M. Adams and R. Deadman
GE Healthcare, Piscataway, NJ, US
GE Healthcare offers a range of DNA amplification products to meet all genomic DNA and sequencing template preparation needs. A combination of these kits with the Genesis Freedom™ Robotic Sample Processor (RSP) from Tecan (Fig 1) creates a total solution for high-throughput DNA preparation.
Traditional DNA purification methods often use organic solvents, are difficult to automate, or produce variable yields. The minimal number of liquid transfers in the TempliPhi™ and GenomiPhi™ DNA amplification procedures are easy to automate, with each reaction producing the same amount of high quality DNA. TempliPhi DNA Amplification Kit and TempliPhi Large Construct DNA Amplification Kit are used to prepare small (plasmids/M13) or large (fosmids/BACs) circular DNA templates, respectively, for sequencing. GenomiPhi DNA Amplification Kit prepares genomic DNA from various sources that can be directly used for genetic analyses. The flexibility of the Genesis Freedom workstation also allows easy reaction setup for downstream applications such as sequencing or PCR.
TempliPhi DNA Sequencing Template Amplification Kit offers the simplest solution to plasmid DNA preparation. Microgram quantities of DNA can be prepared from picogram amounts of purified DNA, colonies, or bacterial cultures using Phi29 DNA polymerase and rolling circle amplification. This unique approach removes the need for culture growth, resulting in high quality DNA templates with less than 20 min of hands-on time.
Amplified DNA can be used directly for sequencing with any chemistry and sequencing platform. TempliPhi Large Construct Kit has been specifically optimized for amplifying large constructs such as fosmids or BACs for sequencing. Uniform quantities of DNA are amplified in all samples, eliminating the need for quantitation.
GenomiPhi DNA Amplification Kit representatively amplifies single- or double-stranded linear genomic DNA using Phi29 DNA polymerase to perform strand displacement amplification. Microgram quantities of representative DNA are produced from small amounts of starting material that can be used immediately in downstream applications.
For all kits, DNA amplification is achieved by combining the starting DNA with random hexamer primers, Phi29 DNA polymerase, dNTPs, and buffer components optimized for DNA synthesis. This reaction mixture is incubated overnight at 30 °C, during which time the available nucleotides are consumed and converted into high molecular weight fragment copies of the input DNA.
The amplification protocols are simple and allow straightforward automation. The Genesis Freedom series of RSPs from Tecan are ideal for automating DNA amplification reactions and downstream applications. The arm of the Tecan liquid handler is equipped with four or eight tips and can perform all required liquid handling steps with high sensitivity and accuracy. When the amplification reaction stops because the nucleotides in the reaction are exhausted, DNA yields are equivalent across all samples (Fig 2). Disposable tips eliminate cross-contamination concerns. With an eight-channel liquid handling arm, multiple 96-well plates of TempliPhi or GenomiPhi DNA amplification reactions can be prepared in half the time of manual pipetting. Whatever the post-amplification application (Fig 3), the open platform can be adapted. The Tecan Gemini software system allows easy protocol setup and operation of the workstation.
The combination of GE Healthcare’s DNA amplification products and Tecan’s Genesis Freedom workstation produces a high-performance platform for automating the labor-intensive steps traditionally associated with manual methods of DNA preparation. The simplicity of the protocols and the robotics system allow a short setup time. Amplification reactions produce consistent, high quality DNA across all samples, eliminating time-consuming quantitation and purification steps. DNA can be used directly in downstream applications with no further purification.
For more information, including protocols, please refer to the application note Automation of TempliPhi and GenomiPhi DNA Amplification Kits on the Genesis Freedom Robotic Sample Processor (63-0056-16) which is available at: www.amershambiosciences.com/promo_dm203
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