what is the texture and consistency of dna extracted according to the method used in this lab

A simple and efficient method for Dna extraction from grapevine cultivars, Vitis species and Ampelopsis.

Lodhi, Muhammad A., Guang-Ning Ye, Norman F. Weeden and Bruce I. Reisch. 1994.

Plant Molecular Biology Reporter 12(one): 6-thirteen.

Department of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456.

Primal Words: Dna extraction, Vitis sp., polyphenols, polysaccharides, RAPD, restriction digestion.

A quick, uncomplicated and reliable DNA extraction method for grapevine species, hybrids and Ampelopsis (Vitaceae) has been developed. This method is a modification of Doyle and Doyle (1990). It is a CTAB-based extraction procedure modified past the use of NaCl to remove polysaccharides and PVP to eliminate polyphenols during Dna purification. The method as well has been used successfully for extraction of total DNA from other fruit species such every bit apple (Malus domestica), apricot (Prunus armeniaca), cherry (Prunus avium), peach (Prunus persica), plum (Prunus domestica) and raspberry (Rubus idaeus). Deoxyribonucleic acid yield from this procedure is high (upwardly to 1 mg/g of leaf tissue). Deoxyribonucleic acid is completely digestible with restriction endonucleases and amplifiable in the polymerase chain reaction (PCR), indicating freedom from common contaminating compounds.

Vitis vinifera and related species accept been the subject of extensive genetic studies due to their worldwide cultivation and importance. Recently this institute has been used for gene mapping (Yamamoto et al., 1991; Mauro et al., 1992; Weeden et al., 1992; Lodhi et al., 1992a; 1992b;1993; Hain et al., 1993), genetic transformation (Baribault et al., 1989; Baribault et al., 1990; Hébert et al., 1993), and Dna fingerprinting (Striem et al., 1990; Bourquin et al., 1991). The relatively small genome size of Vitis vinifera (0.50 pg/C) compared to many other perennial plant species (Arumuganathan and Earle, 1991) should facilitate molecular genetic studies of Vitis. However, Deoxyribonucleic acid extraction from grapevine has been difficult due to the presence of contaminants such as polyphenols and polysaccharides. These compounds have besides been reported to crusade difficulty in DNA purification in other plant species; polysaccharides (Murray and Thompson, 1980; Fang et al., 1992); polyphenolic compounds (Katterman and Shattuck, 1983; Couch and Fritz, 1990; Howland et al. 1991; Collins and Symons, 1992); and pasty and resinous materials (Webb and Knapp, 1990). The presence of these contaminants in Dna preparations ofttimes makes the samples viscous and renders DNA unrestrictable in endonuclease digestion and unamplifiable in PCR. The existing DNA extraction protocols frequently produce unsatisfactory yields and/or quality (Bourquin et al., 1991; Collins and Symons, 1992).

Here we report a uncomplicated, inexpensive and quick DNA extraction procedure for grapevine Vitis species, hybrids and Ampelopsis. This procedure purifies greater amounts of clean Dna which tin exist amplified via PCR or digested with endonucleases.

See Table I for the source of plant material used in this study.

Extraction buffer: xx mM sodium EDTA and 100 mM Tris-HCl, adjust pH to 8.0 with HCl, add one.iv Yard NaCl and 2.0% (w/v) CTAB (cetyltrimethylammonium bromide). Dissolve CTAB past heating to threescore�C. Shop at 37�C. Add 0.2 % of ß-mercaptoethanol merely before use.

Chloroform:octanol 24:i (v/v)

5 M NaCl

TE buffer: 10 mM Tris-HCl and 1 mM EDTA, adjust pH to 8.0 and autoclave

RNAase A (Sigma R9009: ten mg/mL)

Protocol

• Collect unexpanded young leaves in liquid nitrogen or on ice and store at or beneath -70°C until used. Avert thawing earlier grinding the leaf tissue. Grind 0.5 thou of leaves using mortar and pestle in the presence of liquid nitrogen. Although leaves should be thoroughly crushed before adding extraction buffer, information technology is important not to grind the leaves into a very fine powder as it results in shearing of DNA.

• Add v mL of extraction buffer to the basis leaves and mix in the mortar.

• Cascade the slurry into clean 15 mL polypropylene centrifuge tubes (Laboratory Product Sales, Rochester, New York; Threescore 4109), rinse the mortar and pestle with 1 mL of extraction buffer and add to the original extract.

• Add together l mg polyvinylpolypyrrolidone (PVP), (Sigma, P6755) and invert the tubes several times to mix thoroughly with the leafage slurry (100 mg PVP/grand leafage tissue).

• Incubate at 60°C for 25 minutes and cool to room temperature.

• Add half dozen mL of chloroform:octanol and mix gently by inverting the tubes xx to 25 times to form an emulsion.

• Spin at 6000 rpm for fifteen minutes in a table meridian centrifuge at room temperature.

• Transfer the top aqueous phase to a new fifteen mL centrifuge tube with a broad-bore pipette tip. A 2nd chloroform:octanol extraction may exist performed if the aqueous stage is cloudy due to the presence of PVP.

• Add 0.v book of 5M NaCl to the aqueous solution recovered from the previous footstep and mix well.

• Add together two volumes of cold (-20°C) 95% ethanol and refrigerate (four to 6°C) for 15-20 minutes or until DNA strands begin to appear. The solution can be left for one hour or more than if necessary.

• Spin at 3000 rpm for three minutes and and then increment speed to 5000 rpm for an boosted three minutes at room temperature. This differential spinning step helps to go along DNA at the bottom of the centrifuge tube.

• Cascade off supernatant and wash pellet with common cold (0 to four°C) 76% ethanol. Completely remove ethanol without drying the DNA pellet by leaving the tubes uncovered at 37°C for 20 to 30 minutes.

• Dissolve in 200 to 300 �Fifty TE.

• Treat with i �L RNAase A per 100 �50 DNA solution and incubate at 37°C for fifteen minutes.

• Quantify DNA in a spectrophotometer at A 260.

• Continue Dna at -70�C for long term and -twenty°C for short term storage.

We take obtained higher yields of clean DNA from grapevine leaves past using the modified Deoxyribonucleic acid extraction procedure outlined above. The process used for Deoxyribonucleic acid extraction is CTAB-based and is modified from Doyle and Doyle (1990). NaCl has been used to remove polysaccharides (Fang et al., 1992), and PVP to purge polyphenols (Maliyakal, 1992). This procedure does not involve CsCl density gradient purification steps.

Deoxyribonucleic acid yields from Vitis species, Ampelopsis and other woody perennial plant species by the above mentioned procedure range from 0.5 to 1.0 mg/m fresh leaf tissues with A 260 /A 280 between 1.8 and 2.0 (Table 1). The procedure is fast and simple and xxx to xl Dna samples may be processed in a single day. Results of Dna restriction digestion with three endonucleases (EcoRI, EcoRV and HindIII) showed complete digestion (Fig. 1a). It is also evident that the uncut DNA exhibits piddling shearing and is suitable for Southern (1975) hybridization (Fig. 1b). The Dna is likewise amplifiable in PCR using the RAPD technique (Williams et al., 1990) (Fig. 2).

Proper choice of the foliage tissue is very of import for DNA extraction. The apply of very young leafage tissues has resulted in poor yields. We found that partially expanded leaves are the all-time material. This is consequent with the results reported by Mauro et al. (1992), in which the all-time results were obtained from apace expanding leaves, i to 2 nodes from the shoot tip. With fully expanded leaves the yield was low and the Deoxyribonucleic acid was not completely digestible. However, we were able to get equally practiced results with fully expanded leaves when PVP was added to the extraction buffer. PVP has been used to remove polyphenols from mature, damaged and improperly stored leaf tissues (Rogers and Bendich, 1985; Doyle and Doyle, 1987, Howland et al., 1991). PVP forms complex hydrogen bonds with polyphenolic compounds which can be separated from DNA past centrifugation (Maliyakal, 1992). The presence of polyphenolic compounds can be reduced by keeping found material frozen before extraction and by using PVP in the DNA extraction procedure. The developmental phase of the constitute is also important. The optimal time for leaf collection was during the flow of active shoot elongation post-obit bud pause. Later in the flavor Deoxyribonucleic acid extraction was difficult and the DNA obtained was unstable for long term storage.

Complete digestion with restriction endonucleases and amplification in PCR point the absence of polysaccharides. Polysaccharides are hard to separate from Deoxyribonucleic acid (Murray and Thompson, 1980). These compounds are easily identifiable in the Deoxyribonucleic acid preparations every bit they impart a sticky, mucilaginous consistency to the DNA preparations dissolved in TE buffer. Polysaccharides interfere with several biological enzymes such as polymerases, ligases and restriction endonucleases (Shioda et al., 1987; Richards, 1988). We found that when polysaccharides were not removed the DNA would non amplify. PCR amplification of the Deoxyribonucleic acid with several ten base of operations long oligonucleotides and consummate DNA restriction results are consequent with these results. DNA amplification was possible due to the absence of contaminants (Webb and Knapp, 1990; Fang et al., 1992). Fang et al. (1992) institute that 1 Thou NaCl facilitated the removal of polysaccharides by increasing their solubility in ethanol and then that they did non co-precipitate with the Dna. Yet, we found higher concentrations of NaCl (more than than ii.five M) were more than effective with the species under study.

The simplicity of the procedure makes it very applied for Deoxyribonucleic acid extraction specially from Vitis species, hybrids and Ampelopsis and generally from other plant species such every bit apple, apricot, peach, plum and raspberry. Moreover, DNA yield is higher compared with other procedures used for DNA extraction from grapevines (Bourquin et al., 1991, 5-20 �g nuclear Dna /grand FW; Collins and Symons, 1992, 10-30 �g Deoxyribonucleic acid/g FW; and Thomas et al., 1993, 25-150 �one thousand Dna/grand FW). Doyle and Doyle (1987) reported Deoxyribonucleic acid yields upwardly to 1 mg/g of fresh leaf tissues from dissimilar plant species and this procedure was used by Mauro et al. (1992) for extraction of grapevine Deoxyribonucleic acid. We have non been able to obtain such a loftier yield when this procedure was used on grapevine (data non shown). Notwithstanding, we found that DNA extracted by that procedure was occasionally brownish in color and difficult to assimilate with restriction endonucleases. Such samples also were establish to have a shorter storage life. Likewise, the procedure of Doyle and Doyle (1987) gave similar results for dissimilar Vaccinium sp. (Rowland and Nguyen, 1993). Our modification of Doyle and Doyle (1990) consistently produces high quality Dna which remains usable for at least two years when stored at -20°C.

Acknowledgments: We are thankful to Dr. Philip L. Forsline, USDA, ARS, National Clonal Germplasm Repository, Geneva, New York for providing us with Vitis species establish fabric, Dr. Robert Fifty. Andersen for ruddy, apricot, plum and peach, and Mr. Kevin E. Maloney for raspberry. Nosotros wish to thank Drs. John C. Sanford and Susan K. Brown for their critical review and valuable suggestions.

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Table I. Sources and Dna yield of the plant material used for Dna extraction.

Genotype

Source

DNA yield

(�g/g leaf)

Vitis sp. Aurore

NYSAES a

1,130�167 c

Vitis acerifolia

USDA, ARS b

914�427 c

Vitis berlandieri

USDA, ARS

ane,040�39.vi c

Vitis cinerea

USDA, ARS

796�153 c

Vitis labrusca

UDSA, ARS

542�60.1 c

Vitis rupestris

USDA, ARS

594�116 c

Vitis vinifera cv. Cabernet Sauvignon

NYSAES

546�xix.8 c

Ampelopsis brevipedunculata

USDA, ARS

850�48.one c

Apple tree (Malus domestica cv. Red Delicious)

NYSAES

830

Apricot (Prunus armeniaca cv. NY 500)

NYSAES

935

Cherry (Prunus avium cv. NY 6476)

NYSAES

665

Peach (Prunus persica cv. Rutgers Red Foliage)

NYSAES

805

Plum (Prunus domestica cv. NY 65.363.1)

NYSAES

1,055

Raspberry (Rubus idaeus cv. NY 83)

NYSAES

one,135

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Source: http://hort.cornell.edu/reisch/grapegenetics/DNA_Protocol.html

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