Isolation and PCR Amplification of Genomic DNA from ... - Springer Link

Plant Molecular Biology Reporter 17: 171–178, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.

Protocols

Isolation and PCR Amplification of Genomic DNA from Market Samples of Dry Tea MAHIPAL SINGH∗ , BANDANA and P.S. AHUJA Division of Biotechnology, Institute of Himalayan Bioresource Technology, Palampur (HP), 176061, India

Abstract. A simple procedure for DNA isolation from processed dried commercial samples of tea is described. The method involves a modified CTAB procedure employing extensive washing, use of 1% PVP to remove polyphenolics and a single phenol:chloroform extraction step. The average yield ranges from 164–494 µg/g tea sample for various market samples. The DNA obtained from 11 different brands of tea using this procedure were consistently amplifiable (using both RAPD primers as well as defined sequences as primers) and digestible with restriction endonucleases. Key words: Camellia sinensis, DNA isolation, PCR, RAPD, Tea Abbreviations: CTAB, cetyl trimethyle ammonium bromide; EDTA, ethyle diamine tetra acetic acid; PCR, polymerase chain reaction; PVP, poly vinyl pyrrolidone; RAPD, randomly amplified polymorphic DNA; RT, room temperature; Taq, Thermus aquaticus.

Introduction Tea is a woody beverage crop with high polyphenolic contents. Isolation of tea genomic DNA using the basic CTAB procedure (Takeuchi et al., 1994; Matsumoto et al., 1994) and silica gel dried tea leaves (Wachira et al., 1995) has been described. All the above methods, however, were used to isolate the DNA from fresh young leaves of tea plants. Isolation of DNA from herbarium specimens (Rogers et al., 1988) and dehydrated plant tissues (Tai and Tanksley, 1990) have also been described. However, none of these methods could provide DNA from market samples of processed tea, suitable for downstream applications. In molecular diagnostic studies involving identification of cultivars and varieties used by a tea manufacturer in preparation of a particular lot or brand of tea and also in diagnosis of adulteration of tea samples ∗ Author for correspondence. e-mail: [email protected]

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Mahipal Singh , Bandana and P.S. Ahuja

by plants other than Camellia sinensis (L) O. kuntze, isolation of quality DNA is a basic and pre requirement. We describe here a protocol to obtain quality DNA from processed dry market samples of tea. The protocol is mainly based on the protocol of Saghai-Maroof and his colleagues (1984).

Materials and Methods Plant materials The fresh leaf material of Camellia sinensis (L) O. kuntze (Upasi-9) used in this study was obtained from ‘Banuri Experimental Tea Garden’ of IHBT. Different brands of dry commercial tea samples available in the open market in packed forms were obtained from Palampur market and are listed in Table 1. Equipment and chemicals High speed refrigerated centrifuge (Hitachi) Robocycler (Stratagene) Mortar and pestle Liquid nitrogen Water bath Extraction buffer: 2% (w/v) CTAB, 100 mM Tris-HCl (pH 8.0), 20 mM EDTA, 1.4 mM NaCl, 1% PVP 1 × TE buffer pH 8.0 (10 mM Tris, 1 mM EDTA) 0.1 × TE buffer pH 8.0 (10 mM Tris, 0.1 mM EDTA) Chloroform:isoamyl alcohol (24:1) Isopropanol Phenol:chloroform (1:1) Absolute ethanol 70% ethanol (v/v) PVP Taq polymerase (Bangalore Genei) DNA isolation • •

Soak the tea samples overnight in sterile distilled water, cleanse 4 times with double distilled water and dry the samples in an oven (60 ◦ C) for about an hour. Grind 1 g of dried tea samples in a mortar and pestle in the presence of liquid nitrogen.

S.N.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Brand Name#

Lazeez Premium Brook Bond A1 Tea city Birla Premium Gold New Lipton Richbrue Lipton taaza Sargam Taj Mahal Green tea Lazeez masala

Market price∗ (Rs/kg)

DNA extraction +/− µg/gm

OD260/280

Status

PCR amplification RAPD primer Defined primer

152 126 180 160 142 166 183 183 208 110 134

+ + + + + + + + + + +

1.72 1.73 1.73 1.72 1.84 1.83 1.79 1.62 1.78 1.90 1.76

Degraded Degraded Degraded Degraded Degraded Degraded Degraded Degraded Degraded Partially degraded Degraded

+(3) +(3) +(3) +(2) +(3) +(4) +(3) +(4) +(4) – –

494 278 451 444 461 460 164 489 287 240 275

+(5) +(5) +(5) +(5) +(5) +(5) +(5) +(5) +(5) +(5) +(5)

Isolation and PCR Amplification from Tea

Table 1. DNA extraction status and PCR amplification of market samples of tea.

Note: The oligonucleotide used in this study as a RAPD primer was 50 -AAGACCCCTC-30 and as defined primers were: Forward primer (50 TTTAGTGCTGGTTGTCGC-30), Backward primer (50 -TGGGAAGTCCTCGTGTTGCA-30). Numbers in parentheses represents number of PCR bands. # All teas here are black except number 10. ∗ Prices for January 1998.

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174 • • • • • • • • • • • • • •


Transfer the tea powder into a clean autoclaved centrifuge tube and add 4 mL of warm extraction buffer. Mix well and incubate at 65 ◦ C for 1 h with occasional mixing. Cool the mixture to RT and then add 8 mL of chloroform:isoamyl alcohol. Centrifuge at 10,000 rpm for 10 min at RT. Transfer the clear supernate to a fresh tube and precipitate the DNA by adding 0.6 vol of isopropanol. Centrifuge the precipitated DNA at 12,000 rpm for 20 min at RT. Dry the pellet at RT for about 30 min and then dissolve in 2 mL of 1 × TE. Treat the DNA with RNase (10 mg/mL stock) for 1 h at 37 ◦ C. Extract once with an equal volume of phenol:chloroform. Transfer the aqueous phase to a new tube, add 2 volumes of chilled ethanol and keep at −20 ◦ C for at least 1 h. Centrifuge at 12,000 rpm for 20 min at 4 ◦ C. Wash the pellet with 70% ethanol for 5 min. Dry the pellet for 30 min at RT. Dissolve the purified DNA in an appropriate volume of 1 × TE and store at −20 ◦ C until used.

Notes 1. Soaking of tea samples in water for 3–4 h and then extensive washing (5–6 times) removes much of the brownish colour. 2. The extraction buffer works well if kept warm at 65 ◦ C prior to adding the ground sample.

PCR amplification • • • •

Dilute the DNA in 0.1 × TE to 10 ng/µL. Assemble the PCR reaction in a 25 µL final vol containing 100 µM each of the four dNTPs, 100 µM primer, 20 ng of genomic DNA and 0.3 units of Taq polymerase. Program the robocycler for 1 cycle of 4 min at 94 ◦ C and then 45 cycles of 1 min at 94 ◦ C, 1 min at 37 ◦ C and 2 min at 72 ◦ C. The last extension cycle was programmed at 72 ◦ C for 7 mins. Run the amplified products in a 1.4% agarose gel, stain in ethidium bromide and photograph with the help of an image analyser.

Note The annealing temperature for conserved rRNA gene primers was 68 ◦ C instead of 37 ◦ C and it was run for 40 cycles only (instead of 45 cycles).


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Figure 1. Comparison of genomic DNA extracted from black and green tea samples: Lanes 1, 2 and 3 show DNA isolated from fresh leaves of tea plant, dried commercial green tea and black tea samples respectively. M is mol. wt. marker λ Hind III digest.

Results and Discussion We procured samples of 10 different black and 1 green tea brands from the Palampur market and isolated their DNA using the above protocol. The DNA was recovered in all cases, however, it was degraded (Table 1). The average DNA extracted using this protocol was 164–494 µg/g dry weight of tea sample and the OD 260/280 was 1.63 to 1.84. We could not isolate undegraded DNA from any of the samples tested except green tea. The DNA isolated from tea samples without prior soaking in water was brownish, comparatively less in amount and highly degraded. It could not be amplified by PCR. Soaking tea samples in water and then washing them several times in sterile water removed much of the brownish colour and resulted in better yield and quality of DNA. The degraded nature of DNA is probably due to the high temperature and pressure during processing of black teas, which distorts the cells, nucleus and ultimately the genomic material. This is supported by our results, which indicated that DNA isolated from fresh leaves or from green tea, which is processed differently, was undegraded or partially degraded (Figure 1). It has been reported earlier that after cell lysis polyphenolics bind to DNA and cause its degradation (John, 1992). It has also been reported that PVP can remove a lot of the polyphenolics (Kim et al., 1997). We, therefore, replaced mercaptoethanol with PVP in Saghai-Maroof’s protocol and this improved the DNA

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Figure 2. PCR amplification profile of the market samples of tea: Lanes 1 to 11 represent amplified products of DNA isolated from: Lazeez Premium, Brook Bond A1, Tea city, Birla, Premium Gold, New Lipton Richbrue, Lipton taaza, Sargam, Taj Mahal, Green tea and Lazeez masala brands of tea respectively (as in Table 1). Lane 12 is the PCR product of DNA from fresh leaves of a tea clone, ‘Upasi 9’. Lane B is a control lane without template DNA. M is PCR mol. wt. marker from Sigma. Panel A shows RAPD patterns using a single 10 mer oligo (Table 1) as a primer while panel B shows PCR amplification using a set of two primers (Table 1) designed from conserved sequences of plant 5 s rRNA gene so as to amplify variable spacers (Cox et al., 1992). Note that the reaction did not work in lane 10 and 11 in panel A in this gel.

quality. 1% PVP was found optimal for quality DNA. Using this protocol, we also isolated DNA from the discarded samples of teas after brewing. The DNA isolated from post brewed tea samples was relatively more degraded compared to pre-brewed tea samples and was not suitable for PCR (data not shown). To evaluate the suitability of the isolated DNA in downstream applications, we amplified the DNA through PCR using a 10 mer random primer from Operon Biotechnologies Inc. and also using a defined set of primers from conserved 5 s rRNA gene sequences (Table 1). As shown in Figure 2, panels A and B, the PCR product was in nearly all cases comparable to that obtained from undegraded DNA isolated from fresh leaves of a tea clone. It demonstrates that isolated DNA is suitable for any diagnostic purpose employing PCR as a technique. Figure 2, panel A also shows that the PCR products from successful amplification consist of 3 major bands and some minor bands. These include even DNA isolated from fresh leaves of Upasi 9 clone, thus the tea samples contain genuine tea plant material. The upper


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band in lane 4 of Figure 2A is very faint and could represent a mutation at primer annealing sites. The 3rd band (from top) is prominent in lanes 6, 8 and 9 (Figure2, panel A) in addition to 3 major bands in all samples. Since this band is also a prominent band of the control lane (Figure 2 panel A, lane B), it seems to be an artifact and does not represent the true PCR product. The control lane (Figure 2, panel A, lane B ) also shows some bands. It has been reported earlier that sometimes a RAPD primer folds back on itself, resulting in artifactual bands, but this usually does not occur when the DNA template is added to the reaction mixture (Williams et al., 1992). Figure 2, panel B also shows greater similarity of amplification products obtained from various tea samples. However, minor differences in band sizes, was also present and these might be due to minor sequence length differences, in the amplified spacer regions in different tea cultivars. The isolated DNA from tea samples could also be digested with restriction endonucleases Hind III, Sau IIIAI, Hinf I and Hae III (data not shown). To our knowledge, this is the first report of DNA isolation from market samples of tea, their PCR amplification, and restriction endonuclease digestion. In summary, good quality DNA from commercially dried tea samples was isolated and used successfully for down stream applications including restriction digestion and PCR amplification. This method has the potential for development of technology for testing the originality of commercial teas and for the identification of cultivars/varieties used by a tea manufacturer in production of a particular brand.

Acknowledgements Department of Biotechnology, Government of India, is acknowledged for the financial support for this work. This is IHBT communication No-9823.

References Cox AV, Bennett MD and Dyer TA (1992) Use of the PCR to detect spacer size heterogeneity in plant 5 s rRNA gene clusters and to locate such clusters in wheat (Triticum aestivum). Theor Appl Genet 83: 684–690. John ME (1992) An efficient method for isolation of RNA and DNA from plants containing polyphenolics. Nuc Acids Res 20: 2381. Kim CS, Lee CH, Shin JS, Chung YS and Hyung NI (1997) A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP. Nuc Acids Res 25: 1085–1086. Matsumoto S, Takeuchi A, Hayatsu M and Kondo S (1994) Molecular cloning of phenyle alanine ammonia lyase cDNA and classification of varieties and cultivars of tea plants using the tea PAL cDNA as a probe. Theor Appl Genet 89: 671–675.

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Rogers SU and Bendich AJ (1988) Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol 5: 69–76. Saghai-Maroof MA, Soliman KA, Jorgenson RA and Allard RW (1984) Ribosomal DNA spacer length polymorphism in Barley: Mendalian inheritance, chromosomal location and population dynamics. Proc Natl Acad Sci (USA). 8: 8014–18. Tai TH and Tanksley SD (1990) A rapid and inexpensive method for isolation of total DNA from dehydrated plant tissue. Plant Mol Biol Reptr 8: 297–303. Takeuchi A, Matsumoto S, Hayatsu M (1994) Chalcone synthase from C. sinensis: isolation of cDNA and the organ specific and sugar responsive expression of genes. Plant Cell Physiol 35: 1011–18. Wachira FN, Waugh R, Hackett CA and Powell W (1995) Detection of genetic diversity in tea using RAPD markers. Genome 38: 201–210. Williams JGK, Kubelik AR, Livak KJ, Rafalski JA and Tingey SV (1990) DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nuc Acids Res 18: 6531–35.