Low-Cost DNA Extraction

Abstract

The methods described in this chapter were developed to avoid toxic organic phase separation utilized in many low-cost DNA extraction protocols such as the CTAB method. The steps involve: (1) lysis of the plant material, (2) binding of DNA to silica powder under chaotropic conditions, (3) washing the bound DNA, and (4) elution of DNA from the silica powder. This method has been tested in several plant species and the applicability of such DNA preparations for molecular marker studies in barley is shown in Chap. 8.

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4.1 Materials

Chemicals, enzymes, and equipment are listed in Table 4.1, and working stocks in Table 4.2.

Table 4.1 Chemicals, enzymes and equipment for low-cost DNA extraction

Table 4.2 Working stocks for DNA extraction

4.2 Methods

4.2.1 Preparation of Silica Powder DNA Binding Solution

• 1. Transfer silica powder (Celite 545 silica) into a 50-ml conical tube (fill to the 2.5 ml line = approximately 800 mg).

• 2. Add 30 ml dH₂O.

• 3. Shake vigorously (vortex and invert 15 times or until a hydrated slurry forms).

• 4. Let the slurry settle for approximately 15 min.

• 5. Remove (pipette off) the liquid.

• 6. Repeat steps 2–5 an additional two times for a total of three washes. After the last washing step suspend the hydrated silica in a volume of water equal to the volume of silica (typically up to the 5-ml mark on the conical tube). This is the liquid silica stock (LSS) and can be stored at RT for up to 1 month.

• 7. Prior to use, suspend LSS by vortexing for 30 s or until a homogenized slurry is formed. Transfer 50 μl into 2-ml tubes. Prepare one tube per tissue sample.

  ATTENTION: try to keep the silica suspended when transferring to tubes to ensure that all tubes receive the same amount of LSS.

• 8. Add 1 ml H₂O per tube to perform a final wash step.

• 9. Mix by vortexing for 15 s or until silica is fully suspended.

• 10. Centrifuge at full speed (16,000×g) for 20 s.

• 11. Pipette off the liquid.

• 12. Add 700 μl DNA binding buffer (6 M potassium iodide or alternatively 6 M guanidine thiocyanate).

• 13. Suspend the silica in DNA binding buffer by vortexing for 15 s.

• 14. The Silica Binding Solution (SBS) is now ready for use.

4.2.2 Low-Cost Extraction of Genomic DNA

• 1. Prepare an ice bath.

• 2. Label 2-ml tubes containing three metal tungsten carbide beads with sample names.

• 3. Add the dried tissue to the appropriate tube.

• 4. Tape the tubes onto a vortex mixer (Fig. 4.1) and vortex on high setting for 30 s or until the material is ground to a fine powder. NOTE: If the tissue is not fully ground, grinding is facilitated by addition of 0.2 g of purified sea sand per tube. It is common for some tissues to not be completely ground to a powder. High quality DNA can still be produced from such samples. Sample degradation can occur after prolonged vortexing. It is therefore suggested to test different grinding times to find proper conditions to maximize both genomic DNA yield and quality.

  Fig. 4.1

  

  Sample grinding is accomplished by combining desiccated leaf material and metal beads into a 2-ml tube (left panel) and taping sample tubes to a standard vortex mixer (middle panel). Grinding is complete when a find powder is produced. The presence of unground tissue with the powder does not affect the quality of extracted DNA (right panel, and example data in Chap. 8)

• 5. Add 800 μl of Lysis Buffer and 4 μl RNAse A (10 μg/ml) to each tube. NOTE: See Sect. 4.3 for alternative buffers.

• 6. Vortex at a high speed for approximately 2 min until the powder is fully hydrated and mixed with buffer.

• 7. Incubate for 10 min at RT.

• 8. Add 200 μl 3 M sodium acetate (pH 5.2). Mix by the inversion of tubes and incubate on ice for 5 min.

• 9. Centrifuge at 16,000×g for 5 min at RT to pellet the leaf material.

• 10. Label the tubes with aliquots of silica binding solution (SBS, 700 μl) with the sample name.

• 11. Transfer the liquid into appropriately labelled SBS-containing tubes. DO NOT TRANSFER THE LEAF MATERIAL!

• 12. Completely suspend the silica powder by vortexing and inverting the tubes (approximately 20 s).

• 13. Incubate for 15 min at RT (on a shaker at 400 rpm, or invert tubes every 3 min by hand).

• 14. Centrifuge at 16,000×g for 3 min at RT to pellet the silica.

• 15. Remove the supernatant with a pipette and discard (the DNA is bound to the silica at this stage).

• 16. Add 500 μl of freshly prepared wash buffer to each tube.

• 17. Completely suspend the silica powder by vortexing or inverting the tubes (approximately 20 s).

• 18. Centrifuge at 16,000×g for 3 min at RT to pellet the silica. Remove the supernatant and keep the pellet.

• 19. Repeat steps 16–18.

• 20. Centrifuge the pellet for 30 s and remove any residual wash buffer with a pipette.

• 21. Open the lid on tubes containing silica pellet and place in a fume hood for 30 min to fully dry the pellets (NOTE: This can be done for a longer period on the bench top if a fume hood is not available).

• 22. Add 200 μl TE buffer to each tube to elute the DNA. The DNA is now in the liquid buffer. A buffered solution is preferred over water to prevent degradation.

• 23. Completely suspend the silica powder by vortexing and inversion of tubes (approximately 20 s).

• 24. Incubate at RT for 5 min.

• 25. Centrifuge at 16,000×g for 5 min at RT to pellet the silica.

• 26. Label new 1.5-ml tubes with sample numbers/codes.

• 27. Collect the liquid containing genomic DNA and place into new tubes.

• 28. Store DNA temporarily at 4 °C before checking the quality and quantity.

• 29. Evaluate the quality and quantity of the extracted DNA. While fluorometric and spectrophotometric methods have their advantages, it is suggested that samples are evaluated using agarose gel electrophoresis and a quantitative marker so that sample degradation and the presence of any RNA can be monitored. See Chap. 8 for example data.

4.3 Alternative Buffers for DNA Extraction

The main areas for the optimization of DNA-extraction methods include increasing sample yield, reducing co-purification of unwanted components (e.g., polysaccharides, and polyphenols), and reducing sample degradation. To a large extent, providing the starting tissues are of good quality, all three areas can be influenced by the sample lysis procedure. Table 4.3 lists four lysis buffers to optimize the isolation of DNA from grapevine and sorghum. A more thorough compilation of buffer components and additives to enhance DNA isolation in the presence of secondary compounds can be found in Weising et al. (2005). Data from buffer optimizations are shown in Chap. 8.

Table 4.3 Alternative lysis buffers for DNA extraction