Isolation and PCR Amplification of Genomic DNA from Market Samples of Dry Tea

Plant Molecular Biology Reporter17: 171–178, 1999.© 1999Kluwer Academic Publishers. Printed in the Netherlands.

Protocols

Isolation and PCR Amplification of Genomic DNAfrom Market Samples of Dry Tea

MAHIPAL SINGH∗, BANDANA and P.S. AHUJADivision of Biotechnology, Institute of Himalayan Bioresource Technology, Palampur(HP), 176061, India

Abstract. A simple procedure for DNA isolation from processed dried commercial samplesof tea is described. The method involves a modified CTAB procedure employing extensivewashing, use of 1% PVP to remove polyphenolics and a single phenol:chloroform extractionstep. The average yield ranges from 164–494µg/g tea sample for various market samples.The DNA obtained from 11 different brands of tea using this procedure were consistentlyamplifiable (using both RAPD primers as well as defined sequences as primers) and digestiblewith restriction endonucleases.

Key words: Camellia sinensis, DNA isolation, PCR, RAPD, Tea

Abbreviations: CTAB, cetyl trimethyle ammonium bromide; EDTA, ethyle diamine tetraacetic acid; PCR, polymerase chain reaction; PVP, poly vinyl pyrrolidone; RAPD, randomlyamplified polymorphic DNA; RT, room temperature;Taq, Thermus aquaticus.

Introduction

Tea is a woody beverage crop with high polyphenolic contents. Isolation oftea 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 theDNA from fresh young leaves of tea plants. Isolation of DNA from herbar-ium specimens (Rogers et al., 1988) and dehydrated plant tissues (Tai andTanksley, 1990) have also been described. However, none of these methodscould provide DNA from market samples of processed tea, suitable for down-stream applications. In molecular diagnostic studies involving identificationof cultivars and varieties used by a tea manufacturer in preparation of a par-ticular lot or brand of tea and also in diagnosis of adulteration of tea samples

∗Author for correspondence. e-mail: [email protected]

172 Mahipal Singh , Bandana and P.S. Ahuja

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

Materials and Methods

Plant materials

The fresh leaf material ofCamellia sinensis (L) O. kuntze(Upasi-9) used inthis study was obtained from ‘Banuri Experimental Tea Garden’ of IHBT.Different brands of dry commercial tea samples available in the open mar-ket in packed forms were obtained from Palampur market and are listed inTable 1.

Equipment and chemicals

High speed refrigerated centrifuge (Hitachi)Robocycler (Stratagene)Mortar and pestleLiquid nitrogenWater bathExtraction buffer: 2% (w/v) CTAB, 100 mM Tris-HCl (pH 8.0), 20 mMEDTA, 1.4 mM NaCl, 1% PVP1× 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)IsopropanolPhenol:chloroform (1:1)Absolute ethanol70% ethanol (v/v)PVPTaqpolymerase (Bangalore Genei)

DNA isolation

• Soak the tea samples overnight in sterile distilled water, cleanse 4 timeswith double distilled water and dry the samples in an oven (60◦C) forabout an hour.

• Grind 1 g of dried tea samples in a mortar and pestle in the presence ofliquid nitrogen.

Isolationand

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

S.N. Brand Name# Market price∗ DNA extraction PCR amplification

(Rs/kg) +/− µg/gm OD260/280 Status RAPD primer Defined primer

1. Lazeez Premium 152 + 494 1.72 Degraded +(3) +(5)

2. Brook Bond A1 126 + 278 1.73 Degraded +(3) +(5)

3. Tea city 180 + 451 1.73 Degraded +(3) +(5)

4. Birla 160 + 444 1.72 Degraded +(2) +(5)

5. Premium Gold 142 + 461 1.84 Degraded +(3) +(5)

6. New Lipton Richbrue 166 + 460 1.83 Degraded +(4) +(5)

7. Lipton taaza 183 + 164 1.79 Degraded +(3) +(5)

8. Sargam 183 + 489 1.62 Degraded +(4) +(5)

9. Taj Mahal 208 + 287 1.78 Degraded +(4) +(5)

10. Green tea 110 + 240 1.90 Partially degraded – +(5)

11. Lazeez masala 134 + 275 1.76 Degraded – +(5)

Note:The oligonucleotide used in this study as a RAPD primer was 5′-AAGACCCCTC-3′ and as defined primers were: Forward primer (5′-TTTAGTGCTGGTTGTCGC-3′), Backward primer (5′-TGGGAAGTCCTCGTGTTGCA-3′).Numbers in parentheses represents number of PCR bands.#All teas here are black except number 10.∗Prices for January 1998.


• Transfer the tea powder into a clean autoclaved centrifuge tube and add4 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 alco-

hol.• 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.Notes1. 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 25µL final vol containing 100µM each

of the four dNTPs, 100µM primer, 20 ng of genomic DNA and 0.3 unitsof Taqpolymerase.

• Program the robocycler for 1 cycle of 4 min at 94◦C and then 45 cyclesof 1 min at 94◦C, 1 min at 37◦C and 2 min at 72◦C. The last extensioncycle was programmed at 72◦C for 7 mins.

• Run the amplified products in a 1.4% agarose gel, stain in ethidiumbromide and photograph with the help of an image analyser.

NoteThe 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).

Isolation and PCR Amplification from Tea 175

Figure 1. Comparison of genomic DNA extracted from black and green tea samples: Lanes1, 2 and 3 show DNA isolated from fresh leaves of tea plant, dried commercial green tea andblack tea samples respectively. M is mol. wt. markerλ Hind III digest.

Results and Discussion

We procured samples of 10 different black and 1 green tea brands from thePalampur market and isolated their DNA using the above protocol. The DNAwas recovered in all cases, however, it was degraded (Table 1). The averageDNA extracted using this protocol was 164–494µg/g dry weight of tea sam-ple and the OD 260/280 was 1.63 to 1.84. We could not isolate undegradedDNA from any of the samples tested except green tea. The DNA isolated fromtea samples without prior soaking in water was brownish, comparatively lessin amount and highly degraded. It could not be amplified by PCR. Soakingtea samples in water and then washing them several times in sterile waterremoved much of the brownish colour and resulted in better yield and qualityof DNA. The degraded nature of DNA is probably due to the high temperatureand pressure during processing of black teas, which distorts the cells, nucleusand ultimately the genomic material. This is supported by our results, whichindicated that DNA isolated from fresh leaves or from green tea, which isprocessed differently, was undegraded or partially degraded (Figure 1). It hasbeen reported earlier that after cell lysis polyphenolics bind to DNA and causeits degradation (John, 1992). It has also been reported that PVP can removea lot of the polyphenolics (Kim et al., 1997). We, therefore, replaced mercap-toethanol with PVP in Saghai-Maroof’s protocol and this improved the DNA


Figure 2. PCR amplification profile of the market samples of tea: Lanes 1 to 11 representamplified 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 Lazeezmasala brands of tea respectively (as in Table 1). Lane 12 is the PCR product of DNA fromfresh leaves of a tea clone, ‘Upasi 9’. Lane B is a control lane without template DNA. Mis PCR mol. wt. marker from Sigma. Panel A shows RAPD patterns using a single 10 meroligo (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 variablespacers (Cox et al., 1992). Note that the reaction did not work in lane 10 and 11 in panel A inthis 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. TheDNA isolated from post brewed tea samples was relatively more degradedcompared to pre-brewed tea samples and was not suitable for PCR (data notshown).

To evaluate the suitability of the isolated DNA in downstream applica-tions, we amplified the DNA through PCR using a 10 mer random primerfrom Operon Biotechnologies Inc. and also using a defined set of primersfrom 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 thatobtained from undegraded DNA isolated from fresh leaves of a tea clone. Itdemonstrates that isolated DNA is suitable for any diagnostic purpose em-ploying PCR as a technique. Figure 2, panel A also shows that the PCRproducts from successful amplification consist of 3 major bands and someminor bands. These include even DNA isolated from fresh leaves of Upasi 9clone, thus the tea samples contain genuine tea plant material. The upper

Isolation and PCR Amplification from Tea 177

band in lane 4 of Figure 2A is very faint and could represent a mutation atprimer annealing sites. The 3rd band (from top) is prominent in lanes 6, 8 and9 (Figure2, panel A) in addition to 3 major bands in all samples. Since thisband 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. Thecontrol lane (Figure 2, panel A, lane B ) also shows some bands. It has beenreported earlier that sometimes a RAPD primer folds back on itself, resultingin artifactual bands, but this usually does not occur when the DNA templateis added to the reaction mixture (Williams et al., 1992). Figure 2, panel Balso shows greater similarity of amplification products obtained from varioustea samples. However, minor differences in band sizes, was also present andthese might be due to minor sequence length differences, in the amplifiedspacer regions in different tea cultivars. The isolated DNA from tea samplescould also be digested with restriction endonucleasesHind III, Sau IIIA-I, Hinf I and Hae III (data not shown). To our knowledge, this is the firstreport of DNA isolation from market samples of tea, their PCR amplification,and restriction endonuclease digestion. In summary, good quality DNA fromcommercially dried tea samples was isolated and used successfully for downstream applications including restriction digestion and PCR amplification.This method has the potential for development of technology for testing theoriginality of commercial teas and for the identification of cultivars/varietiesused by a tea manufacturer in production of a particular brand.

Acknowledgements

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

References

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Isolation and PCR Amplification of Genomic DNA from Market Samples of Dry Tea

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