DNA banking for plant breeding, biotechnology and biodiversity evaluation

Abstract

The manipulation of DNA is routine practice in botanical research and has made a huge impact on plant breeding, biotechnology and biodiversity evaluation. DNA is easy to extract from most plant tissues and can be stored for long periods in DNA banks. Curation methods are well developed for other botanical resources such as herbaria, seed banks and botanic gardens, but procedures for the establishment and maintenance of DNA banks have not been well documented. This paper reviews the curation of DNA banks for the characterisation and utilisation of biodiversity and provides guidelines for DNA bank management. It surveys existing DNA banks and outlines their operation. It includes a review of plant DNA collection, preservation, isolation, storage, database management and exchange procedures. We stress that DNA banks require full integration with existing collections such as botanic gardens, herbaria and seed banks, and information retrieval systems that link such facilities, bioinformatic resources and other DNA banks. They also require efficient and well-regulated sample exchange procedures. Only with appropriate curation will maximum utilisation of DNA collections be achieved.

Appendices

Appendix 1

Protocol for DNA extraction using hot CTAB

The following protocol, (adapted from Doyle and Doyle 1987), will work well for a wide range of plant material and is particularly well suited to low sample throughput (typically 8–32 samples per day). If a large number of samples is to be extracted, then several improved grinding methods have been developed to speed up extractions. For example, Karakousis and Langridge (2003) used liquid nitrogen and ball bearings in a high throughput plant DNA extraction process. Other methods have used glass beads in microtitre plates (Steiner et al. 1995). Several commercially available kits allow partial or full automation and higher throughput. For example, McGrath et al. (2006) successfully developed a high throughput extraction protocol that used plant material ground with a mill (Retsch bead mill) or pestle and mortar and the magnetic bead-based method (Qiagen MagAttract Plant DNA core kit) that had been modified for use on a Hamilton MicrolabStar robotic system.

Materials

2× CTAB buffer, (100 mM Tris–HCl pH 8.0 (use Tris base and set pH using HCl); 1.4 M NaCl; 20 mM EDTA (Na ethylene-diamine-tetra-acetate); 2% CTAB (hexadecyl-trimethyl-ammonium bromide, w/v)

CI, 24:1 chloroform:isoamyl alcohol

Method

Caution: gloves should be worn at all times.

• 1. Preheat 5 ml of 2× CTAB extraction buffer and 20 μl mercapto-ethanol (in a 12 ml chloroform-resistant capped centrifuge tube) and a pestle and mortar at 65°C in a water bath.

• 2. Weigh out approximately 0.3–0.5 g of fresh leaf or 0.05–0.1 g dry leaf (or other plant tissue).

• 3. Cut leaf into small pieces using scissors or razor blade.

• 4. Grind leaf material in the pre-heated mortar using a small amount of extraction buffer (sterile sand may be added to aid grinding or liquid nitrogen used prior to grinding).

• 5. Add the remaining buffer, grind further, and swirl to suspend the slurry.

• 6. Pour the slurry back into the 12 ml centrifuge tube. Screw on the lid and then incubate the mixture at 65°C for at least 10 min with occasional mixing.

• 7. Add 5 ml of CI. Replace the lid and mix. Release the lid briefly to release gas and then tighten the lid.

• 8. Place on the shaker in a horizontal position for approximately 30 min.

• 9. Centrifuge the tube at 4,000 relative centrifugal force (rcf) for 10 min.

• 10. Gently remove the tube from the centrifuge, taking care not to disturb the separation. Remove the aqueous (upper) phase containing the DNA, using a transfer pipette, into a 50 ml conical-base tube. Ideally, the upper phase will be clear and colourless, but this is often not the case and does not interfere with the latter stages of the protocol.

• 11. Add an equal volume of isopropanol and invert the tube gently to precipitate the DNA. You may see the DNA precipitate at this stage.

• 12. Place the sample into the −20°C freezer to further precipitate the DNA (sometimes it is necessary to leave the sample overnight or longer).

• 13. Centrifuge the sample at 2,000 rcf for 5 min to pelletise the DNA.

• 14. Pour off the supernatant and add 1.5 ml of the wash buffer. Mix gently.

• 15. Centrifuge the sample at 2,000 rcf for 3 min to pelletise the DNA once more.

• 16. Pour off the supernatant and then gently place the tube upside down for 5 min on a paper towel to let the excess wash buffer drain away.

• 17. Turn the tube the right way up and let the pellet dry further for about 20 min (it is important to remove all traces of ethanol).

• 18. Dissolve the pellet in 0.5 ml of TE buffer (considerable mixing with a transfer pipette may be necessary to dissolve the pellet).

• 19. Transfer the DNA solution from the 50 ml tube into the labelled 1.5 ml centrifuge tube and store DNA in a freezer until required (preferably at −80°C).

The DNA is often of sufficient purity for many applications, but samples can be purified further if necessary using caesium chloride gradient centrifugation, genomic DNA purification kits, or selective binding of DNA to a silica matrix in the presence of a chaotrope (see Appendix 2).

Appendix 2

DNA purification

Crude purification can often be achieved by precipitation/washing (e.g. the ethanol washing steps of Appendix 1) and may yield DNA of sufficient purity for the required application. However, such DNA may need to be further purified. One of the best methods to purify DNA from previous extractions, or crude homogenates, is to use equilibrium gradient centrifugation using caesium chloride (Sambrook et al. 1989; Dowling et al. 1996; Ausubel et al. 2002). However, this procedure requires long ultracentrifugation and large amounts of toxic ethidium bromide. However, many other laboratories prefer faster, cheaper and less toxic methods. These sometimes compromise purity or stability but are generally adequate for most applications.

A standard method to remove protein from DNA extracts is to extract first with phenol:chloroform and then with chloroform (to remove any traces of phenol; Sambrook et al. 1989) or proteolytic enzymes such as pronase or proteinase K before extracting with organic solvents (Sambrook et al. 1989). Powell and Gannon (2002) describe a method where DNA extracts are purified with phenol extraction and ethanol precipitation. Dialysis can also be applied to remove salt, detergents (such as SDS and CTAB) and even some enzyme inhibitors (see Sambrook et al. 1989 and Ausubel et al. 2002 for dialysis procedures; or Powell and Gannon 2002 for a simple drop dialysis method). Another method of removing cellular proteins and polysaccharides is to precipitate them prior to precipitating the DNA. Some of these methods have the advantage of not requiring organic solvent extractions. These methods are often used in rapid miniprep methods (Milligan 1992; Edwards 1998).

Other methods of DNA purification have been developed that are based on selective binding of DNA to a silica matrix in the presence of a chaotrope (Vogelstein and Gillespie 1979; Marko et al. 1982; Gilmore et al. 1993). In these methods DNA is selectively bound to a silica matrix in a solution of chaotrope, washed with chaotrope to remove unbound contaminants, including RNA, carbohydrates and proteins, washed with ethanol to remove the chaotrope, and then eluted from the matrix. Modifications of these methods are used in various commonly used commercial genomic DNA purification kits. Even PCR purification spin columns can be used for genomic DNA purification if the investigator is not concerned about losing some high molecular weight DNA. The method of Gilmore et al. (1993) is an efficient purification technique that involves the selective binding of DNA to diatomite (diatomaceous earth) in the presence of a chaotrope.