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Plant Molecular Biology Reporter 15 (1) 1997

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Commentary

Modification of a CTAB DNA Extraction Protocol for Plants Containing High Polysaccharide and Polyphenol Components
Sue Porebski, L. Grant Bailey, and Bernard R. Baum
Agriculture Canada, Eastern Cereal and Oilseed Research Centre, Ottawa, Ontario, K1A 0C6, Canada
Key Words: Fragaria,DNA extraction, RAPD, strawberry Abstract: A relatively quick, inexpensive and consistent protocol for extraction of DNA from expanded leaf material containing large quantities of polyphenols, tannins and polysaccharides is described. Mature strawberry leaves, which contain high levels of these secondary components, were used as a study group. The method involves a modified CTAB extraction, employing high salt concentrations to remove polysaccharides, the use of polyvinyl pyrrolidone (PVP) to remove polyphenols, an extended RNase treatment and a phenol-chloroform extraction. Average yields range from 20 to 84 ~tg/g mature leaf tissue for both wild and cultivated octoploid and diploid Fragariaspecies. Results from 60 plants were examined, and were consistently amplifiable in the RAPD reaction with as little as 0.5 ng DNA per 25-~tLreaction. Presently, this is the first procedure for the isolation of DNA from mature strawberry leaf tissue that produces consistent results for a variety of different species, both octoploid and diploid, and is both stable and PCR amplifiable before and after extended storage. This procedure may prove useful for other difficult species in the family Rosaceae.

NA extraction from berry plants and other plants containing )sticky and resinous materials has been difficult in the past (Webb & Knapp, 1990; Varadarajan & Prakash, 1991; Fang et al., 1992; Collins et al., 1992). In strawberries, like other berry plants, the presence of secondary metabolites, such as polyphenols, tannins, and polysaccharides, inhibit enzyme action. Polysaccharides are visually evident in DNA extracted by their viscous, glue-like texture and make

Abbreviations: CTAB,hexadecyltrimethylammonium bromide; EtOH, ethanol; PVP, polyvinylpyrrolidone.

DNA Extraction in Presence of Polysaccharides and Polyphenols


the DNA unmanageable in pipetting and unamplifiable in the polymerase chain reaction (PCR) by inhibiting Taq polymerase activity (Fang et al., 1992). When young, expanding leaf and shoot material is limited or the plant is not undergoing active shoot elongation at time of collection, DNA purity becomes increasingly difficult. DNA obtained from material past the budding stage has been shown to be difficult to extract and unstable for long-term storage (Lodhi et al., 1994). With maturity, leaves contain increased quantities of polyphenols, tannins, and polysaccharides. Dealing with such components in mature leaves becomes necessary when younger expanding leaves and shoots are not available during the time of collection. We were uniformly unsuccessful in our attempts to amplify Fragaria DNA by PCR using other reported methods, including those of Dellaporta et al. (1984), Saghai-Maroof et al. (1984), Doyle & Doyle (1990), LaRoche (1992), Oard & Dronavalli (1992), Wang et al. (1993), Richards et al. (1994), and Davis et al. (1995). As a result, we found it necessary to devise a protocol for DNA extraction from fully developed expanded leaf tissue that would yield DNA suitable for PCR, especially for RAPDs, and that would be consistent for many different wild and cultivated species of strawberries, both diploid and octoploid. The protocol described here is relatively quick and inexpensive, and provides clean DNA, consistently amplifiable in polymerase chain reaction using the RAPD technique (William et al., 1990) for all Fragaria spp. assayed. Fragaria tissue and DNA stored for 21 months at -20 ~ has also been shown to be stable and amplifiable.

Material and Methods Solutions Required:


chloroform:octano124:1 (v/v) 5 M NaC1 TE buffer: 10 mM tris-HC1, i mM EDTA, pH 8.4 RNase A: 10 m g / m L proteinase K: 1 m g / m L 1 chloroform-phenol saturated in TE polyvinylpyrrolidone (PVP) Sigma P -9003-39-8 (40,000) extraction buffer: 100 mM tris, 1.4 M NaC1, 20 mM EDTA, pH 8.0, 2% CTAB, 0.3% ~-mercantoethanol2 PCR reaction buffer: 100 mM tris-HC1, 100 mM KC1, pH 8.3 primer: set #8 UBC, University of British Columbia Stoffel fragment: reagents supplied by Perkin Elmer

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Notes: 1. Made fresh before use. 2. Added just before use.


Sixty strawberry plants of both wild and cultivated Canadian octoploid species (2 n = 56) including F. chiloensis (L.) Dcne., F. virginiana Dcne, F. virginiana forma multicipita (Fern.) Catling & Cayouette, F. X ananassa nm. cuneifolia (Nutt.) Staudt, and one diploid species (2 n = 14), F. vesca L., were used, 5-12 plants per species. The modification of the CTAB extraction procedure of Doyle & Doyle (1990) includes high NaC1 concentrations in the buffer to remove polysaccharides (Lodhi et al., 1995), and polyvinyl pyrrolidone (PVP) to remove polyphenols (Maliyakal, 1992). An extended, one-hour RNase treatment was required for the DNA product to be RNA free and PCR amplifiable, with an additional phenol-chloroform step (Saghai-Maroof et al., 1984) to remove any excess proteins. These steps provide the principal key to success for collecting DNA from mature strawberry leaves. Examination of polymorphism through use of primers with arbitrary sequences (RAPD) was done successfully with 16 different primers after initial screening of approximately 50 primers. The 25 ~tL volume PCR reactions contained PCR reaction buffer, 2 mM MgC12, 0.1 mM dNTPs, 2.4 mM primer, 2 units of Stoffel fragment, I ng DNA, and were placed in a MJ Research Inc. PTC100 Thermocycler. The thermal cycling included one cycle at 5 minutes of denaturation at 94 ~ one minute annealing at 47 ~ two minutes of primer extension at 72 ~ then 35 cycles of one minute each at 94, 47, and 72 ~ with minimum ramp time. PCR amplicons were then run on a 1.5 % 3- to 4-ram thin agarose gel for best results.

The Protocol D N A Extraction


9 Collect leaves of Fragaria, freeze in liquid nitrogen, and store at -70 ~ until use. Grind 0.5 g of leaves using mortar and pestle in the presence of liquid nitrogen until finely ground. Transfer frozen ground leaf tissue to 15-mL polypropylene centrifuge tubes. 9 Add 5 mL of 60 ~ extraction buffer and 50 mg PVP/0.5 g leaf tissue. Mix by inversion and incubate in 60 ~ oven (with shaking) for 25 to 60 minutes.

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9 Remove from heat, and let cool to r o o m t e m p e r a t u r e for 4 to 6 minutes. 9 A d d 6 m L of chloroform:octanol (24:1) and mix b y inversion to form an emulsion. 9 After mixing thoroughly, spin at 3000 r p m for 20 minutes in a tabletop centrifuge at r o o m temperature. 9 Transfer top aqueous solution to n e w 15-mL centrifuge tubes using wide-bore pipette tip. Repeat chloroform-octanol extraction to r e m o v e cloudiness (PVP) in aqueous phase. 1 9 A d d 1 / 2 v o l u m e of 5 M NaC1 to the final aqueous solution recovered. 2 Mix well. A d d two volumes of cold (-20 ~ 95% ethanol. Mix b y inversion. If required, place in freezer (-20 ~ for 10 minutes to accentuate precipitation. The solution m a y be left at 4 to 6 ~ to precipitate overnight. 9 Spin at 3000 r p m for 6 minutes. 3 9 Pour off supernatant, and wash pellet with cold (0 to 4 ~ 70% v / v ethanol. Dry pellet in 37 ~ oven or v a c u u m until d r y (~1 hour). 9 Dissolve in 300 ~tL TE overnight at (4 to 6 ~ 4 Transfer to 1.5-mL E p p e n d o r f tubes, s 9 A d d 3 ~tL RNase A (10 m g / m L ) and incubate in 37 ~ water bath for approximately I hour. A d d 3 ~tL proteinase K ( l m g / m L ) , and incubate at 37 ~ for 15 to 30 minutes. 9 A d d 150 ~tL of p h e n o l and 150 ~tL of chloroform to each E p p e n d o r f tube. 9 Vortex briefly, and spin (in microfuge) at 14,000 r p m for 10 to 15 minutes. Collect u p p e r layer in n e w 1.5-tube. A d d 50 ~L TE to phenol phase. Vortex, spin, r e m o v e u p p e r layer, and a d d to sample. 9 A d d 1/10 vol. 2 M Na acetate and 2 vol. absolute ethanol and mix. 9 Leave overnight in freezer (-80 ~ Spin at 14,000 r p m for 10 to 20 minutes. Drain and wash with 70% v / v EtOH. R e m o v e EtOH. 9 V a c u u m - d r y tubes. A d d 100 to 200 ~tL TE. Allow time for complete resuspension. 6 9 D N A concentrations w e r e m e a s u r e d using a H o e f e r Quanta 200 Fluorometer and 0.5 ng D N A dilutions were p r e p a r e d for PCR use.
Notes

1. Be careful to remove only top aqueous solution and not white band at interface. (Repeat this step until no longer cloudy due to the presence of PVP bound polysaccharides. Twice is usually sufficient. 2. In some plants a second procedure of salt precipitation may be useful. 3. DNA pellets should now be visible in all tubes. 4. When dissolved, some DNA may still be slightly viscous but easily pipetted. 5. Products at this point in the protocol are usually not PCR amplifiable. 6. Recommended overnight at 4 ~

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Results and Discussion


DNA concentrations were measured for the sixty samples, and the average yields and ranges in ~tg/g leaf tissue are:
F. vesca F. multicipita F. virginiana Gasp6 Petawawa F. chiloensis V.I. B.C. F. ananassa wild cultivated

20 30 20 84 71 52 32 28

10-30 15-45 10-25 20-120 35-90 10-90 30-45 15-45

PCR was successful using quantities as small as 0.5 ng in a 25-~tL reaction. When amounts were increased beyond 5 ng, DNA bands became less distinct. Although secondary metabolites may not be completely removed, the DNA template was pure enough to allow PCR amplification. In addition, such DNA yields are large enough to permit as many as 1.4 x 105 PCR reactions or a number of RFLP applications. This DNA extraction protocol is relatively fast, since a lengthy ultracentrifugation is not required, as in some other PVP protocols (Maliyakal, 1992). PVP, a solid polymer that has high molecular weight and is watersoluble and chemically inert, was shown to remove the polyphenols, while maintaining a higher yield than polyvinylpolypyrrolidone (PVPP), an insoluble cross-linking polymer. PVP forms a complex with polyphenols through hydrogen bonding, allowing them to be separated from the DNA, and reducing levels of polyphenol in the product (Maliyakal, 1992). Although PVPP is useful in improving stability of enzymes by removing phenolic impurities (Sigma Chemical Co., 1993), it yields significantly less DNA than PVP from fully expanded leaves. The technique of Lodhi et al. (1994) utilizing PVPP gave very low DNA yields, 2 to 3 ~tg/g leaf tissue, with fully expanded leaves, and PCR amplification was not consistently successful for all strawberry species. Even when young expanding leaves and shoot material were examined using this protocol, yields reported by Lodhi et al. (1994) for other berry plants (546-1130 ~tg/g) were not reached. The method of Jobes et al. (1995) utilizing PVP, NaC1, LiC1, and SDS is time consuming. We were able to obtain equivalent results with the exclusion of the SDS cleaning and LiC1, which is used for RNA removal

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Fig. 1. PCR amplimers on a 1,5% agarose gel with primer UBC8-751.(left to right). K+ O, ~ digested with Hind III+ ~X 174 digested with Hae III; vesca, F. vesca L.; virginiana-multicipita, F. virginiana forma multicipita (Fern.) Cafling & Cayouette - from Gasp6; virginiana Gasp6, F. virginiana Dcne., from Gasp6; virginiana Petawawa, F. virginiana Dcne., from Petawawa; chiloensis sVI, F. chiloensis (L.) Dcne., from S. Vancouver Island; chiloensis mBC, F. chiloensis (L.) Dcne., from mid BC coast; ananassa wild, X F. ananassa rim. cuneifolia (Nutt.) Staudt, from V.I., wild; ananassa cult, F. ananassa rim. cuneifolia (Nutt.) Staudt, from V.I., cultivated.

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and to prevent residual ribonucleosides from acting as primers in the thermal reaction (Storts, 1993). We found an extended RNase treatment of i hour was sufficient to degrade the RNA into small ribonucleosides that were not detectable by gel electrophoresis. Remaining ribonucleosides would be in very small amounts, and would have to compete with large quantities of added primers in the RAPD reaction, making interference by ribonucleoside primer highly unlikely. Other protocols proved unsuccessful in removing secondary inhibiting components in all species tested, including a NaOH technique (Wang et al., 1993), a protocol developed for maize (Dellaporta et al., 1984; Oard & Dronavalli, 1992), a CsC1 purification (Richards et al., 1994), the EluQuik (Scheicher & Schuell) method (LaRoche, 1992), and various CTAB extractions (Doyle & Doyle, 1990; Saghai-Maroof et al., 1984; Davis et al., 1995)). Unlike all other protocols mentioned above, our relatively fast, consistent protocol produced very acceptable DNA yields from extracted mature leaf tissue for all Fragaria species assayed. All DNA was stable and could be amplified by PCR, requiring only 0.5 ng DNA in 25-~tL reactions, both before and after 21 months of storage. In addition, RAPD profiles were shown to be reproducible for at least five plants examined from each location for each species. This technique has potential to be an effective protocol for DNA extraction using mature leaf tissue for strawberries, and perhaps for other species in the family Rosaceae with high polysaccharides and polyphenols, when sufficient young leaf tissue is not available.

Acknowledgments: Special thanks to Paul Catling and Linda Bonen for their comments and to Brian Wollenschlager for his graphical support. References
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