Agriculture Victoria, Private Bag 7, Werribee, Victoria 3030, Australia (E. 0., ... 16 Gipps Street, Collingwood, Victoria 3066, Australia (S. C.); and ANM Forest.
In VitroCell.Dev.Biol.--Plant34:108-111,April-June1998 9 1998Societyfor In VitroBiology 1071-2690/98 $05.00+ 0.00
TECHNICAL REVIEW OPTIMIZING CONDITIONS FOR DNA ISOLATION FROM PINUS RADIATA EWA OSTROWSKA,MORLEY MURALITHARAN,1 STEPHEN CHANDLER, PETER VOLKER, SANDRAHETHERINGTON, ANDFRANK DUNSHEA Agriculture Victoria, Private Bag 7, Werribee, Victoria3030, Australia (E. 0., F. D.); Department of Chemical Sciences, Victoria University of Technology, St. Albaus, Victoria3021, Australia (E. 0., M. M.); School of Agriculture, Charles Sturt University, PO Box 588, Wagga Wagga, NSW 2678, Australia (M. M.); Florigene, 16 Gipps Street, Collingwood, Victoria3066, Australia (S. C.); and ANM Forest Management, New Norfolk, Tasmania 7140, Australia (P. E, S. H.) (Received 22 February 1997; accepted 17 September 1997; editor C. L. Armstrong)
SUMMARY Genomic DNA was isolated from in vitro Pinus radiata seedlings with five DNA isolation protocols commonly used for pines. The methods described by Jobes et al. (1995) and Nelson et al. (1994) utilize sodium dodecyl sulfate, whereas those of Murray and Thompson (1980), Doyle and Doyle (1990), and Devey et al. (1996) use cetyltrimethyl ammonium bromide for cell lysis. The quality and quantity of the isolated DNA was measured and compared. Lithium chloride was found to be more effective than RNase for minimizing the amount of RNA present in the solution. Protocols described by Jobes et al. (1995) and Devey et al. (1996) yielded a large quantity of pure DNA which was suitable for restriction enzyme digestion and polymerase chain reaction amplification. With these methods, 37 to 79 txg of DNA with an A26o/28o ratio between 1.7 and 1.9 was obtained from 1 g of Pinus radiata seedlings grown in vitro. Key words: conifer, genomic DNA, purity, needles, CTAB, SDS. from conifers (Murray and Thompson, 1980; Doyle and Doyle, 1990; Nelson et al., 1994; Jobes et al., 1995; Devey et al., 1996).
INTRODUCTION The isolation of conifer plant genomic DNA, which has a larger genome (approximately 2 X 10 l~ base pairs in the case of Pinus radiata) than nonwoody plants, requires a method for which extra care must be taken to minimize DNA sheafing during disruption of the cells (Keller and Manak, 1993; Muralitharan et al., 1994). Vailous DNA isolation methods, mainly from needles and haploid megagametophytes, have been used in conifers (Guiles et al., 1978; Nelson et al., 1994; Devey et al., 1996). Most of these methods are based on the cetyltrimethyl ammonium bromide (CTAB) extraction procedure which was initially developed by Murray and Thompson (1980). CTAB, a cationic detergent, complexes with cell walls, protein, and polysaccharides including starch and allows them to be precipitated from a solution. This procedure has been modified by Wagner et al. (1987), Doyle and Doyle (1990) and Bousquet et al. (1990) and more recently by Devey et al. (1996) to optimize the yield and purity of DNA isolated from different types of conifer tissue. Other DNA isolation procedures utilize sodium dodecyl sulfate (SDS) to promote cell lysis (Nelson et al., 1994; Jobes et al., 1995). The aim of the present study was to determine the best DNA isolation conditions for Pinus radiata with the objective of yielding a large amount of pure DNA suitable for polymerase chain reaction, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP). This was achieved by screening the more commonly used protocols currently used to isolate DNA
MATERIALSAND METHODS Seed germination. Open pollinated seeds of Pinus radiata were obtained from Australian Newsprint Mills (ANM)Forest Management,Tasmania, Australia. Seeds were pretreated by soaking in 50 mg gibberellic acid (GA3) (Sigma Chemical Co., St. Louis, MO) per L for 24 h, followedby sterilization in 30% (vol/vol)sodium hypochlorite (available chlorine 37.5 g/L) containing two drops of Tween 80 (Sigma) for 30 min. Seeds were then placed in 40 ml sterile distilled water in a 50-ml container and left on a shaker for 12 h, washed in 30% (vol/vol) sodium hypochlorite (available chlorine 37.5 g/L) supplemented with two drops of Tween 80 for 30 min, rinsed in doubledistilled sterile water three times, and incubated in 50-ml containers at 4~ C for 48 h. Seeds were then resterilized in 20% (vol/vol)sodium hypochlorite (available chlorine 25.0 g/L), rinsed three times in double-distilled sterile water, and germinated on half-strength LP (VonArnold and Eriksson, 1977) basal medium [containing30 g sucrose (Sigma)per L and 3.2 g gelrite (Sigma) per L, pH adjusted to 5.6 by adding drops of KOH] at 16:8 h light and dark cycle and temperature of 24~ C (day) and 22~ C (night). DNA isolation. Material was ground in liquid nitrogen (-194 ~ C) by a pestle and mortar and temporarily stored on ice. One gram of needles obtained from 3-wk-old in vitro Pinus radiata seedlings was used, with 20 replicates for each method. Method 1: Jobes et al., 1995. Ground Pinus radiata needles were incubated in 7 ml of preheated extraction media at 55~ C for 1 h. Extraction media comprised 100 mM sodium acetate (pH 4.8), 100 mM ethylenediaminetetraacetic acid (EDTA;pH 8.0) (Sigma),500 mM sodium chloride (Sigma), 10 mM dithiothreitol (Sigma), and 2% (wt/vol) polyvinylpyrrolidone (PVP) (Sigma). The pH was adjusted with KOH and 100 ~g proteinase K (Sigma) per ml was added to the extraction media shortly before use. SDS (Sigma) was added to a final concentration of 1.5% (wt/vol),followed by another hour of incubation at 55~ C. Tubes were centrifuged at 6000 • g for 10 rain at 22~ C and the supernatant was transferred to a new sterile tube. Potassium acetate
1To whom correspondence should be addressed. 108
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DNA ISOLATION FROM P. RADIATA TABLE 1 AMOUNT AND A26o,~soRATIOS OF DNA AND THE EFFECT OF LICL AND RNASE TREATMENTS
Method Jobes et al., 1995 Doyle and Doyle 1990 Murray and Thompson, 1980 Devey et al., 1995 Nelson et al., 1994
DNAyield" 0tg,'g) 37-46 48-67 16-19 57-79 21-27
Appearanceof RNAon the gels Mean 42.54 59.66 18.22 70.41 25.48
--- 5.32 + 8.27 + 2.36 + 12.67 --- 4.22
A~onao"
AfterLiCLtreatment
AfterRNAsetreatment
1.8-1.9 1.6-1.8 1.4-1.6 1.7-1.9 1.3-1.6
No
No
Very small Yes No Yes
Yes Yes No Yes
aMeasurements were taken after lithium chloride treatment.
(5 M, pH 4.8) was added (1/3 of the supernatant volume), incubated at - 20~ C for 30 min, following which the tube was centrifuged at 6000 • g for 10 min at 4 ~ C. The aqueous phase was then transferred to a new sterile tube and 2/3 of its volume of cold (4~ C) isopropanol was added. The tubes were then incubated at - 2 0 ~ C for 30 min. This was followed by centrifugation at 6000 • g for 10 min. The pellet was dissolved in 50 ~tl of TE buffer [10 mM Tris-HC1 (Sigma) and 1 mM EDTA, pH 8.0], and two sequential extractions with 100 ~tl of phenohchloroform (1:1; vol/vol) were performed, followed by one chloroform extraction (100 ILl). We precipitated DNA by adding 2/3 volume of cold (4~ C) isopropanol and the pellet was washed in 100 ~1 of 70% (vollvol) ethanol. Method 2: Doyle and Doyle, 1990. Ground Pinus radiata needles were incubated in 7 ml of preheated CTAB extraction media [2% (wt/vol) CTAB; 1.4 M NaC1; 0.2% (vol/vol) 13-mercaptoethanol;20 mM EDTA; 100 mM TrisHC1 (pH 8.0)] at 60~ C for 30 min with occasional gentle swirling. This was followed by extraction with 4 ml of chloroform:isoamyl alcohol (24:1, vol/vol) and centrifugation (4000 • g) at 22~ C for 10 min to separate organic solvents from the aqueous layer. The aqueous phase was removed and 2/3 of its volume of cold (4~ C) isopropanol was added. The content was centrifuged at 4000 • g for 10 min and the pellet was washed in 100 ILl of 70% (vol/vol) ethanol. Method 3: Murray and Thompson, 1980. Ground Pinus radiata needles were incubated in 7 ml of preheated CTAB extraction media [0.7 M NaC1; 1% (wt/vol) CTAB; 50 mM Tris-HCl (pH 8.0); 10 mM EDTA; 1% (vol/vol) 13-mercaptoethanol] at 60~ C for 30 rain with occasional gentle mixing. This was followed by two (4 ml) chloroform:octanol (24:1, vol/vol) extractions and centrifugation at 6000 • g for 10 min. The aqueous phase was removed, 1/ 10 volume of 10% (wt/vol)CTAB, and 0.7 M NaC1was added, and the chloroform:octanol treatment was repeated. Seven milliliters of 1% (wt/vol) CTAB, 50 mM Tris-HC1 (pH 8.0), and 10 mM EDTA was added, and after 30 min at 22~ C the DNA was recovered by centrifugation at 4000 • g for 5 min, followed by washing in 100 ~tl of 70% (vol/vol) ethanol. Method 4: Devey et al., 1996 (modifiedfor DNA isolationfrom 1 g of tissue). Ground Pinus radiata needles were incubated in 7 ml of extraction buffer at 22 ~ C for 15 min. Extraction buffer comprised 50 mM Tris (pH 8.0), 5 mM EDTA, 0.35 M sorbitol, 0.1% (wt/vol) bovine serum albumin (Sigma), 0.1% (vol/vol) 13-mercaptoethanol,10% (wt/vol)polyethylene glycol (mol. wt. 8000) (Sigma), and N-laurylsarcosine (Sigma) at a concentration of 1% (wt/vol).The suspension was brought to a final concentration of 0.7 M NaC1, 1% (wt/vol) CTAB and incubated at 60~ C for 60 min. Following the incubation, an organic extraction with 4 ml of chloroform:octanol (24:1, vol/vol)was performed, and the aqueous layer was precipitated with 2/3 volume of cold (4~ C) isopropanol. The DNA was recovered by centrifugation at 4000 • g for 5 min and the pellet was rinsed in 100 ill of 70% (vol/vol) ethanol. Method 5: Nelson et al., 1994 (modified for DNA extractionfrom needles). Ground Pinus radiata needles were incubated in 7 ml extraction buffer [50 mM Tris-HC1 (pH 7.2); 50 mM EDTA (pH 8.0); 3% SDS; 1% (vol/vol) 13mercaptoethanol] at 65 ~ C for 1 h, extracted with 7 ml of phenol:chloroform:isoamyl alcohol (25:24:1, vol/vol/vol), and reextracted with 7 ml of chloroform:octanol (24:1, vol/vol). Nucleic acids were precipitated with 0.7 ml of 3.0 M sodium acetate (pH 5.2) and 15 ml of 100% ethanol (4~ C), incubated at - 70~ C for 30 rain, pelleted by centrifugation at 4000 X g for 5 rain, and rinsed with 100 ~tl of 70% (vol/vol) ethanol. Ten of the samples from each DNA isolation method were treated with lithium chloride (Sigma) and the remaining 10 samples were treated with the enzyme RNase (Sigma). Lithium chloride and RNase treatments were carried
out according to protocols of Jobes et al. (1995) and Doyle and Doyle (1990), respectively. We assessed the purity of the DNA solution spectrophotometrically with Cary, UV-Visible Spectrophotometer, Varian) by measuring absorbances at 260 and 280 nm. The quantity of DNA was calculated by BeerLambert Law and values outlined in Current Protocols in Molecular Biology (Ausubel et al., 1987). Undigested genomic DNA obtained from 1 g of Pinus radiata needles was diluted in 60 I.tl of TE buffer (pH 7.2) and electrophoresed in 0.8% (wt/vol) agarose gels in 0.5 • TBE (Tris-borate-EDTA) buffer and 50 ~tl of ethidium bromide per L (stock solution 10 mg/ml) at 12V for 12 h. We investigated purity again by recording the presence of RNA on gels. Gels were viewed under a UV transilluminator (Pharmacia, manufactured by UVP Inc., USA) and were photographed with Polaroid DS-34 Direct Screen instant camera using Polaroid 667 film. RESULTS AND DISCUSSION DNA yield and purity is strongly influenced by the type, age, and quality of plant tissue used, but perhaps more important is the DNA isolation procedure (Muralitharan et al., 1994). Polysaccharides, polyphenolics, tannins, and lignin cause major problems in the isolation of DNA from woody plants because these compounds are difficult to separate from the genomic DNA (Murray and Thompson, 1980). If not removed, these contaminants inhibit DNA digestion with restriction endonucleases or amplification by PCR. A high purity of DNA is also vital for production of specific probes because 100% of the DNA labeled will represent the sequences of interest (Keller and Manak, 1993). DNA isolation protocols designed for woody species aim at removing contaminants such as polysaccharides, tannins and lignin by addition of polyvinyl pyrrolidone or polyethylene glycol and 13-mercaptoethanol or dithiothreitol. The concentration of chemicals in the solution and the order of the addition of the chemicals are vital. For example, Jobes et al, (1995) found that adding SDS after 1 h of incubation in extracting buffer instead of adding it directly to the buffer can improve the efficiency at which PVP binds to polyphenolics. Salt concentration (sodium chloride) of the extraction solution must be kept above 0.5 M to ensure that the nucleic acids do not complex with CTAB (Keller and Manak, 1993) and that there is no coprecipitation of polysaccharides and DNA (Jobes et al., 1995). The total yield of DNA isolated from Pinus radiata seedlings with the protcinase K-SDS extraction method (Jobes et al., 1995) was in the range of 37-46 ~g tissue per g (Table 1). Ratios of A26o/280between 1.8 and 1.9 were obtained. The SDS method described by Nelson et al. (1994) was less successful, yielding 21-27 ttg of DNA per g with A26o/28o ratios between 1.3 and 1.6. The solution appeared viscous, therefore indicating the presence of high levels of polysaccharides (Jobes et al., 1995). With the Murray and Thompson (1980) protocol, yield of DNA ranged from 16-19 I.tg tissue per g and A26o/2so ratio was between 1.4 and 1.6. The CTAB procedures described by Doyle and Doyle (1990) and Devey et al. (1995) yielded the highest levels of DNA (48-67 and 57-79 ~tg tissue per g, respectively). The A26o/
110
OSTROWSKA ET AL.
1 ,t. ab
3 Xabc
cxa
bc
4 abc
FIG. 1. Agarose gel (0.8%, wt/vol) of undigested DNA isolated from Pinus radiata needles. Lanes marked "~." are lambda/EcoRI molecular weight markers. Lanes 1,2,3,4, and 5 represent the DNA isolated according to Jobes et al. (1995), Nelson et al. (1994), Murray and Thompson (1980), Doyle and Doyle (1990), and Devey et al. (1996), respectively. Lanes marked "a" show untreated DNA; lanes marked "b" show RNase treated DNA; and lanes marked "c" show lithium chloridetreated DNA.
2
5 ;
2so ratio was between 1.6 and 1.8 with the Doyle and Doyle (1990) method and 1.7-1.9 for the Devey et al. (1995) method. The quality of isolated DNA from fresh needles was qualitatively determined by separating undigested DNA according to molecular weight by gel electrophoresis (Fig. 1). Low molecular weight contaminants, mainly RNA and to lesser extent, proteins, were visible in the form of a smear in the lower part of the gel (Fig. 1). Before lithium chloride and RNase treatments RNA content was high in most of the samples, with the lowest amount in DNA isolated by the Jobes et al. (1995) method. However, the content of RNA was reduced by treating the DNA with lithium chloride or RNase, which either precipitate or degrade RNA respectively. Both treatments successfully obtained pure DNA by the Jobes et al. (1995) and Devey et al. (1995) methods. However, a substantial amount of RNA was still present in the DNA samples isolated by the remaining three protocols, indicating the requirement for further purification with RNase or LiC1.
abc
DNA isolated by the Jobes et al. (1995) and Devey et al. (1996) methods before and after RNase and LiC1 treatments appeared more "intact" (compact bands) than DNA isolated by the remaining three protocols (Fig. 1). The intactness of the DNA was the indicator of less severe disruption of long fragments of DNA during the process of DNA isolation in these two protocols. This is very important in RAPD and RFLP analysis, in which it can have an effect on the reproducibility of RAPD and RFLP bands (Muralitharan et al., 1994). Both the protocols of Jobes et al. (1995) and Devey et al. (1996) have proven to be very effective in extracting large quantities of pure genomic DNA from Pinus radiata needles. DNA isolated by these protocols was found to be very suitable for RAPD and RFLP analysis in our laboratory. Arbitary Bresatec and Operon primers were screened for polymorphism with DNA isolated by the methods from Jobes et al. (1995) and Devey et al. (1996). Both DNA isolation
DNA ISOLATIONFROM P. RADIATA methods produced clear RAPD bands; however, the sharpness was higher with the method of Jobes et al. (1995) (unpublished results). Further, DNA was digested with EcoRI and HindlII and were probed with 32p-labeled chloroplast probe as outlined in Muralitharan et al. (1994). Both DNA isolation methods produced similar results with this labeled probe (unpublished results). ACKNOWLEDGMENTS We thank Mr. Phillip Holgate, Mrs. Bozena Kuszyk, and Ms. Colleen MacMillan for their technical assistance and also Dr. Simon Stuart for his critical comments on the manuscript. This work was supported by an ANM Forest Management Research Grant awarded to Dr. Morley Muralitharan. REFERENCES Ausubel, F. M.; Brent, R.; Kingston, R., et al. Current protocols in molecular biology. New York: John Wiley and Sons; 1987:12-19. Bousquet, J.; Simon, L.; Lalonde, M. DNA amplification from vegetative and sexual tissues of trees using polymerase chain reaction. Can. J. For. Res. 20:254-257; 1990. Devey, M. E.; Bell, J. C.; Smith, D. N., et al. A genetic linkage map forPinus radiata based on RFLP, RAPD, and microsatellite markers. Theor. Appl. Genet. 92:673~79; 1996.
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Doyle, J. J.; Doyle, J. L. Isolation of plant DNA from fresh tissue. Focus 12:12-15; 1990. Guries, R. P.; Friedman, S. T.; Ledig, F. T. A megagametophyte analysis of genetic linkage in pitch pine (Pinus rigida Mill.). Heredity 40:309314; 1978. Jobes, D. V.; Hurley, D. L.; Thien, L. B. Plant DNA isolation: method to efficiently remove polyphenolics, polysaccharides and RNA. Taxon 44:379-389; 1995. Keller, G. H.; Manak, M. M. DNA probes. Macmillan Publishers, New York, NY; 1993:70-150. Muralitharan, M. S.; Stuart, S.; Graham, M. Methods in plant molecular biology techniques. Proceedings of Molecular BiologyWorkshop, Launceston, Tasmania. Published by Applied Biology,University of Tasmania, Launceston; 1994. Murray, M. G.; Thompson, W. E Rapid isolation of high molecular weight DNA. Nucleic Acids Res. 8:4221--4235; 1980. Nelson, C. D.; Kubisiak, T. L.; Stine, M., et al. A genetic linkage map of longleaf pine (Pinus palustris Mill.) based on random amplified polymorphic DNAs. J. Hered. 85:433439; 1994. Von Arnold, S.; Eriksson, T. A revised medium for growth of pea mesophyll protoplasts. Physiol. Plant. 39:257-260; 1977. Wagner, D. B.; Furnier, G. R.; Saghai-Maroof, M. A., et al. Chloroplast DNA polymorphisms in lodgepole and jack pines and their hybrids. Proc. Natl. Acad. Sci. USA 84:2097-2100; 1987.
