Whatman
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Florham Park, NJ, 07932
Website: http://www.whatman.com
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Collection, Transport, Purification And Storage Of PCR-ready Plant DNA
by Tracey Long, Renate Karle, Frank Igoe and Martin Smith
Introduction
The number of PCR-based methods for plant molecular research has increased dramatically. Application of these methods alone, or in combination with other tools, range from marker assisted selection, molecular mapping, varietal identification and phylogeny research to genomics and chip technology. PCR is also routinely used in detection of genetically modified (GM) organisms. This screening can be done at various stages starting with testing of callus cells, then small seedlings and finally with propagated plants. We describe a simple, non-organic matrix-based DNA purification procedure using FTA™ card technology (Whatman, Inc., Clifton, NJ) and demonstrate it’s suitability in a number of PCR based applications.
FTA technology
FTA technology offers a system that protects the nucleic acids of a sample as soon as it is applied to the coated matrix. The patented FTA formula lyses cell membranes on contact so that nucleic acids are immediately immobilized and stabilized within the matrix. The coating contains denaturing agents, a chelating agent and a free radical trap, which together permit long-term storage of double-stranded DNA at room temperature without degradation. The coating is non-toxic and non-hazardous, thus the cards are safe to handle. Pathogens are inactivated and the nucleic acids are protected from environmental assault (Seah and Burgogne, 2000).
Sample collection
Both plant material and plant homogenate are suitable for use with FTA. The method of sample application depends on whether the sample is solid or liquid. Liquid plant homogenate samples are directly pipetted onto the matrix. These samples are applied evenly over the defined sample area to avoid overloading the matrix in any one location. Collection of plant tissue DNA is performed by physically crushing the tissue on the card, a simple procedure that can be done in field conditions. Sample transfer can be checked by removing the tissue to visualize the matrix; if required, the sample is reapplied with increased pressure. The use of a blunt instrument such as a pestle to apply sufficient pressure can aid good cell transfer (Lin et al., 2000). In all cases, any residual tissue is discarded and spotted cards are dried at room temperature prior to storage or analysis.
Sample storage
Once dry, samples are either purified immediately or stored in a dark environment at room temperature. The chemical coating that is impregnated within the FTA card inactivates pathogens, protects the DNA from degradation and allows the cards to be stored at room temperature until ready to be tested or for more extended periods of time. DNA has been stored and successfully amplified over 14 years later. For such long-term storage a desiccant pouch is recommended.
Sample purification
The following is the general outline of the FTA purification protocol:
The sample is applied to the FTA Card — SPOT
A disk of the FTA matrix is removed from the card — PUNCH
The punched disks are washed, rinsed and dried — PURIFY
Amplification
To prepare the sample for PCR, disks (1.2 or 2 mm) are punched out from the applied sample spot and transferred to individual amplification tubes. Disks are washed 3 times with 200 ml FTA Purification Reagent (non-toxic and nonorganic) and twice with 200 ml TE-1 Buffer. Wash solutions are removed and discarded after each wash. Disks are dried at room temperature for 1 hour or at 56 C for 10 minutes. DNA remains bound to the matrix and the small disk (1.2- or 2-mm in diameter) provides enough DNA to be used as a template for PCR analysis. Double stranded DNA is immobilized within the matrix and hence cannot be quantified prior to PCR. For other analyses requiring DNA in solution or DNA quantitation, GenSpin Plant Purification kit columns incorporate the same FTA technology. These columns use a different matrix, which facilitates the recovery of PCR-ready DNA in solution while having all the inactivation and protection properties of FTA Cards. If large amounts of liquid DNA are required, samples collected on the cards make templates for use in whole genome amplification.
PCR procedure
PCR master mix is added directly to each tube and amplification performed as normal. A 1.2 mm disk was used for 5-15 ml reactions, and a 2 mm disk for 10-50 ml reactions. Volumes were adjusted according to the sensitivity, type of the assay, or predicted DNA concentration in the samples.
Applications and results
Plant samples collected on FTA Cards have been used for a range of PCR based applications (Table 1). Examples are shown in Figures 1-6. Collected DNA samples can be tested right away or after storage for several months at room temperature.
High throughput DNA purification of archived samples
Whatman has automated purification of DNA stored on FTA Cards using a Beckman Coulter Biomek® 2000 Liquid Handler (Pierce et al., 2002). Processing takes approximately one hour for 96 samples. Resultant DNA is suitable for PCR-based downstream applications.
Conclusion
Suitable for use with a range of sample types, FTA cards provide a pure template from small sample volumes for analyses requiring single and double stranded DNA. Genomic DNA remains immobilized, enabling successive amplifications to be performed on the same disk (Del Rio et al, 1996). DNA integrity is maintained for years at room temperature. Cards are safe to handle pre and post sample application. The solid phase format enables out of lab collection and simplifies DNA storage and shipping. Whatman, Inc.
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