Evaluation of methods for DNA extraction from must and wine

Transcription

1 Research article Received: 3 December 2012 Revised: 2 February 2014 Accepted: 28 February 2014 Published online in Wiley Online Library: 15 April 2014 (wileyonlinelibrary.com) DOI /jib.129 Evaluation of methods for DNA extraction from must and wine Burçak Işçi, 1 * Hatice Kalkan Yildirim 2 and Ahmet Altindisli 1 The quality of wine depends on many factors. One of the most important is the selection of appropriate and defined grape varieties. The analysis of phenolic compounds, amino acids, trace elements and isotopes of wines, used for the identification of grapes varieties, is not sufficient and requires a lengthy analysis period. The development of molecular techniques such as restriction fragment length polymorphism, random amplified polymorphic DNA and microsatellites provides opportunities for the differentiation of grape varieties. In this regard, the use of DNA extracted from must and wine appears to be agoodmarkerfortheidentification of grape varieties used in wine production. In this study, DNA was extracted from grape, leaf, must and wine samples of Vitis vinifera L. cv. Cabernet Sauvignon, Merlot and Sauvignon Blanc origin and examined using different extraction methods. Of the DNA extraction methods tested, the method using absorption at 260/280 nm (with values of 0.19 and 1.92) was considered the method of choice. Copyright 2014 The Keywords: DNA; phenol; microsatellites; must; wine 238 Introduction In order to adhere with Appellation d origine contrôlée (AOC) wine reliability, the origin of the grape variety used in wine production should be stated on wine bottle labels; this labelling is required for both consumers and wine producers. Using identified grape varieties during the production of blended wines is of importance (1). With the development of molecular methods, there is the possibility to adapt these methods for the differentiation of grape varieties used in grape juice, must and wine production. Among these methods, the most appropriate are restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and microsatellites (SSR). The first step in all of these methods is the extraction of DNA from the test material, thus DNA preparation is a crucial point in the assessment. In order to apply the molecular marker methods, isolation of DNA in the appropriate amount and quality is required; however, DNA of sufficient quality and quantity cannot be consistently obtained owing to differences in the plant part and leaf structure, leaf age or chemicals (2). Purification of plants with a high polysaccharide content can be difficult (3,4) and over the last few decades many investigators have attempted to solve this problem (5). The results of studies performed in recent years have demonstrated that the extraction of gdna, of high yield and purity, from matrices rich in polyphenols and polysaccharides could be accomplished even from matrices such as, vinegars, cider, cereals and fresh and dried fruits (6,7). The success of the DNA extraction depended on the restriction and ease-of-use of enzymes such as polymerase and ligase. If the DNA was not pure enough, contaminants inhibited polymerase chain reaction (PCR) amplification (5). In the current study, different DNA extraction methods were used for the isolation of pure DNA. A range of sample volumes from must and wines produced with Cabernet Sauvignon, Merlot and Sauvignon Blanc grape varieties were used for PCR amplification. Materials and methods Plant material Tests were performed with grapes (100 kg; Vitis vinifera L. Cabernet Sauvignon, Merlot and Sauvignon Blanc) hand-harvested at the appropriate maturity (Cabernet Sauvignon at 23 Brix, Merlot at 23 Brix and Sauvignon Blanc at 18 Brix) from the Menderes- Gölcükler region of Izmir (Sevilen Winery vineyards). Prior to the harvest period, young leaf samples were collected from each grape variety and stored at 80 C until analysis. Wine production All grapes were transported to the Food Engineering Department of Ege University and crushed within 24 h of harvest. White grapes (V. vinifera L. Sauvignon Blanc) were crushed, destemmed and pressed immediately with a hydraulic press (Com. Fad. Type G, Rural Companion, Italy). Portions of the juices were collected, treated with SO 2 (50 ppm) and held for 6 h. The juice portions were then inoculated with Fermivin yeast (2.0% Saccharomyces cerevisiae 7013 INRA, Gist-Brocades Co.); pectolytic enzyme was then added at 2.0 g/hl (Rapidase-Ex-Color DSM Food Specialties, Delft) and the must was allowed to ferment at 25 C, until the fermentation was complete. Red grapes (V. vinifera L. Cabernet Sauvignon and Merlot) were crushed, destemmed and prepared for the skin fermentation. The * Correspondence to: Burçak Işçi, Department of Horticulture, Agriculture Faculty, Ege University, Bornova, Izmir, Turkey. burcak. isci@ege.edu.tr 1 Department of Horticulture, Agriculture Faculty, Ege University, 35100, Bornova, Izmir, Turkey 2 Department of Food Engineering, Ege University, 35100, Bornova, Izmir, Turkey J. Inst. Brew. 2014; 120: Copyright 2014 The

2 Evaluation of methods for DNA extraction crushed grapes were treated with SO 2 (50 ppm), inoculated with 2.0% Fermirouge yeast (Saccharomyces cerevisiae 7000 INRA, Gist- Brocades Co.), 4.0 g/hl of pectolytic enzyme (Rapidase-Ex-Colour) was added and the suspension was allowed to ferment for 5 days at 25 C. The pomace was stirred and pressed twice daily. Bentonite (150 g/hl) and gelatin (250 g/hl, Merck Darmstadt, Germany) were used as fining agents for the white wines; only gelatin (250 g/hl) was used for the red wines. After filtration (Seitz plate filter, D-6800 Mannheim, Germany) and bottling, the wines were stored at 15 C. Must production Must samples were analysed using methods 3 5 (described below), throughout the 8 days of fermentation of the Cabernet Sauvignon grapes, 15 days of fermentation of the Merlot grapes and 7 days of fermentation of the Sauvignon Blanc grapes. The extractions were performed on the solid portions of the suspension as well as on the aqueous fraction. DNA extraction DNA was extracted using the following five methods: from the young leaf, the DNA was extracted using methods 1 and 2; from must and wine, the DNA was extracted using methods 3 5. In this study, there were some modifications to the volume and centrifugation speeds of methods 3 5. Method 1 The DNA extraction from the young leaf was performed using the method described by Lodhi et al. (8) and the protocols of Lefort et al. (9), incorporating some modifications. The young leaf (0.5 g) was quickly ground with liquid nitrogen using a sterilized mortar and pestle. The powder obtained (0.1 g) was transferred into 1 ml of extraction buffer [1% hexadecyl trimethyl ammonium bromide (CTAB), Sigma; 0.4 M LiCl, 50 mm Tris HCl, ph 8.0, 50 mm EDTA, 2% PVP, 0.5 ml TWEEN 20 and 10% 2- mercaptoethanol]. After 60 min of gentle agitation, samples were incubated in a water bath at 65 C for at least 15 min. A half-volume of chloroform isoamyl alcohol (24:1) was added and the sample was shaken for 30 min on broken ice. Samples were centrifuged at 1200g for 5 min (Hettich, Universal 30 RF, E1175 rotor). The resulting supernatant (0.8 ml) was transferred into a clean microtube. One volume of ice-cold 2-propanol was added to the resulting aqueous phase and the tubes were incubated at 20 C for at least 30 min. Tubes were then centrifuged at 14,000g for 3 min, the liquid phase was discarded and the resulting pellet was vacuum-dried at room temperature. The pellet was then resuspended with 100 μl of diluted TE [10 mm Tris HCl and 1 mm EDTA (ph 8.0)] (0.1 ). Finally, 2 μl RNAase-A (Sigma R6513) was added to each microtube and the sample was incubated at 37 C for at least 15 min. Method 2 (Dneasy Plant Minikit; Qiagen, Hilden, Germany) The young leaves (0.1 g) were quickly ground with liquid nitrogen using a sterilized mortar and pestle. The resulting powder was transferred into 400 μl of AP1 buffer and 4 μl RNAase-A was added. The samples were incubated at 65 C for 10 min, transferred into 130 μl of AP2 buffer and gently shaken for 30 min on broken ice. The tubes were centrifuged at 14,000g for 3 min (Hettich, Universal 30 RF, E1175 rotor). The liquid phase was discarded using a Mini spin column (2 ml) and the sample was centrifuged again at 20,000g for 2 min; a 1.5 volume of AP3 buffer was then added. The mixed sample (650 μl) was transferred into a 2 ml microtube and centrifuged at 6000g for 1 min. After the addition of 500 μl AW buffer into the microtubes, the samples were centrifuged at 6000g for 1 min. The 500 μl liquid phase was discarded using a Mini spin column (2 ml) and the sample was centrifuged again at 20,000g for 2 min. The AE buffer (100 μl) was transferred into a clean microtube, incubated at room temperature for 5 min and centrifuged at 6000g for 1 min. Finally, 40 μl of DNA solution was obtained. Method 3 (10) Samples (100 ml) were taken daily and centrifuged (10,000g for 20 min at 4 C; Hettich centrifuge). The pellets (solid portions in suspension) were resuspended in 20 ml of TEX buffer (1 M Tris-HCl, ph 8, 1.4 M NaCl, 20 mm EDTA, 3% CTAB and 1% β-mercaptoethanol) and homogenized using a vortex. DNA extractions were carried out according to the method of Siret et al. (10) with the following modifications: the samples were incubated in a water bath at 65 C for 60 min; chloroform isoamyl alcohol (24:1) was added and, after 5 min of gentle shaking, the samples in the microtubes were centrifuged at 5000g for 20 min at 4 C; the supernatants were transferred into clean microtubes and 0.1 volume of 10% CTAB and chloroform isoamyl alcohol (24:1) was added; the tubes were shaken for 5 min and centrifuged (5000g for 20 min at 4 C). The supernatants were transferred into new microtubes and one volume of ice-cold 2-propanol was added. Samples were incubated at 70 C for 30 min and centrifuged at 15,000g for 20 min at 4 C. After the addition of 10 mm Tris HCl (ph 8.0; 0.5 ml), 1 mm EDTA and 40 μl proteinase K (20 mg/ml, Sigma) to the pellet, samples were incubated at 50 C for 30 min. One volume of phenol chloroform (25:25) and 1 volume of chloroform isoamyl alcohol (24:1) were added to the aqueous phase. Supernatants were transferred into clean microtubes, and 2.5 M ammonium acetate was added along with two volumes of icecold 95% ethanol. Each sample in a microtube was centrifuged at 15,000g for 30 min at 4 C (11). Centrifugation was carried out according to the Dneasy Plant Minikit (Qiagen, Hilden, Germany) method for the final DNA extraction (12,13). Method 4 (14) Samples (45 ml) were homogenized with 5 ml of NaCl (1.2 M) and stored at 20 C for one week. The samples were then centrifuged at 2500g for 30 min at 4 C and the liquid phase was discarded. Finally, 750 μl of CTAB buffer [25 mm EDTA, 1 M Tris HCl, 8 ph, 2 M NaCl and 3% (w/v) CTAB] was added to the pellet. Samples were vortexed, transferred into 0.2% (v/v) 2-mercaptoethanol and 1% (w/v) polyvinylpyrrolidone, and incubated in a water bath at 65 C for 60 min. One volume of phenol chloroform isoamyl alcohol (25:24:1) and a 0.6 volume of 2-propanol were added. Samples in tubes were incubated at 20 C and centrifuged at 13,000g for 30 min at 10 C. The liquid phase was discarded, the pellet was washed with 70% ethanol 239 J. Inst. Brew. 2014; 120: Copyright 2014 The wileyonlinelibrary.com/journal/jib

3 and the resulting pellet was vacuum-dried at room temperature for 30 min. The pellet was then dissolved in 50 μl of diluted sterile deionised water. Method 5 (12) Samples (750 ml) were centrifuged (15,300g for 10 min at 4 C; Sigma 6K15 centrifuge) and the procedure was then carried out as described in method 2 (Dneasy Plant Minikit; Qiagen, Hilden, Germany). The concentration and purity of each DNA sample (three replicates were performed for each method) were determined using a NanoDrop ND-1000 model spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA), which measures the concentration in 1 2 μl of sample with good accuracy and reproducibility, and was verified using a 1% agarose gel stained with ethidium bromide. The DNA was stored at 20 C until analysis. Results DNA analysis results Each product, obtained from the DNA extractions of all of the samples, was checked using a 1% agarose gel. Not all the DNA products extracted from the must and wine were displayed with EtBr in the 1% agarose gel, which concurs with the reports of Faria et al. (11) and Garcia-Beneytez et al. (12). The production of wine includes many stages, such as crushing, pressing, fermentation, filtration, fining and ageing; each of these steps affects the quality and quantity of the DNA material, until it reaches the final stages. Limitations of the extraction methods are related to the composition of the wines, for example, in red wines there are tannins and polysaccharides that could be responsible for impurities in the DNA (13). The results indicate that the ratio of the DNA levels in the young leaf, taken from each grape variety, ranged between 1.74 and 1.86 using the A 260/280 ratio, when determined using methods 1 and 2 (Table 1). It is known that a ratio of OD 260 /OD 280 between 1.8 and 2.0 is accepted as a pure state of DNA. Alower value can indicate the presence of contaminants. The molecular weights of DNA isolated using method 1 were greater than the molecular weights of DNA extracted using method 2. The purity, quality and amount of DNA obtained using method 1 were determined to be the most appropriate for use in the PCR studies (Table 1). The DNA results of the Sauvignon Blanc samples from different dates showed values between and Methods 4 and 5 allowed the extraction of the highest amount (ng/μl) of DNA from must samples of Sauvignon Blanc. DNA concentrations (ng/μl) were obtained in the required amount using method 5; however, the OD 260 /OD 280 ratios of DNA were poor. Methods 4 and 5 were determined to be appropriate for the PCR studies, hence, during extraction of DNA from Sauvignon Blanc must, methods 4 and 5 were preferred (Fig. 1). The DNA extraction from Merlot must samples was performed using three different methods, namely methods 3 5. The results displayed purity values between 1.04 and. The values of DNA (ng/μl) and OD 260 /OD 280 ratios were lower than when using method 4. Methods 3 and 4 were more effective than method 5 for the DNA extraction from Merlot must samples. Owing to the quality and amount of DNA, method 3 was determined to be appropriate for the PCR studies. Comparing the two aforementioned methods, method 3 was preferred for the extraction of DNA from Merlot must (Fig. 2). The DNA extraction from Cabernet Sauvignon samples using methods 3 5 exhibited DNA purity, represented by OD 260 /OD 280 values, of between and The results obtained using method 4 were the best for both the quantity of DNA (ng/μl) and OD 260 /OD 280 ratios (Fig. 3). The OD 260 /OD 280 values of the blended samples were in the range of 0.19 and. As seen from Fig. 4, the best results for the DNA extraction procedure for A1 (100% Cabernet Sauvignon) and A5 (100% Merlot) samples were obtained The of Sauvignon Blanc must samples at different dates The DNA absorbance ratio (OD 260 /OD 280 ) of Sauvignon Blanc must samples at different dates. B. Işçi et al. Figure 1. The DNA concentration (ng/μl) and (OD 260 / OD 280 )of Sauvignon Blanc must samples at different dates. Table 1. The DNA concentration (ng/μl) and DNA absorbance ratio (OD 260 /OD 280 ) of the young leaves of tested grape varieties extracted by methods 1 and 2 Sample Method 1 Method DNA concentration (ng/μl) DNA absorbance ratio (OD 260 /OD 280 ) DNA concentration (ng/μl) DNA absorbance ratio (OD 260 /OD 280 ) Cabernet Sauvignon Merlot Sauvignon Blanc wileyonlinelibrary.com/journal/jib Copyright 2014 The J. Inst. Brew. 2014; 120:

4 Evaluation of methods for DNA extraction The of Merlot must samples at different dates /22/2008 8/23/2008 8/24/2008 8/25/2008 8/26/2008 8/27/2008 8/28/2008 8/29/2008 8/30/2008 8/31/2008 9/1/2008 9/2/2008 9/3/2008 9/4/2008 9/5/2008 The of blended wine samples A1 A2 A3 A4 A5 B1 B2 B3 B4 C1 C2 C3 Da1 Da2 Da3 Db1 Db2 Db3 Dc1 Dc2 Dc3 Blended wine sample The DNA absorbance ratio (OD 260 /OD 280 ) of Merlot must samples at different dates. 8/22/2008 8/23/2008 8/24/2008 8/25/2008 8/26/2008 8/27/2008 8/28/2008 8/29/2008 8/30/2008 8/31/2008 9/1/2008 9/2/2008 9/3/2008 9/4/2008 9/5/2008 The DNA absorbance ratio (OD 260 /OD 280 ) of blended wine sampes A1 A2 A3 A4 A5 B1 B2 B3 B4 C1 C2 C3 Da1 Da2 Da3 Db1 Db2 Db3 Dc1 Dc2 Dc3 Blended wine sample Figure 2. The DNA concentration (ng/μl) and (OD 260 / OD 280 )of Merlot must samples at different dates. The of Cabernet Sauvignon must samples at different dates The DNA absorbance ratio (OD 260 /OD 280 ) of Cabernet Sauvignon must samples at different dates. Fermantation day Fermantation day Figure 3. The DNA concentration (ng/μl) and (OD 260 / OD 280 )of Cabernet Sauvignon must samples at different dates. using method 5. In the case of the B4 (100% Sauvignon Blanc) sample, the preferred method was method 4. The highest concentrations of DNA (ng/μl) for A4 (25% Cabernet Sauvignon. + 75% Merlot) and B1 (75% Cabernet Sauvignon + Sauvignon Blanc) blended wine samples were also obtained using method 4. Evaluating all the methods, the best Figure 4. The DNA concentration (ng/μl) and (OD 260 /OD 280 ) of blended wine samples. results were found using method 4, which gave a high quantity (ng/μl) and quality (component composition) of DNA. The superiority of this method was confirmed by the results of the OD 260 /OD 280 ratios obtained for the blended wine samples. The most difficult step during the extraction of DNA from must and wine was found to be the separation of phenolic compounds and certain polysaccharides. The OD 260 /OD 280 values were found to vary depending on the origin of the grape variety and blending procedure employed (10,11,13,14). Discussion As DNA is specific to each organism, DNA-based methods are beneficial in differentiating the grape varieties used in wine production. Methods based on molecular techniques (RFLP, RAPD and microsatellites) strengthen the possibility of identifying grape varieties, employed in wine production, using molecular markers. SSR, in particular, has opened new possibilities for grape genotype characterization; however, this method requires the use of pure and high-quality DNA for PCR studies. The extraction of DNA from must and wine is difficult owing to the presence of large quantities of polyphenols and polysaccharides (15). Therefore, our experiments have attempted to optimize different protocols of DNA extraction from must and wine. 241 J. Inst. Brew. 2014; 120: Copyright 2014 The wileyonlinelibrary.com/journal/jib

5 242 In this study, it was possible to partially display DNA extracts using EtBr on a 1% agarose gel. Similarly, Siret et al. (13) and Daniela et al. (15) also partially displayed the DNA in the samples, obtained during fermentation stages, with EtBr on a 1% agarose gel; the explanation of this has also been elucidated in other studies. According to one study, the presence of a low-molecularweight band or smear, consisting essentially of degraded RNA, is useful in evaluating the efficiency of the extraction procedure, as a rough direct relationship between DNA and RNA band intensities can be established in musts and wines (15). In the present study, the DNA extracted from leaf, must, vine and wine was quantified using a 1% agarose gel and Spectrophotometer ND Daniela et al. (15) determined the quality and quantity of must DNA using the above-mentioned procedure and Spectrophotometer ND-1000 (NanoDrop software), and obtained satisfactory results. In this study, using a similar procedure we obtained the largest amount of DNA extract from Merlot must reaching ng/μl. Evaluating all our results, employing different DNA extraction methods led to DNA values ranging between 4.00 and ng/ μl and OD 260 / OD 280 purity values between 0.76 and. Savazzini et al. (14) stated that the OD 260 /OD 280 values of the DNA obtained in their study differed, at approximately 1.2, and depended upon the must samples obtained from local grape varieties of the Trentino Südtirol region. Drabek et al. (16) tested three different DNA extraction methods and demonstrated the DNA profile of two white, one rose and one red wine. Using must and wine samples (106 days) they obtained DNA extracts with values of more than 1.6 using a CTAB-based DNA extraction method. Siret et al. (13) evaluated the DNA products obtained during the fermentation stage of wine production. During this stage, some transformation and biochemical modification occurs which probably affects the DNA content. The results obtained from samples taken daily during fermentation demonstrated a significant decrease in DNA obtained in the pellet. The presence of DNA even after 13 days of fermentation of Muscat Blanc was seen as a positive result. Faria et al. (11) obtained DNA concentrations varying between 23 and 52 μg/ml from the must samples of V. vinifera L. Tinta Roriz, V. vinifera L. Tinto Cão, V. vinifera L. Touriga Francesa, V. vinifera L. Touriga Nacional and V. vinifera L. Tinta Baroca. The authors reported that they could not display the DNA samples using EtBr on an agarose gel owing to the high ratio of RNA. DNA extraction performed using method 5 gave a greater amount (750 ml) of wine where the A 260/280 values obtained were at a minimum of 1.21/23.94 ng/μl and a maximum of 1.70/48.37 ng/μl for V. vinifera L. Sauvignon Blanc; 1.04/9.74 ng/μl and /13.47 ng/μl for V. vinifera L. Merlot; and /12.07 ng/μl and 1.65/66.33 ng/μl for V. vinifera L. Cabernet Sauvignon, respectively. Using different grape varieties for the production of different wines caused large ranges in the DNA concentration (10 12,17). In our study, samples volumes (100 ml) and centrifugation parameters (30 min at 10,000g) were changed owing to the small amounts of DNA obtained. The purpose of this study was to set up and evaluate DNA extraction procedures from grape, leaf, must and wine. This was performed by amplification using PCR with SSR markers employed in the European Project GENRES #08, and 2 nd Edition of the OIV Descriptor list for grape varieties and Vitis species ( First, the amplification products were controlled using electrophoresis on a 2% agarose gel with EtBr and PCR products were silver stained on a 6% (w/v) polyacrylamide gel (Promega Silver Staining kit). The obtained DNA extracts were low in quantity, so the parameters were changed. Biochemical modification of the fermentation media is known to be a factor that interferes with DNA efficiency and PCR bias (6,7). Garcia-Beneytez et al. (12) performed DNA analyses of a must mixture using seven microsatellite markers and starting from the fermentation stage. Different grape varieties and wine samples were used to evaluate the STMS (transferability of sequence tagged microsatellite sites) analysis in order to define the varieties used. The researchers used an increased amount of sample (750 ml) and applied more accurate methods, such as an automatic sequencer to separate the amplification fragments corresponding to the microsatellite markers. Rodrigez-Plaza et al. (17) proved that the grape type and variety could be determined in must samples using microsatellite markers with capillary electrophoresis. Spectrometric measurements performed for DNA evaluation were used to compare the 260 nm OD (A 260/280 ratios of DNA) value with the 280 nm OD measurement, providing information regarding the DNA (presence of protein or RNA) purity. Usually, the OD 260 /OD 280 ratio is expected to be between 1.8 and 2.0. An OD 260 /OD 280 ratio over 2.0 indicates RNA contamination, whereas an OD 260 /OD 280 under 1.8 shows protein is the cause of the contamination. The PCR studies performed, where the DNA was beyond these values, did not provide fully accurate results. There is a significant decline in the amount and quality of wine DNA as a result of some production procedures, such as filtration and wine ageing. The richness of DNA in phenols and polysaccharides is another restrictive factor during the PCR stage of DNA (10,13,12,16). According to our results, methods 1 4 could only be used for DNA extraction after the modification of these methods, taking into account the grape varieties and sample categories (white, red). The identification of grape variety origin in the leaf and vine is possible using the proposed modification to method 4, but for must and wine evaluation further optimization studies are needed. Acknowledgements We express our deep gratitude to TÜBİTAK (Scientific and Technological Research Council of Turkey) for the support of our project, and Sevilen Winery of the İzmir Higher Institute of Technology Molecular Biology, Professor Sami Doğanlar of the Genetic Department and the technical staff for permitting the use of the Sigma 6K15 centrifuge. We also thank Ege University Science Technology Research and Practice Center (EBİLTEM) and Dr Şevket Karaçancı for the use of the Hettich centrifuge; TARIS- ARGE Directorate and Dr Mübeccel Topuzoğlu for supplying the ultra-pure water; Ege University, Medicine Faculty, Medical Genetic Research and Practice Center staff for allowing the use of the Nanodrop ND-1000 for the measurement of DNA quality and quantity; and Professor Zeki Topçu of Ege University Pharmacology Faculty Pharmaceutic Biotechnology Department and the laboratory staff. References B. Işçi et al. 1. Siret, R. (2001) Study of the genetic polymorphism of the cultivated grapevine (Vitis vinifera L.) by means of microsatellite markers: Application to the characterisation of grape varieties in wines. Postgraduate Thesis, University of Montpellier, School of Pharmacy. 2. Doyle, J. J. and Doyle, J. L. A. (1988) Rapid DNA isolation from small amount of fresh leaf tissue. Phytochem. Bull. 19, wileyonlinelibrary.com/journal/jib Copyright 2014 The J. Inst. Brew. 2014; 120:

6 243 Evaluation of methods for DNA extraction 3. Do, N. and Adams, R. P. (1991) A simple technique for removing plant polysaccharide contaminants from DNA. Biotechniques 10, Fang, G., Hammar, S., and Rebecca, R. (1992) A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biotechniques 13, Luro, F., Laigret, F., Bove, J. M., and Ollitrault, P. (1992) Application of random amplified polymorphic DNA (RAPD) to Citrus genetics and taxonomy. Proc. Int. Soc. Citriculture 1, Giudici P., Gullo, M., Solieri, L., and Falcone, P. M. (2009). Technological and microbiological aspects of traditional balsamic vinegar and their influence on quality and sensorial properties. Adv. Food Nutr. Res. 58, Mamlouk, D., Hidalgo, C., Torija, M.J., and Gullo, M. (2011). Evaluation and optimisation of bacterial genomic DNA extraction for no-culture techniques applied to vinegars. Food Microbiol. 28(7), Lodhi, M. A., Weeden, N. F., and Reisch, B. I. (1994) A Simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Mol. Biol. Rep. 12(1), Lefort, F., Lall, M., Thompson, D., and Douglas, G. C. (1998) Morphological microsatellite fingerprinting and genetic relatedness of a stand of elite oaks (Q. robur L.) at Tullynally, Ireland. Silvae Genetica 47, Siret, R., Boursiquot, J. M., Merle, M. H., and Cabanis, J. C. (2000) Toward the authentication of varietal wines by the analysis of grape (Vitis vinifera L.) residual DNA in must and wine using microsatellite markers. J. Agric. Food Chem.. 48, Faria, M. A., Magalhaes, R., Ferreira, M. A., Meredith, C. P., and Ferreira Monteiro, F. (2000) Vitis vinifera must varietal authentication using microsatellite DNA analysis (SSR). J. Agric. Food Chem. 48, Garcia-Beneytez, E., Moreno-Arribas, M. V., Borrego, J., Polo, M. C., and Ibanez, J. (2002) Application of a DNA analysis method for the cultivar identification of grape musts and experimental and commercial wines of Vitis vinifera L. using microsatellite markers. J. Agric. Food Chem.. 50(21), Siret, R., Gigaud, O., Rosec, P. J., and This, P. (2002) Analysis of grape Vitis vinifera L. DNA in must mixtures and experimental mixed wines using microsatellite markers. J. Agric. Food Chem. 50, Savazzini, F. and Martinelli, L. (2005) DNA analysis in wines: Development of methods for enhanced extraction and real-time polymerase chain reaction quantification. Anal. Chim. Acta 563, Briciu, D., Pamfil, D., Briciu, A., Curticiu, D., Balazs, E., Taoutaou, A., Pop, I., and Coia, L. (2010) Development of methods for DNA extraction from leaves and must grapes. Bulletin UASVM. Anim. Sci. Biotechnol. 67(1), Drabek, J., Stavek, J., Jaluvkova, M., Jurcek, T., and Frebort, I. (2008) Quantification of DNA during winemaking by fluorimetry and Vitis vinifera L. Specific quantitative PCR. Eur. Food Res. Technol. 226, Rodrigez-Plaza, P., Gonzalez, R., Moreno-Arribas, M. V., Polo, M. C., Bravo, G., Martinez-Zapater, J. M., Martinez, M. C. and Cifuentes, A. (2006) Combining microsatellite markers and capillary gel electrophoresis with laser-induced fluorescence to identify the grape (Vitis vinifera) variety of must. Eur. Food Res. Technol. 223, J. Inst. Brew. 2014; 120: Copyright 2014 The wileyonlinelibrary.com/journal/jib