Plant BIOTECHNOLOGY

Experiment 1

Principle

Isolating DNA from plant tissues can be very challenging as the biochemistry between divergent plant species can be extreme. Unlike animal tissues where the same tissue type from different species usually have similar characteristics, plants can have variable levels of metabolites and structural biomolecules. Polysaccharides and polyphenols are two classes of plant biomolecules that vary widely between species and are very problematic when isolating DNA. Contaminating polysaccharides and polyphenols can interfere with manipulations of DNA following isolation. Methods are available that effectively remove polysaccharides and polyphenols from plant DNA preparations. The use of CTAB (cetyl trimethylammonium bromide), a cationic detergent, facilitates the separation of polysaccharides during purification while additives, such as polyvinyl pyrrolidone ,can aid in removing polyphenols. CTAB based extraction buffers are widely used when purifying DNA from plant tissues.

One option for purifying DNA using CTAB exploits that polysaccharides and DNA have different solubility in CTAB depending on the concentration of sodium chloride. At higher salt concentrations, polysaccharides are insoluble, while at lower concentrations DNA is insoluble. Consequently, by adjusting salt concentration in lysates containing CTAB, polysaccharides and DNA can be deferentially precipitated.

Polyphenols are compounds that contain more than one phenolic ring (e.g., tannin), a structure that binds very efficiently to DNA. They are naturally occurring in plants, but are also generated when plants have tissue damage (browning). Upon the homogenization of plant tissues, polyphenols are synthesised by liberated polyphenol oxidase. The addition of polyvinyl pyrrolidone prevents the interaction of DNA and phenolic rings by binding up the polyphenols.

Materials required

CTAB buffer: 2% cetyl trimethylammonium bromide, 1% polyvinyl pyrrolidone,100 mM Tris-HCl,1.4 M NaCl, 20 mM EDTA, or CTAB Extraction Buffer

Centrifuge (up to 14,000 x g)

Isopropanol

70% Ethanol

2 ml centrifuge tubes

TE Buffer (10 mM Tris, pH 8, 1 mM EDTA)

Agarose,

TAE Buffer

Procedure

1. Plant samples can be prepared by cryogenically grinding tissue in a mortar and pestle after chilling in liquid nitrogen. Freeze dried plants can be ground at room temperature. In either case, a fine powder is best for extracting DNA.

2. For each 100 mg homogenized tissue use 500 µl of CTAB Extraction Buffer. Mix and thoroughly vortex. Transfer the homogenate to a 60°C bath for 30 minutes.

3. Following the incubation period, centrifuge the homogenate for 5 minutes. at 14,000 x g.

4. Transfer supernatant to a new tube. Add 5 µl of RNase solution A and incubate at 32°C for 20 minutes

5. Add an equal volume of chloroform/isoamyl alcohol (24:1). Spin at 14,000 x g to separate the phases. Transfer the aqueous upper phase to a new tube. Repeat this extraction until the upper phase is clear.

6. Transfer the upper aqueous phase to a new tube. Precipitate the DNA by adding equal volume cold isopropanol and incubate at -20°C for 15 minutes.

7. Centrifuge the sample at 14,000 x g for 10 minutes. Decant the supernatant without disturbing the pellet and subsequently wash with 500 µl ice cold 70% ethanol. Decant the ethanol.Remove residual ethanol by drying.

8. Dry the pellet long enough to remove alcohol, but without completely drying the DNA. Dissolve DNA in 50 µl TE buffer (10 mM Tris, pH 8, 1 mM EDTA). The pellet may need warming in order to dissolve.

9. Run the isolated DNA in agarose gel electrophoresis.

10. Prepare a 0.8 to 1 % solution of agarose by melting 1 g of agarose in 100 mL of 0.5x TBE buffer in a microwave for approximately 2 min. Allow to cool for a couple of minutes then add 2.5 μl of ethidium bromide, stir to mix.

11. Cast a gel using a supplied tray and comb. Allow the gel to set for a minimum of 20 min at room temperature on a flat surface.

12. Load the following into separate wells

13. 10 μL 1kb ladder

14. 10 μL sample + 4 μL 6x Loading Buffer

15. Run the gel for 30 min at 100 V

16. Expose the gel to UV light and photograph (demonstration)

17. Confirm DNA quality, presence of a highly resolved high molecular weight band indicates good quality DNA, presence of a smeared band indicates DNA degradation.

Result

The bands can be observed on gel as seen in figure 1.

Reference Material

1. T.A. Brown- Gene Cloning Book.

2. https://bio-protocol.org/bio101/e30

Questions

1. Enlist the chemicals used in this protocol and write their functions.

2. How one can quantify the isolated DNA? explain.

3. For what purposes a researcher isolates DNA?

4. Are there any other chemical methods of isolating DNA from plants? Name those methods.

5. Difference in units of centrifuge machine ie 14000g and 14000 rpm. Are they both same?

6. How many dyes are used in Agarose gel electrophoresis