Extraction/purification of nucleic acids.
Plant geneticists of Montpellier, working on Mediterranean and tropical plants, used many DNA extraction protocols adapted to various situations. A compilation and critical analysis of protocols for plant DNA extraction have been conducted, in order to compare to main technics: SDS/AcK protocol and CTAB/Chloroform protocol. We chose to focus on the development of optimized protocols that uses the principles of method SDS/AcK without using organic solvent and purifying DNA by capture on silica.
We have developed a set of protocols for plant DNA extraction written according to the standards of Quality Assurance in Research adaptable to a great diversity of plant samples and able to produce DNA of very high quality. The protocols are organized into a decision support that allows us to choose the most suitable one. To date, 18 specific DNA extraction protocols have been developed and validated. It would be possible for a person to extract in a day, high purity DNA from 400 samples previously crushed. An accurate assessment of the various total plant RNA extraction protocols is underway. The quality of RNA extracts and conditions of use (convenience, danger, etc.) are taken into account simultaneously.
Secure storage of DNA.
The DNA extracts should be stored to ensure maximum safety. We have conducted a comparison of different conditions of storage of DNA: for average duration at -18°C and for a long term at room temperature.
- For regular use of DNA or for storage of short period (<5 years), we use specific polypropylene tubes (0.6 to 2.2 ml) and screw caps (Abgene or Matrix) that avoid any risk of evaporation and contamination, ordered in dedicated racks of 48 or 96 places. They are stored in secure freezers (-20°C) (ARCAD SP6 budget). Mechanisms of degradation of frozen DNA are already described. They only occur during multiple episodes of freezing/de-freezing that we will reduce and manage.
- For very precious samples and for long term storage (> 5 years), we plan to use the system of protection of DNA under controlled atmosphere in airtight metal capsule (Imagène, Process for long term preservation of DNA at room temperature, www.imagene.fr ) compatible with a storage at room temperature. It is the only system which can guarantee the integrity of the DNA molecule stored for a minimum period of 100 years
Management of DNAs in the DNA bank
The chain of custody between samples of plants and DNA samples, using bare-coded labels and appropriate readers is particularly careful. The tubes or storage capsules are barcoded (2D DataMatrix). The building of a specific database for the DNA samples, linked to the database of plant genetic resources is underway. A first DNA bank containing 40,000 DNA samples from different plants (wheat, corn, olive, fig, apricot and grape) is being set up.
Transfer of DNA / relationships with partners
Reception of DNA from foreign partners, brought into conformity with the standards of the DNA bank. Re-purification protocols by capture on silica of DNAs from different origins and purified using different techniques are validated.
Most MTA applying to seeds can be generalized to transfer DNA or any unmodified derivatives when the material is not subject to any industrial or intellectual property (exchange through the International Treaty on Plant Genetic Resources for Food and Agriculture, for example). For all other cases, a specific MTA should be used. A reflection concerning the regulatory conditions to transfert nucleic acids of plants is under progress at national scale.