ISSN 2320-5407 International Journal of Advanced Research (2015), Volume 3, Edisi 7, 30-34

homepage jurnal: http://www.journalijar.com JURNAL INTERNASIONAL

PENELITIAN ADVANCED

ARTIKEL PENELITIAN

Tinggi berat molekul ekstraksi DNA dari enam Quercus spesies Kumaun Himalaya,
India
1 Aseesh Pandey * dan 2 Sushma Tamta

1. GB Pant Institut Himalaya Lingkungan dan Pembangunan, Kosi-Katarmal, Almora-263.643 Uttarakhand, India

2. kultur jaringan tanaman dan Laboratorium Biologi Molekuler, Departemen Bioteknologi, Bhimtal Kampus, Kumaun University, Nainital- 263.136,
Uttarakhand

Info naskah Abstrak

Naskah Sejarah: Quercus is an economically and ecologically important globally distributed genus of family
fagaceae comprises of trees and shrubs. For genetic diversity, phylogenetic and evolutionary
Diterima: 15 Mei 2015 Akhir
Diterima: 15 Juni 2015 Diterbitkan
study of the species of this genus along their distribution, molecular tools can be beneficial.
Online: Juli 2015 For them isolation of good quality DNA is prerequisite. High phenolic content of leaf tissue of

Kata kunci: Quercus makes it difficult to isolate good quality DNA free from phenol, lignin and pigments by
conventional CTAB method. A reliable protocol for DNA isolation from frozen leaves of six Quercus
Quercus, polifenol, studi molekuler, species of Kumaun Himalaya has been standardized. The isolated DNA was found suitable
cpDNA, Kumaun Himalaya for molecular studies and can be used to assess genetic diversity, phylogeny, and other
molecular marker based studies.
* Penulis yang sesuai

Copy Right, IJAR, 2015,. All rights reserved

Aseesh Pandey

INTRODUCTION

The isolation of pure, high-molecular-mass genomic DNA is essential for many molecular biology applications (Nagori and Purohit, 2012).
There are a number of protocols available for the DNA extraction from plant material, but often it is difficult in many plants due to presence of
metabolites that interfere with DNA isolation procedures and subsequent applications (Arbi et al., 2009). Extraction of DNA is generally
hindered by polyphenols and their quinone oxidation products and by carbohydrate polymers, isolation of DNA using the CTAB complex
gives a product of inadequate purity (Murray and Thompson, 1980; Manning, 1981).

Quercus is an economically and ecologically important genus of family fagaceae comprises of trees and shrubs. In India the oak flora
is distributed in the sub Himalayan region from Jammu and Kashmir in the Northwest, Garhwal and Kumaun hills of Uttarakhand and Himanchal
Pradesh in the Central zone, to Manipur and Arunanchal Pradesh in the Northeast (Pandey and Tamta, 2014) and molecular tools may be
beneficial for genetic diversity, phylogenetic and evolutionary study of its species along their distribution. The target six Quercus species includes
5 evergreen species, namely Q. glauca ( phaniyat oak), Q. leucotrichophora ( banj oak), Q. floribunda ( tilonj oak), Q. lanuginosa ( rianj oak) and Q.
semecarpifolia ( brown oak) in the Central Himalaya between 1000 to 3600 m asl (Champion and Seth, 1968) and one deciduous species i.e. Q.
serrata ( Manipuri oak) which is planted in an altitude of 1370 m asl (Pandey and Tamta, 2012).

For molecular studies isolation of good quality DNA is prerequisite. However due to high phenolic content, tannins and
polysaccharides in leaf tissues of Quercus ( Beldeanu, 2008), it is very difficult to isolate good quality DNA free from phenol, lignin and pigment
by conventional methods. These metabolites cannot be eliminated during DNA isolation and can inhibit the DNA polymerase activity during
polymerase chain reaction (PCR) (Pandey,
1996). A good quality of DNA corresponds to a high specificity of PCR amplified products. Therefore, in the present study attempts were
made and a simple, reliable and reproducible protocol for isolation of DNA from frozen leaves of six Quercus spesies Kumaun Himalaya,
India telah dibakukan. Ada terbatas / tidak ada laporan yang

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tersedia mengenai studi molekuler dalam genus Quercus dari negara dan upaya ini akan bekerja sebagai cetak biru molekuler untuk genus Quercus
serta jenis pohon lainnya.

Material dan metode

bahan tanaman. daun muda secara acak dikumpulkan dari pohon elit dewasa tumbuh dalam populasi liar diidentifikasi berbeda dari bukit Kumaun. Daun
dipetik disimpan dalam -20 Hai C mini-chiller dan dibawa ke laboratorium dan disimpan pada -20 0 C sampai digunakan.

Solusi. Extraction buffer: 2% (w/v) CTAB, 100 mMTris-HCl (pH 8), 1.4 M NaCl, 20 mM EDTA (pH 8), 0.5% 2-mercaptoethanol (v/v), 1%
PVP (w/v); chloroform-isoamylalcohol- 24:1 (v/v); TE buffer- 10 mMTris-HCl (pH 8); 1 mM EDTA; Isopropanol (Stored at -20 o C); 70%
ethanol; 5M NaCl; 1xTAE- Tris base, Glacial acetic acid, 0.5M EDTA (pH 8.0) were prepared.

DNA extraction. Frozen leaf tissues of Quercus species kept at -20 0 C for five days were surface sterilized with 70% ethanol and leaf-midrib
was removed by using a sterile blade. Pre-chilled mortar and pestle were used to grind the sample in the presence of liquid nitrogen and PVP
(1:1; ratio of tissue: PVP). The frozen powdered sample was transferred to 5ml of extraction CTAB buffer containing 0.5% β-mercaptoethanol
(added freshly) in 50-ml centrifuge tube. It was mixed vigorously by vortexing and incubated at 60°C for 60 min in water bath followed by
treatment with equal volume of chloroform: isoamyl alcohol (24:1) and mixed for 2 min. The upper aqueous phase obtained by centrifugation
at 15000 rpm for 15 min at room temperature, was transferred to a fresh autoclaved centrifuge tube by using wide-bore pipette, chloroform
extraction was repeated if extract still contain color. DNA was precipitated by gently mixing with equal volume of chilled isopropanol to obtain
fibrous DNA and incubated for 30 min at 4 0 C for complete precipitation. DNA fibers were spooled out using bent capillary tube and transferred
to new autoclaved centrifuge tube having 70% ethanol for 30 min for washing, this step was repeated if there is any visible adhering
contaminant remaining. DNA pellet was air dried and suspended in 50-200 μl of 1xTris EDTA (TE) buffer, if necessary brief heating in water
bath at 65°C was provided to dissolve pellet in TE and stored at -20 o C till use.

DNA Qualitation and Quantification. The quality of DNA was checked by agarose (Promega) gel electrophoresis (GeNei). Samples were
prepared by taking 5.0 μl of DNA and 0.5μl of 6x bromophenol blue dye (0.25% Bromophenol Blue and 50% glycerol) mixed well with the
help of a micropipette. Samples were electrophoresed in 1xTBE buffer for 1h, initially at 60 V until DNA comes out from the well and then
increased up to 80 V in 0.8% agarose gel matrix having 10mg/ml ethidium bromide (EtBr). Gel was photographed under UV light using Gel
Documentation System (Bio Red). Quantity and purity of DNA was estimated using UV–vis Spectrophotometer (Thermo Scientific, UV1) by
measuring the absorbance at 260 and 280 nm. DNA quantity was assessed using the formula:

DNA quantity (µg/µl) = OD 260 X Dilution factorX50

1000

OD: optical density

The absorbance ratio (A 260/ A 280) between 1.6-1.9 was considered as pure and used for PCR amplification.

Reaksi PCR dilakukan di 0,2 ml tabung polypropylene PCR (Bangalore Genei, India) menggunakan Thermal Cycler (Guru Cycler Personal,
Eppendorf). Sampel DNA asli kemudian diencerkan sampai 50 ng / ml untuk campuran reaksi. Setiap campuran reaksi 50 ml terkandung 1x Taq
penyangga A (10mMTris-Cl, 50 mM KCl), 200μM dNTP, 0,2 pM primer oligonukleotida, 1U Taq DNA polymerase (Bangalore Genei, India) dan 50ng
Template DNA, air nuklease bebas digunakan untuk makeup volume akhir sampai 50μl. Pemrograman PCR untuk amplifikasi fragmen cpDNA
adalah sebagai berikut: denaturasi awal pada 95ºC selama 8 menit diikuti oleh 34 siklus denaturasi pada 95ºC untuk 1min, primer langkah anil pada
58 dan / atau 60 º C untuk 1min, perpanjangan langkah pada 72ºC selama 2 menit dan ekstensi akhir langkah pada 72ºC selama 10 menit. Produk
PCR diperkuat dijalankan pada 1,5% agarosa (w / v) gel dan produk diperkuat dibandingkan dengan menggunakan DNA 1kb tangga (Bangalore
Genei, India). Band intensitas divisualisasikan dan difoto di bawah sistem dokumentasi gel. pasangan primer yang mampu menghasilkan band-band
yang jelas dalam setiap sampel dipilih dan dianggap primer terbaik untuk amplifikasi daerah yang ditargetkan (mat k dan intergenic trnL-trnF spacer).

hasil
daun Quercus jenis di pabrik ekstraksi DNA menciptakan masalah karena kehadiran polisakarida dan metabolit sekunder tertentu,
yang telah diamati untuk mengganggu prosedur isolasi DNA dan menghambat

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aktivitas berbagai enzim DNA-memodifikasi, seperti enzim restriksi, polimerase, dan ligases. Dua protokol berbasis CTAB dari ekstraksi DNA
genom, salah satu metode berbasis CTAB dijelaskan oleh Doyle dan Doyle (1990) dan lain metode berbasis CTAB dengan modifikasi diuji
dalam penelitian ini untuk hasil, kualitas dan kesesuaian DNA untuk biologi molekuler. Protokol berbasis CTAB tanpa modifikasi menghasilkan
DNA warna krem ​dengan viskositas tinggi yang tidak dapat dilarutkan dalam TE penyangga dan tidak cocok untuk elektroforesis karena
terjebak ke dalam sumur selama pemisahan elektroforesis (Plat-1c, Lane 1-4). dimodifikasi metode CTAB tidak hanya menghasilkan DNA
berkualitas tinggi di masing-masing sampel tetapi juga menunjukkan kemurnian (Plat-1bLane 1-6). Modifikasi prinsip dalam metode ini
termasuk penggunaan penambahan 2,0% dari PVP, pengulangan kloroform: curah hujan isoamylalcohol, akhirnya elusi serat DNA
menggunakan pipa kapiler bengkok tanpa sentrifugasi. konsentrasi PVP lebih tinggi mencegah browning DNA (karena kemampuannya untuk
membentuk kompleks dengan polifenol melalui ikatan hidrogen) umumnya disebabkan oleh adanya polifenol. Panjang kloroform jangka:
perawatan alkohol isoamil dielusi klorofil dan pigmen lain dan pewarna. Dalam CTAB berdasarkan protokol 'sentrifugasi' mengakibatkan pelet
DNA kompak dengan polifenol dan kontaminan lainnya, yang sulit untuk melarutkan dalam etanol dan TE juga. Oleh karena itu, untuk
mengatasi pembentukan pelet terkontaminasi, protokol ini dimodifikasi dengan menggantikan langkah dengan langsung spooling off serat
DNA. modifikasi telah meningkat kelarutan, karena hanya benang DNA rapuh diambil tanpa sentrifugasi dan ditransfer langsung di 70% etanol
untuk mencuci. Peningkatan kelarutan dalam etanol memfasilitasi penghapusan polisakarida dari DNA, dan kemurnian meningkat karena ini.
Agarose gel elektroforesis diverifikasi tidak ada bukti kontaminasi RNA dan menegaskan bahwa DNA terisolasi yang berkualitas baik
(1,65-2,10). Rentang hasil bervariasi menurut spesies. Quercus glauca yielded maximum

13.17 µg followed by Q. lanuginosa 5.97 µg, Q. floribunda 2.94 µg, Q. semecarpifolia 2.80 µg, Q. leucotrichophora 1.89 µg and Q. serrata 1.68
µg DNA/gram of leaf tissue. The isolated DNA was found suitable for molecular biology experiments and the amplified products of all six Quercus
species with primer pair (forwardAAAGGCCCGTTTGATTCCCCA and Reverse-ACCAGCTGAGCTATCCCGACCA) is depicted in
Plate-1d.

a b

c d
c

Plate-1: DNA extraction and amplification of target cpDNA regions of six Quercus species of Kumaun
Himalaya.

a. Spooled extracted DNA suspended in 70 % ethanol b. lane1-6 DNA extracted through modified CTAB method (1 Q. leucotrichophora 2
Q. glauca 3 Q. floribunda 4 Q. semecarpifolia 5 Q. serrata 6 Q. lanuginosa)
c. lane1-4 DNA extracted through CTAB method without modification (1 Q. glauca 2 Q. serrata 3 Q. semecarpifolia 4 Q. lanuginosa d. Amplification
of 500bp DNA of six Quercus species; M marker (1kb, 100

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jalur tangga) 1 Q. semecarpifolia 2 Q. leucotrichophora 3 Q. serrata 4 Q. floribunda 5 Q. glauca 6 Q.

lanuginosa

Diskusi
To obtain molecular biology study grade genomic DNA several modifications in the original CTAB procedures have been reported from
plants with high polysaccharides and polyphenols compounds (Porebski and Bailey, 1997; Michiels et al., 2003; Haquen et al., 2008; Nagori
and Purohit, 2012). In the present study addition of increased amount of PVP, repeatition of chloroform: isoamylalchol precipitation step
followed by spooling of DNA threads directly, provide good quality DNA from leaves using CTAB based DNA extraction. In normal CTAB
method high viscosity of extracted DNA may be due to high endogenous levels of polysaccharides, phenolics and other organic constituents
(Sarwat et al., 2006). PVP forms complex hydrogen bonds with phenolic compounds and coprecipitates with cell debris upon lysis
(Ghaffariyan et al., 2012). All phenolic compounds as well as polysaccharides, bind firmly to nucleic acids during DNA extraction and
interfere with subsequent reactions (Angeles et al., 2005; Hanania et al., 2004; Puchooa and Khoyratty, 2004). In addition to phenolic
compounds in the leaves, polysaccharides also interfere with biological enzymes such as polymerases, restriction endonucleases and
ligases (Michiels et al., 2003), which results unsuccessful amplification during PCR, PVP has been used to extract genomic DNA from other
polyphenol-rich plants such as cotton, sugarcane, lettuce and strawberry (Aljanabi et al., 1999) and several conifers (Kim, 1997). Since
CTAB binds to fructans and other polysaccharides and forms complexes that removed during subsequent chloroform extraction (Michiels et
al., 2003; Jitu and Kr, 2008) so the repetition of chloroform: isoamyl step produce polysaccharide free DNA.

Conclusions
The method described here is (i) swift- one step (centrifugation) was reduced and pellet solubility time was also shortened, (ii) modest-
concentration of PVP was increased and pellet was directly eluted and (iii) reliable- it has been successfully tested in six evergreen and
deciduous Quercus species. The DNA obtained from described method was free of polysaccharides and secondary metabolites, as shown by
the absence of viscous or brown substances. Therefore this method can be used effectively in molecular and evolutionary research, and can be
applied for the processing of number of samples of Quercus as well as other polyphenols and polysaccharide rich samples.

Acknowledgement
The authors wish to acknowledge to GBPIHED for financial assistance and the Department of Biotechnology, Kumaun University, Nainital,
Uttarakhand, India for providing necessary facilities for this study.

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