA preparation are solid phase reversible immobilization (SPRI) bead technology,1 commercial plasmid kits, or homemade recipes. All of these methods rely on overnight growth of bacterial hosts to amplify the amount of cloned DNA. Popular commercial kits are based on traditional alkaline lysis procedures2 employing filters and separation media that require a vacuum manifold and considerable amounts of reagents and plasticware. The TempliPhi™ DNA sequencing template amplification kit is a novel product that does not require culturing of bacteria or centrifugation steps to prepare sequencing template. TempliPhi amplifies circular DNA directly from colonies, plaques, or microliter volumes of bacterial culture or glycerol stock. Amplified template is directly sequenced without additional purification, thereby saving on the cost of commercial purification columns and plates. Since TempliPhi amplification is isothermal (30°C), the reaction can be performed in an oven, heat block, water bath, or thermocycler.
Early bacteriophage studies revealed that replicating phage DNA in infected bacteria was larger than in phage particles3,4 because of the formation of concatamers5 of genomic phage DNA. Since phage DNA is circular, tandem repeats are produced by rolling circle replication (RCR), in which the phage DNA polymerase replicates and subsequently displaces the newly made strand.6,7 Rolling circle amplification (RCA) is a technique that uses RCR to amplify circularized DNA with a sequence specific primer and a strand displacing DNA polymerase.8,9 TempliPhi is based on RCA using bacteriophage Phi29 DNA polymerase10 and random hexamer primers.11 Random hexamers bind to the denatured circular template allowing Phi29 DNA polymerase to initiate multiple amplification events (Fig.1(Fig.1).). The inherent strand-displacement activity of the enzyme displaces the 5′-ends of downstream strands. As DNA synthesis and displacement continue, the enzyme produces single-stranded, complementary concatamers of the circular template (Fig. 22).). Priming and polymerization directed by the displaced strands produces double-stranded DNA. Approximately 80% of the total DNA produced is double stranded and can be digested by restriction enzymes and cloned.11 Nucleotides in the TempliPhi premix fuel the reaction to produce as much as 5 μg of DNA from as little as 1 pg of template in 4 h.
The rate of the strand synthesis by Phi29 DNA polymerase is approximately 50 nucleotides/second, due in part to the enzyme’s high processivity.12 Phi29 DNA polymerase is able to incorporate greater than 70,000 nucleotides during a single binding event without the aid of accessory proteins. A concern with any method that involves DNA amplification is the insertion of an incorrect nucleotide that changes the DNA sequence. With the polymerase chain reaction, errors are introduced into a synthesized fragment and propagated through amplification by the DNA polymerase. Phi29 DNA polymerase has a proofreading activity with an error frequency of 1 × 10− 6–10− 7.13 This error rate is comparable to that of Pfu (7 × 10− 7) and other proofreading DNA polymerases14 and is significantly lower than that of Taq DNA polymerase (2 × 10− 4).15 Although an error by Phi29 DNA polymerase could occur, the error would not be exponentially amplified as in PCR, since each repeat in the DNA concatamer represents a copy of the plasmid directly from the original template. Once an error has been introduced, all additional amplification of that single tandem repeat would also have the error. However, when random hexamers were used in RCA of 1 ng M13 DNA, a 375-fold amplification of DNA was achieved compared with M13 amplified with a site-specific primer.11 This suggests that many replication sites occur with the use of random hexamers and an error on one tandem repeat could never be amplified to high enough numbers to be seen in an electropherogram of the DNA sequence. Although very rare, a clone from TempliPhi-prepared DNA could contain the error; however, multiple coverage of the clone library would easily reveal the misincorporated base.
A 384-well plate of glycerol stocks from a whole genome shotgun library of the bacterium Thermotoga subterannea (Tsu) was used in this study. Host Escherichia coli (E. coli) strain XL-1blue (Stratagene, La Jolla, CA) were transformed with pUC19 vector containing 2 kb Tsu genomic DNA inserts. The TempliPhi DNA Sequencing Template Amplification Kit and DYEnamic™ ET Terminator Cycle Sequencing Kit were from Amersham Biosciences (Piscataway, NJ). The PicoGreen™ dsDNA Quantitation Kit was from Molecular Probes (Eugene, OR). The AccuGENE™ 1X TE Buffer (0.01M Tris, 0.001M EDTA, pH7.4) was from BioWhittaker Molecular Applications (Rockland, ME).
Employing a Quadra96™ SV (Tomtec, Hamden, CT), 10 μL of Tsu glycerol stock was diluted 1:10 in 90 μL of 1X TE. Ten microliters of each diluted stock was then transferred to the corresponding well of a new 384-well plate and incubated at 95°C for 5 min to partially lyse cells and release and denature the plasmid. After cooling to room temperature, 10 μL of TempliPhi Premix was added to each well and incubated for 4 h at 30°C. Incubation at 95°C for 3 min inactivated the Phi29 DNA polymerase activity. Two microliters of each amplified template were indexed from the 384-well plate to four 96-well plates containing 18 μL of sequencing master mix per well (8 μL of DYEnamic ET terminator premix and 5 pmoles −40 primer in dH2O). Reactions were cycled at 95°C 20 s, 50°C 15 s, and 60°C 60 s 25 times, ethanol precipitated, and resuspended in 10 μL of MegaBACE™ loading buffer. The four plates were analyzed by capillary electrophoresis on a MegaBACE 1000 DNA Analysis System (Amersham Biosciences) and on an ABI PRISM™3700 DNA Analyzer (Applera Corp., Foster City, CA).
TempliPhi reactions were performed as above, except the plate was incubated for 16 h at 30°C and then denatured at 95°C for 3 min. The completed TempliPhi reactions were indexed from 384-well format into four 96-well plates and diluted 1:4 (60 μL 1X TE to completed reactions, VT: 80 μL). From the 1:4 dilution, 6 μL of TempliPhi product were taken from each well and added to sequencing reactions containing 8 μL of DYEnamic ET terminator premix and 5 pmoles −40 primer in 14 μL dH2O. The sequencing reactions were cycled, cleaned, and resuspended as described in the previous section and sequenced on an ABI 3700.
A 10-μg/mL λ DNA dilution from the 100 μg/mL stock provided in the PicoGreen assay kit was prepared by making a 1:10 dilution in 1X TE. The λ dilution was used for the preparation of two sets of assay standards per plate (range 0–800 ng). One hundred microliters of each concentration standard were assayed. Ninety-five microliters of 1X TE were dispensed into four Costar™ 96-well flat-bottom assay plates (Corning Corp., Corning, NY). Five microliters of 1:4 diluted 16 h TempliPhi product from the previous section were added to the TE in each corresponding well of the assay plate. One hundred microliters of a 1:25 dilution of PicoGreen reagent in 1X TE were added to each well. The relative fluorescence of each plate was measured on a HTS 7000 Plus Bioassay Reader (Perkin Elmer, Boston, MA). Excitation was set at 485 nm and emission was measured at 535 nm.
Sequencing reactions of TempliPhi template were analyzed on both the MegaBACE 1000 and the ABI 3700 sequencing instruments. Each processed trace was scored by PHRED16,17 and a “successful” sequence was defined as being at least 300 bases at PHRED 20 cutoff. Similar results were obtained on both instruments (Table 11).). Sequencing of TempliPhi products incubated for 4 h had at least 92% pass rates, greater than 500 bp average read lengths and over 200,0000 total PHRED 20 bases from the 384 samples. For a single 96-well plate, the total time from starting the experiment to having analyzed sequence was approximately 9 h.
Normalization of template concentration can significantly increase sequencing read lengths and pass rates with capillary electrophoresis instruments since consistency is much more important than with slab gel instruments. Capillary instruments are loaded via electrokinetic injection, the efficiency of which is influenced by the composition of the sample. Although TempliPhi amplification can produce sufficient DNA to sequence after 4 h, nucleotides are typically not exhausted until after 6 h and the well-to- well consistency of yield may not be as high as can be achieved.18 Extending the incubation time of the TempliPhi reaction from 4 to 16 h helps normalize the yield, ensuring that all samples proceed to completion and correcting for variable input samples. This also eliminates the need for measuring the concentration by A260 on a plate reader and subsequent normalization steps. Using the TempliPhi reactions of the Tsu library plate incubated at 30°C for 16 h, the concentration of each sample was measured by a PicoGreen assay on a plate reader. The average DNA concentration was approximately 3.6 μg, with 73% of the samples yielding between 3 and 5 μg of TempliPhi product (Fig. 33).). An equivalent of 1.5 μL or 375 ng of each TempliPhi reaction was sequenced with DYEnamic ET terminators and analyzed on an ABI 3700. In this study, the advantage of increased TempliPhi incubation time was apparent. The pass rate increased by 5%, resulting in 20 additional samples with greater than 300 PHRED 20 bases and increasing total PHRED 20 bases by 12%.
The goal of this study was to demonstrate the advantages of TempliPhi amplification over present template preparations (Fig. 44).). Conventional alkaline lysis requires cultivation of individual cultures in 96-well culture boxes containing 1 μL of growth media for approximately 20 h. Cells are pelleted by centrifugation and resuspended before lysis in a solution of sodium hydroxide and sodium dodecylsulfate. After neutralization of the lysate with potassium acetate, the plates are again centrifuged to pellet cellular debris. The supernatant is transferred to a microtiter plate where it can be ethanol precipitated and resuspended in water or buffer or, as in most commercial preps, added to a purification plate. DNA is bound to a solid matrix within the purification plate and washed with ethanol to remove salts and impurities. This typically requires the use of a vacuum manifold to apply pressure for liquid removal or centrifugation. A short drying time is required before water is added to elute the bound plasmid DNA into collection plates for storage. At least 1 L of liquid waste is generated with a 384-well format based on spent media, 100 μL of each lysis reagent, and ethanol. Plasticware waste includes four 96-well growth boxes (that could be reused if washed and autoclaved), four purification plates, and four collection plates. If template is to be stored in a 384-well format, then individual preps have to be indexed into a 384-well plate. Otherwise, template storage will be in four 96-well plates, which can eventually put a constraint on freezer space.
In production sequencing facilities, implementation of TempliPhi has not only increased pass rates and read length but also lowered the cost per base sequenced by reducing staff and floor space dedicated to template preparation.19 Individual colonies can be picked from plates by robot directly into a 384-well plate of buffer. For a large number of plates, ovens can be used to perform incubations, and either a technician or an automated liquid delivery system can pipette the TempliPhi Premix into reaction plates. There is no waste stream. A single 384-well plate is used in the process that also stores the amplified template. Since the TempliPhi product is produced directly from culture material, liquid reagent waste is not generated if colonies or glycerol stock is the starting material. Since DNA in a TempliPhi reaction is amplified until nucleotides are exhausted, template concentration in a multiple reaction plate is effectively normalized to 3–5 μg (150–250 ng/μL). The uniform concentration of template in a 96- or 384-well plate is advantageous for sequencing with capillary electrophoresis instruments where excessive template concentration can result in short sequence reads due to capillary blockage and low concentration to short reads due to low signal.
Several applications using the TempliPhi process are available to core facilities where as much as 66% of templates sequenced are plasmids.20 Many sequencing failures in core labs result from poor quality or quantity of customer DNA. With TempliPhi, template concentration of submitted samples can be uniformly amplified to 3–5 μg. Since amplification occurs with as little as 1 pg of template, TempliPhi Denature Buffer and Premix can be added to the original tube of a failed sample to produce template for another round of sequencing. Using TempliPhi, the turn-around time from a colony or culture to a sample ready to run on a sequencing instrument is 6 h with minimal manipulation, compared with more than 24 h using standard alkaline lysis preparations of overnight cultures. The ease and speed of the DNA amplification allows template preparation to be offered as a service to customers. Culture or colonies can be submitted to the core facility where template DNA can be produced in 4 h with very little hands-on time.