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A Simple and Efficient Method for Isolation of DNA in High Mucilaginous Plant Tissues Ileana Echevarría-Machado, Lucila A

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A Simple and Efficient Method for Isolation of DNA in High Mucilaginous Plant Tissues Ileana Echevarría-Machado, Lucila A DNA Isolation From MalvaceaePROTOCOLS 129 A Simple and Efficient Method for Isolation of DNA in High Mucilaginous Plant Tissues Ileana Echevarría-Machado, Lucila A. Sánchez-Cach, Cecilia Hernández-Zepeda, Renata Rivera-Madrid, and Oscar A. Moreno-Valenzuela* Abstract A protocol is described for rapid DNA isolation from Malvaceae plant species and different tissues of Bixaceae that contain large amounts of polysaccharides, polyphenols, and pigments that interfere with DNA extractions. The method is a modification of Dellaporta et al. The current protocol is simple, and no phenol- chloroform extraction, ethanol, or isopropranol precipitation is required. The method is based in the incuba- tion of soluble DNA with silica, mix in batch during the extraction. The procedure can be completed in 2 h and many samples can be processed at the same time. DNA of excellent quality was recovered and used for polymerase chain reaction (PCR) amplification, restriction enzyme digestion, and Southern blot analysis. The method was used with healthy Bixa orellana and virus-infected Malvaceae plants. Index Entries: DNA extraction; Malvaceae; PCR; Bixa orellana. 1. Introduction Bayer et al. (4) reported that many Malvaceae The extraction of high-quality DNA is an impor- samples contain high amounts of mucilage, and it tant step in many molecular biology protocols, and is difficult to remove the mucilaginous superna- several factors alter the amount, purity, and qual- tant after centrifugation without losing most of the ity of the extracted molecules. The method de- DNA. Jose and Usha (5 ) described a protocol for the scribed is a modification of the one by Dellaporta extraction of geminiviral DNA from Abelmoscus et al. (1). Plant species of the Malvaceae family esculentus, a Malvaceae plant. This method involves are characteristically high in polysaccharides and a combination of Dellaporta et al. (1), the CTAB other secondary metabolites, which interfere with (cetyltrimethylammonium bromide) method (6), effective DNA extractions and polymerase chain and alkali lysis. Other techniques for DNA extrac- reaction (PCR) amplification. Polysaccharides tion from different highly mucilaginous plants, have viscous, glue-like texture and make the whichh include modifications of the CTAB-based DNA unmanageable in pipetting and unsuitable protocols, have been reported (7–9). for PCR because polysaccharides inhibit Taq Similar problems in DNA extraction occurs polymerase activity (2). Many protocols for with Bixa orellana, the only species within the DNA extraction from Malvaceae plant species family Bixaceae. This plant produces a variety of rich in polysaccharides, polyphenols, and latex secondary metabolites in all of its tissues. The high have been described. However, these protocols content of phenols and polysaccharides represents are generally time consuming or expensive (3). a problem in the extraction of high-quality DNA. *Author to whom all correspondence and reprint requests should be addressed. Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, A. C. Calle 43 No. 130, Col. Chuburná de Hidalgo, CP 97200, Mérida, Yucatán, México. E-mail: [email protected]. Molecular Biotechnology © 2005 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2005/31:2/129–136/$30.00 MOLECULAR BIOTECHNOLOGY 129 Volume 31, 2005 129_136_Moreno_MB04_0071 129 8/29/05, 3:28 PM 130 Echevarría-Machado et al. Molecular studies in B. orellana are very scarce 2. Materials despite its economical value because of the 2.1. Plant Material large amounts of bixin found in the seeds. This Malvaceae plant species with viral symptoms apocarotenoid is widely used as a colorant in the were collected in different locations in the Yucatan food industry (10). In this case, the quality of the Peninsula, stored on ice after collection, and stored DNA isolated is a critical step in molecular and at –80°C until used. Dry herbarium material of genetic studies. Narváez et al. (11) used a meth- Malvaceae species were collected from plants that odology for genomic DNA isolation from B. orellana showed possible viral symptoms from the Her- tissues, previously reported by Rogers and Bendich barium CICY. Three leaves of each herbarium (12). This method is based on the CTAB extrac- sample were taken and stored at –80°C until tion procedure. This technique uses extraction DNA was extracted. Different tissues of adult with chloroform/isoamyl alcohol, precipitation plants of B. orellana L. (Annatto) var. Criolla with ethanol, and others steps that are time con- such as leaf, flower buds, open flowers, fruits, and suming. Also, a CTAB extraction procedure does seed were collected during the flowering and fruit- not guarantee the elimination of some contami- ing period (September through October) at “La Ex- nants of DNA such as polysaccharides and lipids. tra” plantation located in Chicxulub Pueblo, When DNA from B. orellana was extracted fol- Yucatán, México. Fresh tissues were immediately lowing Rogers and Bendich’s method (12), a wide frozen in liquid nitrogen and kept at –80°C for variation in the yield of nucleic acids was ob- later use. tained, as a result of the interference by their high polysaccharide content. 2.2. Equipment and Reagents Despite isolation of specific probe for analyz- 1. Mortar and pestle. ing the expression of mRNAs of enzymes related 2. 2.0-mL eppendorf tubes autoclaved. with isoprenoid biosynthesis in B. orellana (13), 3. Extraction buffer: 10 mM Tris-HCl, 50 mM the isolation of complete genes has been very dif- EDTA (ethylendiaminetetraacetic acid), 500 ficult in this species using plant DNA extracted mM NaCl (sodium chloride), 10 mM β-mercap- by reported methods. This is possibly because of toethanol, pH 7.0. the presence of a contaminant in DNA extract that 4. 20% sodium dodecyl sulfate (SDS). interferes with the gene isolation process. 5. 5 M potassium acetate. The protocol reported here produces clean high 6. Silica (Sigma S5631). Resuspended 2 g of molecular weight genomic DNA from fresh or silica in 15 mL of distilled water and spun at herbarium material of Malvaceae plant species 756g for 1 min. Eliminated the milky superna- that presented viral symptoms, without phenol, tant. Repeated twice and resuspended silica in 50 mL of distilled water. Solution could be ethanol precipitation, or cesium chloride gradient. stored at room temperature. Also, the method yields DNA extract from differ- 7. RedTaq polymerase (Bioline). ent healthy tissues of B. orellana, without the vis- 8. Restriction enzymes (Invitrogene). cous appearance that indicates the presence of a 9. dNTPs mix (Bioline). high polysaccharide content. Both DNA extracts 10. PCR thermal cycler (96-well plate format). from B. orellana tissues and Malvaceae species were used in analytical applications, such as re- 3. Methods striction digestion, Southern blot analysis, and 3.1. DNA Extraction PCR, producing a good restriction pattern and The method developed by Dellaporta et al. (1) amplification, as indicated by stain with ethidium was modified to recover DNA from leaves of bromide. Malvaceae plant species: Corchorus siliquosus, MOLECULAR BIOTECHNOLOGY Volume 31, 2005 129_136_Moreno_MB04_0071 130 8/29/05, 3:28 PM DNA Isolation From Malvaceae 131 Malvastrum coromandelianum, Abutilon permolle, 35 cycles of 95°C for 1 min, 55°C for 1 min, and Hibiscus sabdariffa, Sida acuta, Herissantia crispa, 72°C for 1 min, and one final cycle at 72°C for Anoda cristata, Sida rhombifolia, Bastardia 10 min, 25°C for 20 min, and 4°C infinite. viscosa, and Bixaceae spp., B. orellana. The amplified products were assayed by elec- trophoresis on 1% agarose gels and stained with 1. Homogenized 0.1 g of leaf material or others tissues with 1 mL of extraction buffer, trans- ethidium bromide. ferred to a 2.0-mL eppendorf tube, and added 3.2.2. Restriction Analysis 100 µL of 20% SDS. After mixing, incubated ° Ten DNA samples of different Malvaceae spe- at 65 C for 10 min. cies were digested by overnight restriction with µ M 2. Added 500 L of 5 potassium acetate. Shook EcoRI. The susceptibility of genomic DNA from tube vigorously and incubated for 20 min on ice. B. orellana leaf tissue to cleavage by EcoRV, 3. Spun tubes at 16,000g for 20 min. BamHI, HindIII, Xba I, Sac I, and Sau 3A was 4. Transferred supernatant to a new 1.5-mL tube and add 300 µL of silica, mixed manually for determined by overnight restriction with these 3–5 min. enzymes. 5. Spun tubes at 16,000g for 30 s. 3.2.3. Southern Blot Analysis (see Note 5) 6. Washed pellet twice with 70% ethanol. Dried Four hundred nanograms of total DNA from dif- the pellet. ferent Malvaceae species (Corchorus siliquosus, 7. Resuspended the pellet in 50 µL of distilled Malvastrum coromandelianum, Abutilon permolle, water and incubated at 55°C for 5 min. Hibiscus sabdariffa, Sida acuta, Herissantia crispa), 8. Spun tubes at 16,000g for 2 min and transferred supernatant to a new 500-µL eppendorf tube. were electroforesed in 1% agarose gel (without 9. Used an aliquot for quantification of total DNA ethidium bromide), and transferred to Hybond (+) in gel electrophoresis with 1% agarose. nylon membranes (Amersham, Arlington Heights), by standard protocols. Hybridization reaction 3.2. DNA Analysis were performed with a Alkaline phosphatase labeled 3.2.1. Polymerase Chain Reaction (Alkphos Direct Hibridization kit, Amersham) core Genomic DNA from Malvaceae species: 50 ng Coat protein probe of the Pepper golden mosaic of template DNA extract were added to a final virus (576 nt fragment), as recommended by the volume of 25 µL containing a previously stan- supplier. dardized PCR master mix. Core Coat Protein frag- 4. Notes ments of begomoviruses were obtained by PCR 1. In the protocol reported here, no phenol–chlo- amplification, using 20 pmol of degenerate prim- roform, ethanol, or isopropanol precipitation ers AV324 and AC1154, capable of universal were used, and we obtained clean high molecu- amplification of most begomoviruses (14).
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