ISOLATION AND PURIFICATION OF GENOMIC DNA Gurinder Jit Randhawa NRC DNA Fingerprinting, NBPGR, New Delhi For plant cells with a rigid cell wall, the disruption of cells usually requires the tissue to be ground using a pestle and mortar in liquid nitrogen. The powdered plant tissue is then transferred to an extraction buffer that contains detergent to disrupt the membranes. Cetyltrimethyl ammonium bromide (CTAB) is commonly used for this purpose. The extraction buffer also contains a reducing agent (β- mercaptoethanol) and a chelating agent (ethylenediamine tetraacetic acid, EDTA). This helps to inactivate nucleases that are released from the plant cell and can cause serious degradation of the genomic DNA. Keeping the reactions cold, when possible can minimize their effects. Phenolic compounds may also be released on disruption of plant tissues and these may interfere with subsequent uses of the DNA (e.g. if it is to be used in the PCR). Polyvinyl pyrolidone (PVP) can be added to the extraction buffer to remove phenolic compounds. Phenol extraction can be used to remove any traces of proteins and the genomic DNA can be precipitated using either ethanol or isopropanol. Precipitated DNA can be hooked out of the solution or collected by centrifugation. It is important that DNA is not sheared, for this reason the DNA should not be vortexed or pipetted repeatedly using a fine tipped pipette and all manipulations should be as gentle as possible. DNA extraction has three main steps: 1. Lysis of cell walls and membranes to release DNA into solution. 2. Purification of DNA by precipitating proteins and polysaccharides. 3. Precipitation of DNA and resuspension in a buffer. Equipments required 1. 2. 3. 4. 5. 6.

High speed centrifuge Microfuge Auto-pippets 2-20μl, 20-200μl, 200-1000μl Waterbath -20oC Deep freezer Refrigerator.

A number of published methods are available for the extraction of genomic DNA, some of these are discussed here:

1.

Saghai-Maroof et al. (1984) Reagents 1. CTAB Buffer: 1.4 M NaCl 100 mM Tris-Cl 20 mM EDTA 2% β-Mercaptoethanol 1.5% CTAB Adjust pH to 8.0 with HCl and autoclave before use. 2. Isopropanol 3. Saturated phenol 4. Chloroform: isoamyl alcohol (24:1) mixture 5. 10:1 TE: 10mM Tris 1mM EDTA Adjust pH to 8.0 with HCl and autoclave before use. 6. RNase A (10mg / ml): Dissolve RNase A in 10mM Tris-Cl, pH 7.5, 15 mM Na Cl. Heat at 100 oC for 15 min. Cool to room temperature. Store as aliquots at -20 oC. 7. 70% ethanol Protocol 1. Weigh 5 g of clean young leaf tissue and grind to fine powder with a pestle and mortar after freezing in liquid nitrogen. 2. Transfer to 50 ml centrifuge tube with 20 ml CTAB buffer maintained at 60oC in a water bath. Mix vigorously or vortex. 3. Incubate at 60oC for one hour. Mix intermittently. 4. Fill the tube with chloroform: isoamyl alcohol. Mix gently by inverting for 5 min. 5. Spin at 17,000 rpm for 10 min. with ss34 rotor in Sorval RC-5C centrifuge at 25oC. 6. Transfer aqueous phase to a fresh centrifuge tube. Add equal amount of isopropanol and let the DNA to settle down for 20 min. 7. Spool out the DNA. If necessary we can centrifuge in the microfuge for 2 minutes. Drain out the excess chemicals with a pipette. 8. Add 0.5 ml of 70% ethanol. Mix gently and incubate for 30 min. Decant and repeat the 70% ethanol treatment. Decant off and dry the pellet under vaccum. 9. Dissolve DNA in minimum volume of 10 : 1 TE. 10. Add RNase (0.2 ml) and incubate at 37o C for one hour. 11. Add equal volume of phenol: chloroform (1:1), mix properly and spin for 5 min. Take out the DNA supernatant and after this perform two chloroform:isoamyl alcohol extractions as before. Spin after each extraction.

12. Precipitate DNA by adding 1/10 volume of 3M NaOAc and 2.5 times the total volume of chilled ethanol. Mix and spool out the DNA. Remove extra salts by two washings with 70% ethanol. Dry under vacuum. 13. Add minimum volume of TE (10:1). Dissolve at room temperature. Store frozen at -20o C. 2.

Dellaporta et al. (1983) Reagents 1. Extraction buffer 100 mM 50mM 500 Mm 10mM 2. 20% SDS 3. 5M KAc 4. Resuspension buffer I 50mM 10mM 5. Resuspension buffer II 10mM 1mM 6. 3M NaOAc 7. Isopropanol

Tris-Cl (pH-8.0) EDTA (pH-8.0) NaCl β-mercaptoethanol

Tris - Cl (pH-8.0) EDTA (pH-8.0) Tris - Cl (pH-8.0) EDTA (pH-8.0)

Protocol 1. Weigh 0.5 to 1 g of tissue. Freeze the tissue rapidly in liquid nitrogen and grind to a powder with a pestle and mortar as the liquid nitrogen boils off. Add a little more liquid nitrogen, if necessary, to keep the powder from thawing while grinding. It is important not to let the tissue thaw once frozen, until it is added to the buffer. 2. Transfer the frozen powder into a 50 ml centrifuge tube containing 15 ml of extraction buffer (100 mM Tris- HCI pH 8.0; 50 mM EDTA pH 8.0; 500 mM Na CI) and 10mM β-mercaptoethanol using a spatula. The tubes must be placed in an ice bucket. 3. Add 1.0 ml of 20% SDS, mix thoroughly by vigorous shaking and incubate the tubes at 650C for min (a water bath at 650C must be prepared ready). 4. Add 5.0 ml 5M potassium acetate working solution, shake vigorously and incubate at 00C for 20 min (minimum time). Most of the proteins and polysaccharides are removed as a complex with the insoluble potassium dodecyl sulphate precipitate. 5. Balance the tubes by adding extraction buffer and spin at 15000 rpm (20,000g) for 20 min. Pour the supernatant through a sterile small funnel containing two layers of gauze, or mira cloth, into a clean 50ml tube

containing 10ml of cold (-20 0 C) isopropanol kept in an ice bucket. Mix gently, by inverting at –20 0C for 30 min. A DNA clot should appear. 6. Balance the tubes by adding isopropanol or extraction buffer. Produce a pellet of DNA by centrifuging at 15,000 rpm for 15 min. Gently pour off the supernatant and lightly dry the pellets by inverting the tubes on paper towels for 1-2 min. he pellet must be clear; if it is white, it will contain polysaccharides, if dark phenolic compounds. 7. Redissolve the DNA pellet in 0.7 ml of TE 50:10 pH 8.0 at room temperature for 20-30 min or for the time required to be sure that all the pellet is dissolved. Help to resuspend the pellet by pipetting very gently with a blue tip, but do not overdo it. 8. Transfer the DNA to an eppendorf tube. All centrifugation from this point in eppendorf microfuges. Spin the tubes in a microfuge for 12 min at 12 000 rpm to remove the insoluble debris. 9. Transfer the supernatant to a new eppendorf tube and add 75μ of 3M sodium acetate and 500μ l of isopropanol. Mix well without vortexing and pellet the clot of DNA by centrifuging for 1-5 min at 12,000 rpm in microfuge. 10. Discard the supernatant and save the pellet. Add 700μl cold (-200C) 80% ethanol and dislodge the pellet from the bottom of the tube by tapping the tube gently with your fingertips. The diluted ethanol removes salts. 11. Centrifuge for 1-5 min at 12,000 rpm. Discard the ethanol and dry the pellet eliminate to alcohol completely. It can be dried by leaving the tubes open to the air or by using a vacuum desiccator. 12. Add 500 μl sterile distilled deionized water or TE 10:1 pH 8.0 and maintain at room temperature for 1h to redissolve the DNA. It may help to pipette up and down with a blue tip, very gently1 if the DNA cannot be completely dissolved, centrifuge at 12 000 rpm the supernatant. Discard the insoluble debris. There is an option to stop at this point and to store the material at 200C. 13. Add 10μl of RNase solution, incubate for at least 1.5 h at 37 0 C to ensure that all the remaining RNA is digested. 14. Add 500μl (one volume) PCIA (phenol: chloroform: isoamyl alcohol) to eliminate RNase and other contaminants. Shake the solution, do not vortex. Centrifuge at 12, 000 rpm for 10 min to separate the phases. 15. Collect the aqueous phase in a new eppendorf tube, measuring the volume recovered. 16. Add the same volume of chloroform: isoamyl alcohol. Shake the solution, do not vortex. Centrifuge at 12,000 rpm for 10 min. to separate the phases. Any remaining phenol is removed by this procedure. 17. Recover the upper aqueous layer in a new eppendorf tube, measuring the volume recovered. Add 1/10 volume 3M sodium acetate and 2-3 volume of cold 100% ethanol. Maintain for 1 h at –200C. There is an option to stop at this point. 18. Centrifuge for 15 min at 13,000 rpm discard the supernatant. Wash the pellet with 700μl of cold 70% ethanol to dissolve the remaining salts and dislodge from the tube wall by tapping the tube gently with your finger tips. If the

pellet is white, salts may still be present. Try to disaggregate the pellet and wash again with 70% ethanol at room temperature. 19. Centrifuge for min at 12,000 rpm and discard the supernatant. 20. Dry the pellet to eliminate the ethanol. 21. Dissolve the pellet in sterile distilled deionized water or TE 10:1. As the yield should be 50-100 μg per g of tissue, dissolve in 50-100 μl to obtain a final concentration of 1μg / μl DNA. 22. Store at -200C.

Miniprep protocol for the isolation of plant genomic DNA a) CTAB miniprep DNA extraction protocol (Taylor and Powell, 1982) Reagents 1. Extraction buffer: (50 mM Tris HCl pH 8.0, 10mM EDTA, 0.7M NaCl, 1.0% CTAB and 0.1% β-mercaptoethanol) 2. Chloroform 3. 10mg/ml RNAase A 4. Isopropanol 5. 70% Ethanol 6. TE buffer: (10mM Tris-HCL pH 8.0, 1mM EDTA) Protocol 1. Weigh out 0.05 g (dry weight) of freeze-dried tissue powder into a 1.5ml eppendorf tube. 2. Add 500 μl of CTAB extraction buffer, pre-warmed to 600C and disperse the lumps by inverting several times. 3. Incubate at 600C for 15 min with occasional mixing by inversion. 4. Add 500 μl of chloroform and mix by inverting until an emulsion is formed. 5. Centrifuge at 13,000 rpm in a bench top centrifuge for 10 min at room temperature. 6. Place the aqueous phase in a fresh eppendorf tube and mix with 2.5 μl RNAase A mix gently and incubate at 370 C for 30 min. 7. Transfer the aqueous phase, which should be clear, to a fresh tube and add 0.6 volumes of isopropanol, mix by inversion to precipitate the DNA. Wash the pellet with 70% ethanol. 8. Resuspend the pellet in 25 μl of 1 x TE.

b) Isolation of genomic DNA for PCR (Edwards et al. 1991) Reagents 1. Extraction buffer (200mM Tris HCl pH 7.5, 25 mM EDTA, 250 mM NaCl, 0.5% SDS) 2. Isopropanol 3. 1x TE buffer (10 mM Tris-HCl pH 8.0, 1mM EDTA) Protocol 1. Remove leaf samples (a few mg of leaf tissue will be sufficient) in a 1.5 ml eppendorf tube. Leaf samples may be collected using the lid of the tube to punch a disc of leaf material straight into the tube. This not only ensures uniform sample size but is also reduces contamination. 2. Grind the samples in the original eppendorf tube at room temperature, without buffer, for 15 to 20 sec, or until liquid exudes from the issue. 3. Add 400μl of extraction buffer and mix the samples for 5sec. At this stage samples may be left at room temperature until they have all been processed. 4. Centrifuge the samples at full speed (13,000 rpm) for 5 min at room temperature. 5. Remove 300 μl of the aqueous fraction, place in a fresh eppendorf tube and add 300 μl of isopropanol. 6. Mix the samples and leave at room temperature for 30 to 60 min before centrifuging at full speed for 10 min. 7. Remove all of the supernatant from the pellet and vacuum dry for about 20 min. 8. Dissolve the pellet in 100μl of 1x TE. 9. Use 1μl of the above for the usual 25μl PCR. Quantification of DNA DNA quantification is an important step to know the amount of isolated DNA. DNA Quantification by UV Spectroscopy 1. Take 5μl of the DNA sample in a quartz cuvette. Make up the volume to 1ml with distilled water. 2. Measure absorbance of the solution at wavelengths 230, 260, 280 and 300nm. 3. Calculate the ratios A230 / A260 and A280 / A260. 4. A good DNA preparation exhibits the following spectral properties: A300< 0.1 O.D. units A230 / A260 < 0.45 O D units A280 / A260 < 0.55) O D units. 5. Calculate DNA concentration using the relationships for double stranded DNA, 1 O D at 260 nm = 50μg / ml. This estimate is influenced by the contaminating substances like RNA and very low molecular weight DNA in the solution. 6. Prepare a working stock of samples of about 100μl with concentrations of 10 ng/μl.

DNA Quantification by Fluorimetry The DNA extraction procedures do not eliminate RNA. Therefore, the estimatied DNA concentration by UV spectrophotometry may not be very accurate. RNase treatment may help in reducing the errors. However, fluorimetric estimations are more reliable as it measures the fluorescence emitted by the double stranded DNA- Hoechst 33258 dye complex, which is directly proportional to the amount of DNA in the sample. Since Hoechst 33258 dye does not bind to single stranded DNA and very small fragments of DNA this procedure gives more reliable estimates of the DNA concentrations in the sample. Reagents 1. 10x TNE (1000ml, buffer stock solution) 100mM Tris 1M NaCl 10 mM EDTA (Dissolve in 800 ml distilled water. Adjust pH to 7.4 with HCl. Add distilled water to make volume 1000ml. Filter and Autoclave before use. Store at 40C for up to 6 months in an amber bottle.) 2. Hoechst 33258 dye stock Hoechst 33258: 1 mg /ml in distilled water. (Add 10 ml distilled water to 10 mg H33258. Do not filter. Store at 40C for up to 6 months in an amber bottle). Protocol 1. Prepare the assay and DNA standard solutions as described below: DNA assay solution Low range (A): (10-500 ng/ml final DNA concentration) H33258 stock solution: 10.0 μl 10x TNE buffer: 10.0 ml Distilled filtered water: 90.0 ml High range (B): (100-500 ng/ml final DNA concentration) H33258 stock solution: 100.0 μl 10x TNE buffer: 10.0 ml Distilled filtered water: 90.0 ml. 2. Turn on the fluorimeter (Hoefer) at least 15 min before using. 3. Zero the instrument: Prepare an assay blank using 2 ml of appropriate assay solution (A or B for high DNA concentration). Dry the sides of a cuvette. Insert the cuvette into the well, close the lid, and press < ZERO>. After “0” displays, remove the cuvette. 4. Calibrate the instrument: Deliver 2 μl of the appropriate DNA standard solution (low or high range) to 2 ml of assay solution in the cuvette. Mix by pipetting

several times into a disposable transfer pipette. Place cuvette in well, close the lid and press . Enter 100 for the low range assay, 1000 for the high range assay and press < ENTER>. After the entered value displays, remove the cuvette. 5. Zero the instrument: Empty and rinse the cuvette. Dry by draining cuvette and blot in upside down on a paper towel. Add 2 ml of the same Assay Solution used in step 2, insert the cuvette into the well, close the lid, and press . After “0” displays remove the cuvette. 6. Measure the sample and mix well. Place the cuvette in the well, close the lid, and record the measurement. 7. Measure subsequent samples. Repeat steps 5 and 6 for each sample.

References Dellaporta, S.L. Wood, J. and Hicks, J.B. 1983. A plant DNA mini preparation: Version II. Plant Molecular Biology Reporter 1: 19-21. Edwards, K.J., Johnstone, C and Thompson, C.1991. A rapid and simple method for the preparation for plant genomic DNA for PCR analysis. Nucleic Acids Research 19, No.6: 1349. Saghai-Marrof, M.A, Soliman, K.M., Jorgensen, R.A. and Allard, R.W. 1984. Ribosomal DNA spacer-length polymorphism in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA 81: 80148018. Taylor, B. and Powell, A. 1982. Isolation of Plant DNA and RNA. Focus 4 : 4-6.