Analytical Biochemistry 343 (2005) 353–355

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ANALYTICALBIOCHEMISTRY

Notes & Tips

Coisolation of genomic and organelle DNAs from 15 genera and 31 species of plants

Mehmet Karaca ¤, Ayoe Gül Ince, SaWnaz Y. Elmasulu, A. Naci Onus, Kenan Turgut

Faculty of Agriculture, Akdeniz University, 07070 Antalya, Turkey

Received 5 March 2005Available online 30 March 2005

A large number of medicinal and aromatic plant spe-cies naturally grown in the Mediterranean basin of Tur-key contain and produce secondary metabolites such asalkaloids, Xavanoids, phenols, polysaccharides, terpenes,and quinones that are used in the food, pharmaceutical,cosmetic, and pesticide industries. Many medicinal andaromatic plant species are undergoing domesticationand cultivar development. Unfortunately some of theseplant species are among the most endangered specieswhich need to be protected to ensure their sustainableuse in this region. However, medicinal and aromaticplants under study contain exceptionally high amountsof polysaccharides, polyphenols, and other secondarymetabolites. Our preliminary studies showed that mostmedicinal and aromatic plant species were recalcitrant toseveral DNA extraction protocols tested including onecommercial DNA extraction kit (Qiagen, Valencia, CA,USA). Although the kit used produced high-qualityDNA, the amount of DNA was very low for many sam-ples and it could not produce DNA in some samples.Also several other protocols described for plant DNAisolation failed to produce good quality and quantity ofDNA or failed in PCR or restriction enzyme digestionsfrom some of the Origanum, Salvia, Thymus, Sideritis,Saturaja, Calaminta, Rosemarunus, and Nerium spp.,indicating that these plants contain exceptionally highamounts of secondary metabolites that interfere withDNA isolation [1–3]. These situations necessitate thedevelopment of protocols for isolating DNA from diVer-ent plant species. To address this problem we developed

* Corresponding author.E-mail address: mkaraca@akdeniz.edu.tr (M. Karaca).

0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2005.03.021

a suitable DNA isolation method for molecular biologi-cal applications of the plant species depicted in Table 1.

The protocol depicted in Table 2 can be modiWed forsmall-, medium-, and large-scale DNA extraction byincreasing the amount of tissues and chemical/solution ifrequired. We noted that addition of 0.35 M sorbitol toEB1 increased the amount of DNA extracted in compar-ison to sucrose and glucose (data not shown). Additionof lithium chloride (LiCl) into the extraction buVer elim-inated the use of RNase treatment; however, we still rec-ommend RNase treatment to eliminate any trace ofRNAs. The combined use of sodium acetate and isopro-panol during the precipitation of DNA eYcientlyremoved most of the secondary metabolites and polysac-charides from the DNA. However, precipitation of largequantities of sodium salt along with the DNA requiredresuspension of the pellet twice in ethanol.

The amount of extracted DNA varied depending onthe plant species, the physiological age, and the amountof the leaf tissues used. Results indicated that partiallyexpanded leaves were the best material for all of thesamples, consistent with the results previously reported

1 Abbreviations used: PVP, polyvinylpyrrolidone; CTAB, cetyl tri-methyl ammonium bromide; BME, �-mercaptoethanol; EDTA, ethyl-enediaminetetraacetic acid, disodium salt; EB, extraction buVer [0.35M sorbitol, 100 mM Tris–HCl, pH 7.5, 5 mM EDTA, pH, 7.5, 2%Tween, 1% Triton X-100, 1% BME]; LB, lysis buVer [200 mM Tris–HCl, pH 8.0, 50 mM EDTA, pH, 8.0, 2 M NaCl, 2% PVP, 2% CTAB,2% Triton X-100, 2% BME]; TE, [10 mM Tris–HCl, pH 7.5, 1 mM ED-TA, pH 7.5]; SLS, sodium lauryl sarcosinate; CIA, [chloroform:isoam-ylalcohol, 24:1]; NaAc, [sodium acetate, pH 5.2]; PCR, polymerasechain reaction; RAPD, random ampliWed polymorphic DNA; SSRLP,simple sequence repeat length polymorphism; DAMD, directed ampli-Wcation of minisatellite DNA; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism.

354 Notes & Tips / Anal. Biochem. 343 (2005) 353–355

by Mauro et al. [4]. Very old and fully expanded leavesyielded little DNA and in many cases the DNA was notcompletely digestible. However, we were able to getequally good results with old and fully expanded leaveswhen PVP was added during the homogenizing step andto the lysis buVer for older tissues. We recommend 1%increased amount of wetting agent (Tween 20 and TritonX-100), antioxidant (BME), PVP, and CTAB for DNAextraction on very old tissues. However, increasing theconcentration of these chemicals would reduce theamount of DNA.

DNA yield generated by use of the protocol reportedin this note was much higher than that using other pro-cedures for DNA extraction [5,6]. Doyle and Doyle [7]reported DNA yields up to 1 mg/g of fresh leaf tissuesfrom diVerent plant species. We have not been able toobtain such a high DNA yield. We also tried the proto-col of Doyle and Doyle [7]; however, DNA extracted bythat procedure was occasionally brownish in color forseveral species that we studied. We found that the meanvalue of the extracted total DNA was 589.4 �g/g, rangingfrom 370 to 1410 �g/g across the 31 samples belong to

Table 1List of 31 plant species within 15 genera and their DNA purities andextracted DNA amounts

a Based on the absorbance at 260 and 280 nm spectrophotometerreadings (average of two independent DNA isolations).

No. Species A260/280 ratioa DNAa (�g/g)

1 Origanum majorona 1.84 8102 Origanum onites 1.76 5503 Origanum vulgare 1.71 5604 Origanum saccatum 1.75 5105 Origanum spp. 1.66 5706 Origanum hirtum 1.61 5207 Salvia sclarea 1.85 6908 Salvia fritucosa 1.79 7909 Salvia tomentosa 1.82 750

10 Salvia virgata 1.88 53011 Salvia dicroantha 1.89 141012 Salvia spp. 1.83 66013 Phlomis spp. 1.78 77014 Salvia spp. 1.94 59015 Thymus sipleus 1.91 53016 Thymus longicoulus 1.76 57017 Calaminta incona 1.89 52018 Sideritis spp. 1.61 55019 Teucricum chomedris 1.86 98020 Rosemarunus oYcinalis 1.71 56021 Lavandula spp. 1.76 57022 Vicia faba 1.87 67023 Gossypium hirsutum 1.67 52024 Gossypium barbadense 1.76 58025 Nerium oleander 1.71 57026 Liquidamber orientalis 1.75 58027 Satureja thymbra 1.67 57028 Calaminta pomphylica 1.79 59029 Dianthus elegans 1.82 91030 Dianthus orientalis 1.62 60031 Dianthus coleaphalus 1.97 1290

Mean 1.78 673.2

the 15 genera (Table 1). The isolated DNA had normalspectra in which the absorbance ratios at 260/280 wereapproximately 1.8, indicating that large amounts ofcoprecipitants such as peptides, aromatic compounds,and polysaccharide and/or phenolic complexes were notpresent in the samples.

Restriction endonuclease digestions, PCR, and subse-quent agarose gel electrophoresis analyses clearlyindicated that DNA was completely digestible and freefrom common contaminating compounds. Results ofDNA restriction digestion with three endonucleases(EcoRI, EcoRV, and HindIII) clearly showed the

Table 2Step by step DNA extraction protocol for medicinal and aromaticplant species

1. Add 100–200 mg PVP (100 mg PVP per gram leaf tissue) to aprechilled mortar. Add 1–2 g leaf tissues to the mortar and grindto a powder in the presence of liquid nitrogen using a pestle.

2. Pour the slurry powder directly into a 15- or 30-ml polypropylenecentrifuge tube (Nalgene Oak Ridge) and incubate on ice for10 min or until the liquid nitrogen is totally evaporated.

3. To each tube add 6 ml EB and vortex thoroughly, avoidingaggregation of the homogenate for 3–5 min.

4. Mix the homogenate by several inversions and centrifuge for 10min at 8800–9000g (4 °C).

5. Gently pour oV the supernatant and add 4 ml solutions containing2 ml LB and 2 ml 8 M LiCl to the pellet; mix well by thoroughlyvortexing and inversions.

6. Add 1 ml of 5% SLS to the homogenate and mix well by vortexingand inversions.

7. Incubate the samples at 65 °C for 30–35 min with occasionalmixing to avoid aggregation of the homogenate.

8. Transfer the tubes on ice and add 5 ml CIA; mix thoroughly byinversion. Repeat step 4.

9. Transfer the supernatant to a clean tube, add equal volume ofchloroform:isoamylalcohol repeat centrifuge the tubes at 4000–5000g for 5 min to clear the aqueous phase. If the aqueous phase isnot clear, repeat step 8 before DNA precipitation.

10. Transfer the supernatant into a clean tube, add equal volume ofisopropanol and 5 M NaCl to a Wnal concentration of 1/20(v/v);mix well by inversion to precipitate the DNA. At this step tubescan be stored at 4 °C for several weeks. Alternatively, samples canbe kept in a ¡20 °C freezer to allow more precipitation of theDNA.

11. Centrifuge the samples at 4000–5000g for 3 min (4 °C), gently pouroV the supernatant, remove the remaining solution using a pipette,and dry the pellet brieXy in air.

12. Resuspend the pellet in 1 ml TE buVer and 0.1 ml 3 M NaAc. Tothe solution add RNase A to a Wnal concentration of 10 � g/ml andincubate at 37 °C for 30 min.

13. Transfer the tubes on ice and add 3 volumes of absolute ethanol tothe solution; centrifuge at 4000–5000g for 3 min (4 °C).

14. Gently pour oV the supernatant, remove the remaining solutionusing a pipette, and resuspend the pellet in 1 ml TE buVer and0.1 ml of 3 M NaAc.

15. Add 3 volumes absolute ethanol; centrifuge at 4000–5000g for3 min (4 °C). Repeat this step with 3 volumes of 70% ethanol.

16. Gently pour oV supernatant and remove the remaining solutionusing a pipette. Completely remove ethanol without drying theDNA pellet by leaving the tubes uncovered at 37 °C for 20–30 min.

17. Resuspend the pellet in 0.5 or 1 ml TE and store the DNA at 4 °Cfor short-term storage or at ¡20 °C for long-term storage.

Notes & Tips / Anal. Biochem. 343 (2005) 353–355 355

complete digestion, indicating that the uncut DNA issuitable for Southern hybridization and for SSRLP,RFLP, and RAPD (Fig. 1) approaches employed in plantbreeding, genotypic diVerentiation, gene linkage analysis,assignment of evolutionary and taxonomic aYnities andrelatedness, and DNA Wngerprint analysis [8,9].

We used chromosomal-speciWc SSR primer pairs oncotton total DNA to conWrm the existence of ampliWablegenomic DNA. Also PCR analyses indicated thatextracted total DNA contained chloroplast and mito-chondrial DNA (Fig. 1). PCR-based ampliWcation ofDNA is simple but the proWles are highly susceptible tosubtle diVerences in reaction mixture composition, qual-ity and quantity of template DNA, and ampliWcationconditions. To minimize time and eVort, the DNA prep-arations should be identical with respect to purity, integ-rity, and quantity. Thus a suitable DNA extractionmethod for many species is very important for PCRapplications.

In summary a modiWed CTAB-based DNA extractionprotocol to obtain large-scale, high-molecular-weight,

Fig. 1. Demonstration of high molecular mass and restrictable andPCR-ampliWable extracted total DNAs. (A) Lane 1, genomic DNAmarker (30 kb); lanes 2–30 and 1–32 in (B) are undigested and digestedgenomic DNAs from the plant species depicted in Table 1 in the sameorder. (C) Lanes 1–31 are ampliWed products of plant species depictedin Table 1 using chloroplast-speciWc primer pairs. (D) Lanes 1–31 areampliWed products of plant species depicted in Table 1 using mito-chondrial genome-speciWc primer pairs. (E) RAPD (E1), SSRLP (E2),DAMD (E3), and PCR-RFLP (E4) agarose gel electrophoresis.

restrictable, and ampliWable nuclear, mitochondrial, andchloroplast DNAs from several ornamental, medicinal,and aromatic plants along with some other crop specieswidely grown in the Mediterranean region of Turkey isdescribed . Spectrophotometry, restriction enzyme diges-tions, PCR analyses, and agarose gel separation studiesconWrmed that large-scale, high-molecular weight, andrestrictable DNAs were obtained. Mean of the extractedtotal DNA was 673.2�g/g, ranging from 510 to 1410�g/gacross the 31 species belong to 15 genera. Coisolation ofgenomic and organelle DNAs was also conWrmed usingorganelle and nuclear DNA-speciWc primers. Suitabilityof the extraction method for DNA Wngerprinting tech-niques such as RAPD, SSRLP, DAMD, and PCR-RFLPwas conWrmed. This DNA extraction method might beuseful for direct comparison and classiWcation of a vari-ety of plants including the endemic ornamental, medici-nal, and aromatic plant species.

Acknowledgments

This research was funded by the ScientiWc ResearchProjects Administration Unit of Akdeniz University(Project No. 2004.03.0121.009). The authors thank Saa-det Tufrul Ay, a PhD candidate, for collecting andmaintaining the medicinal and aromatic plant species.

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