Plant Molecular Biology Reporter15: 319–325, 1997. 319c 1997Kluwer Academic Publishers. Printed in Belgium.

Commentary

DNA Isolation and Amplification from Cacti

Marlene de la Cruz1;�, Fabiola Ramirez1 and Hector Hernandez2

1Instituto de Ecologia, UNAM Apdo. Postal 70–275 UNAM 04510 Mexico;2Instituto de Biologia, UNAM Apdo. Postal 70–233 UNAM 04510 Mexico;

Current Address: Department of Botany and Plant Sciences, University of California,Riverside CA 92521, USA

Key words: cacti, DNA isolation

Abstract: The cacti family is a morphologically heterogeneous group comprising 100genera and about 1500 species (Hernandez and Barcenas, 1996). With the exception ofone genus, all members of this family are native to America (Hernandez and Barcenas,1996). There are three subfamilies, Opuntioideae, Cactoideae, and Pereskioideae (Gib-son and Nobel, 1986). DNA isolation from cacti is notoriously difficult because theycontain high amounts of polysaccharides and secondary metabolites which form insol-uble complexes with nucleic acids during extraction (Guillemaut and Marechal-Drouard,1992). Like in other groups of plants, the secondary metabolites and polysaccharidesin cacti inhibit enzyme action (Porebski et al., 1997). The polysaccharides are visuallyevident by their viscous, glue-like texture and they make the DNA unmanageable whenpipeting and hard to amplify by the polymerase chain reaction (PCR) (Poresbski et al.,1997). We report an easy and inexpensive protocol to isolate DNA from cacti. We usedthis method to isolate DNA from 85 species (170 individuals) of 39 genera of the sub-families Pereskioideae, Opuntioidea, and Cactoideae. This procedure is a modificationof a protocol described by De la Cruz et al. (1995) for the Cacti family. It requires onlya few grams of tissue and does not require destruction of the whole plant to producehigh molecular weight genomic DNA. The DNA from this procedure can be amplifiedconsistently by PCR and used for RAPD analysis.

�Corresponding author.

Abbreviations: PVP, Polivinilpyrydone 40,000; DIECA, Diethyldithiocarbamic acid;SDS, Disodium Lauryl Sulfate; PCR, Polymerase Chain Reaction.

320 de la Cruz, Ramirez and Hernandez

Material and Methods

The cacti genera used for analysis are listed in Table 1. Plants werecollected from the wild in Chihuahua, San Luis Potosi, Queretaro, andZacatecas in Mexico. Small cacti were transplanted into a greenhousewhere a small portion was sampled. For large cacti, one of the ridgeswas sampled and kept for 8 days at room temperature until use.

Reagents� CTAB extraction buffer (100 mM tris-HCl pH 8, 20 mM EDTA pH

8, 4% CTAB, 1.5 M NACl, 4% PVP-40, 500 g ascorbic acid, 500 gDIECA and 10 mM 2-Mercaptoethanol).

� STE extraction buffer (100 mM Tris-HCl pH 8, 50 mM EDTA pH8, 100 mM NaCl, 10 mM 2-Mercaptoethanol).

� 20% SDS.� 5 M Potassium acetate.� TE buffer (10 mM Tris, 1 mM EDTA).� PCR Reaction buffer (Promega, 50 mM KCl, 10 mM Tris HCl, pH

9, 0.1% Triton X-100).� Primers: Operon and OLIGOToGo (RAPDs and chloroplast DNA).� TaqDNA Polymerase (Gibco).� Nucleotides (Pharmacia).

Protocol

DNA extraction� Grind 3 g of tissue to a fine powder in liquid nitrogen.� Add 4 ml of CTAB extraction buffer with further grinding to produce

a slurry.� Add 15 ml of STE extraction buffer and transfer the solution to a

50 ml Oak Ridge tube.� Add 1 ml of 20% SDS with vigorous shaking for 7 min.� Incubate at 65�C for 10 min.� Add 5 ml of cold 5 M potassium acetate and incubate at 0�C for

40 min.� Spin tubes at 20,000 RPM for 20 min to remove debris and the

supernatant.

DNA Isolation and Ampli�cation from Cacti 321

Table I. Yield of DNA extracted from members of the cacti family.

Subfamily and Genus Number of Species Average (range), ng/�l

Subfamily PereskioideaePereskiospis 1 35 (35)Subfamily OpuntioideaOpuntia 4 540 (250-2000)Nopalea 1 20 (15-20)Subfamily CactoideaeAztekium 1 200 (150-200)Acanthocereus 1 47 (27–47)Ariocarpus 6 168 (30–270)Astrophytum 2 100 (60–100)Cephalocereus 2 80 (80–84)Coryphanta 2 558 (363–933)Echinocereus 3 44 (10–125)Echinocactus 1 89 (10–230)Escobaria 1 860 (600–1125)Epiphyllum 1 15 (10–20)Epythelanta 1 33 (30–40)Escontria 2 130 (130–132)Ferocactus 4 73 (36–130)Geohintonia 1 500 (250–500)Hylocereus 1 20 (15–20)Leuchtenbergia 1 345 (275–415)Lophophora 1 10 (10–27)Mammillaria 19 282 (10–460)Mammylloidea 1 113 (76–120)Melocactus 1 150 (100–200)Myrtllocactus 1 620 (40–1200)Neobuxbamia 1 210 (120–300)Neolloydia 1 10 (10–15)Pachicereus 1 210 (120–300)Pelecyphora 2 480 (322–633)Pilocereus 2 25 (12–35)Poniocereus 1 30 (15–35)Polasfia 1 500 (500)Rhipsalis 1 27 (15–30)Sclerocactus 1 300 (173–428)Selenicereus 2 70 (35–120)Stenocereus 2 25 (25–30)Stenocactus 2 118 (100–240)Strombocactus 1 200 (150–250)Thelocactus 5 195 (20–465)Turbinicarpus 3 303 (33–360)

322 de la Cruz, Ramirez and Hernandez

� Filter the aqueous phase through a Miracloth filter into a clean 50 mlOak ridge tube.

� Add 7/10 vol of cold isopropanol, mix gently and incubate at�20�Cfor 10 min to precipitate genomic DNA.

� Spin the tubes at 20,000 RPM for 15 min and discard the super-natant, air-dry the pellet and resuspended in 1 ml of TE.

� Transfer the solution into a 1.5 ml tube and spin in a microcentrifugefor 10 min.

� Transfer the supernatant into a new 1.5 ml tube and add 65�L of3 M sodium acetate and 600�l of cold isopropanol and gently mix.Incubate at�20 �C for 10 min.

� Spin for 30 s and wash the pellet carefully with 76% ethanol.� Resuspend the pellet in 1 ml of TE.DNA yields were determined using a model TKO100 mini-

fluorometer (Hoefer Scientific Instruments) following the manurac-turer’s protocols. Before the PCR reaction, all samples were treated withRNAse (Cheol-Sik and Dean, 1993). Some genera, such as Mammillariaand Myrtillocactus, contain 90% water (Nobel and Gibson, 1987) andcan be ground with CTAB buffer without liquid nitrogen.

DNA amplificationAmplification of RAPD fragments from genomic DNA was carried outin a total reaction volume of 25�L containing 10 ng of genomic DNA,1X Taq polymerase reaction buffer, 2 mM MgCl2, 0.1 mM each ofdATP, dCTP, dGTP, and dTTP, 0.2�M decamer primer (Operon A10,A11, A12, A13, G7, G10, G12, G13, F03) and 1.5 U ofTaq DNApolymerase. Each reaction was overlaid with 30�l of sterile mineral oil.Amplifications were performed in a model PTC-100 thermocycler (MJResearch) programmed for 45 cycles of 1 min at 94�C, 1 min at 38�C,30 s at 54�C, 2 min at 72�C with a final 15 min extension of 72�C.Amplification products were separated on 2% agarose (GibcoBRL) TAEgels run at 3.2 V/cm for 4 h. Gels were stained with ethidium bromide(0.5 mg/ml) and photographed under UV light.

Amplification of chloroplast DNA fragments from total DNA wascarried out in a 50�L reaction volume containing 5 ng of total DNA,1X Taq polymerase reaction buffer, 2 mM MgCl2, 0.1 mM each ofdATP, dCTP, dGTP, and dTTP, 0.2 mM of primer for chloroplast DNAregion (OLIGOTOGO cp1A-cp1B, cp2A-cp2B, cp3A-cp3B, cp6A-

DNA Isolation and Ampli�cation from Cacti 323

cp6B, cp7A-cp7B) and 1.5 U ofTaq DNA polymerase. Each reactionwas overlaid with 30�L of sterile mineral oil. Amplifications were per-formed in a thermocycler (MJ Research) programmed for 34 cycles of1 min at 94�C, 1 min at 58�C, 2.30 min at 72�C with a final 5 min exten-sion at 72�C. PCR amplification products were cut withDraI, HindIIIandHaeI restriction enzymes for 1 h at 37�C. Products were separ-ated on 1.4% agarose (GibcoBRL) TAE gels run at 3.2 V/cm for 3 h.Gels were stained with ethidium bromide (0.5 mg/ml) and photographedunder UV light.

Results and Discussion

We found the standard procedures e.g., Murray and Thompson 1980;Dellaporta et al., 1983) unreliable for isolation of DNA from cacti.Using the protocol described above, the average DNA yield from allgenera was 179 ng/�L. DNA yields per individual ranged from 60 ng/g(Stenocereus spp.) to 2000 ng/g (Opuntia spp.) of fresh tissue (Table 1).This is the first report of DNA isolation from 39 genera (85 species) ofcacti.

Fresh tissue from cacti present large amounts of polyphenolics andpolysaccharides that co-precipitate with DNA and affect subsequentPCR amplification (Flament, 1989). The method presented here reducesthe amount of these contaminants. We found that the addition of the ini-tial CTAB step significantly reduced the visible amount of polysachar-ides in DNA samples. In all species, all RAPD and chloroplast primersproduced a constant and reproducible banding pattern across all samplesin two independent replicates. (for example, see Fig. 1) Variation in theability to produce RAPD and chloroplast DNA fragments depended onthe primer and the genus.

Significant modifications to the method reported by De la Cruz et al.(1995) include higher CTAB and PVP concentrations to remove poly-phenols, and higher concentrations of ascorbic acid, DIECA, and mercp-atoethanol to reduce oxidation. We also found it important to use freshand young tissues. Our protocol does not require chloroform:phenolextraction or cesium chloride centrifugation. It is simple and easy and isa modification of a protocol used successfully with other plant families

324 de la Cruz, Ramirez and Hernandez

Fig 1. Amplification of a DNA in different cacti species.Lane 1 molecular weighmarker 1 Kb, lane 2, 3Thelocactusp.s, lane 4, 5Echinocactussp., lane 6, 7Ariocarpussp. DNA amplified using RAPDs with primer OPA11.

that have high concentration of polyphenols and polysaccharides (De laCruz et al., 1995).

Acknowledgements:This work was supported by DGAPA (Direccion General de Asun-tos del Personal Academico)/UNAM (IN206495) grant to H. Hernandez and M. de laCruz.

References

De la Cruz, M.R. Whitkus, L.M. Mota-Bravo. 1995. Tropical tree DNA isolation andamplification. Mol. Ecol. 4: 787–789.

Dellaporta, S.J., J. Wood, J.B. Hicks. 1983. A plant DNA minipreparation: Version II.Plant Mol. Biol. Reptr. 1: 19–21.

Flament, I. 1989. Coffee, cocoa and tea. Food Rev. Int. 5: 317–414.Gibson, A.C., P.S. Nobel. 1986.The cactus primer, pp. 18, 188–206. Harvard University

Press.Guillemaut, P., L. Marechal-Drouard. 1992. Isolation of plant DNA: a fast, inexpensive

and reliable method. Plant Mol. Biol. Reptr. 10: 60–65.

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Hernandez, M.H., R.T. Barcenas. 1996. Endangered cacti in the Chihuahuan Desert: II.Biogeography and conservation. Con. Biol. 10: 1200–1209.

Murray, M.G., W.F. Thompson. 1980. Rapid isolation of high molecular weight plantDNA. Nucleic. Acid. Res. 8: 4321–4325.

Porebski, S.L., G. Bailey, R.B. Baum. 1997. Modification of a CTAB DNA extractionprotocol for plants containing high polysaccharide and polyphenol components.Plant Mol. Biol. Reptr. 12: 8–15.