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ISBN No. 978-979-25-1209-0


EDITORS

Prof. Dr. Ir. C. Hanny Widjaja, MSc, Bogor Agricultural University, Bogor, Indonesia

Prof.Dr.Ir. Suminar Setiati Achmadi, M.Sc, Bogor Agricultural University, Bogor, Indonesia

Dr. dr. Irma Herawati Suparto, MS, Bogor Agricultural University, Bogor, Indonesia

Irmanida Batubara, SSi., MSi, PhD, Bogor Agricultural University, Bogor, Indonesia

Dr. Yaya Rukayadi, Universiti Putra Malaysia, Selangor, Malaysia

drh. Sulistiyani, M.Sc, PhD, Bogor Agricultural University, Bogor, Indonesia

Mohamad Rafi, SSi., MSi., Bogor Agricultural University, Bogor, Indonesia

Prof. Dr. Ir. Latifah K Darusman, MS, Bogor Agricultural University, Bogor, Indonesia

Biopharmaca Research Center

Institute of Research and Community Services - Bogor Agricultural University

INDONESIA

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ISBN No. 978-979-25-1209-0


LAY-OUT

Titis Arifiana, SSi

December, 2011

ISBN No. 978-979-25-1209-0

Address

Biopharmaca Research Center

Institute of Research and Community Services - Bogor Agricultural University

Kampus IPB Taman Kencana

Jln. Taman Kencana No. 3, Bogor 16128, INDONESIA

Telp +62-251-8373561 Fax +62-251-8347525, Mobile +62 81311195614

Email: bfarmaka@gmail.com Website: http://biofarmaka.ipb.ac.id

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ISBN No. 978-979-25-1209-0


Genomic DNA Isolation of Three Varieties of Graptophyllum pictum (L.) Griff

- 327 -

Harto Widodo and Dyah Subositi

Medicinal Plant and Traditional Medicine Research and Development Office

Jl. Raya Lawu No.11 Tawangmangu, Karanganyar, Central Java, E-mail: har2wido2@yahoo.com

ABSTRACT

Graptophyllum pictum, in Indonesian is named ‘daun ungu’, is one of the traditional plants usually used as

hemorrhoids medicament. It is claimed that the infusion prepared from the leaf of this plant possess analgesic, antiinflammatory,

and anti-diabetic activities. Medicinal Plant and Traditional Medicine Research and Development Office has

been cultivating three varieties of daun ungu which is lack of data about their characters mainly base on genetic

diversity. High content of gummy polysaccharides, polyphenols and secondary metabolites are critical factor in order to

obtain genomic DNA from medicinal and aromatic plants. For further genetic analysis of plants relies on high yields of

pure DNA samples. The main objective of the study was to provide a simple method of isolation of DNA, using in house

prepared reagents. Here we present the optimization of DNA isolation and PCR conditions for RAPD and ISSR analysis of

G. pictum (L.) Griff. The method involves a modified CTAB extraction employing polyvinylpyrrolidone while grinding,

successive long-term Chloroform: lsoamyalcohol extractions, and an RNase treatment. Thus the results indicate that the

optimized protocol for DNA isolation was suitable for further analysis, i.e. PCR based techniques and other diversity

analyses.

Keywords: DNA isolation, Graptophyllum pictum, RAPD and ISSR.

INTRODUCTION

Graptophyllum pictum, Indonesian named ‘daun ungu

(violet leave)’ is one of the traditional herbal plants

commonly grow in Indonesia as shrubs (Dalimarta,

1999). It grows profusely during the rainy season and

can easily be cultivated by vegetative propagation at this

period. It is mainly used as ornamental plant to adorn

the home gardens (Perry, 1980).

G. pictum was reportedly used in folkloric medicine as

poultice on cuts, wounds and all kinds of swellings and

for the treatment of ulcer, abscess, haemorrhoids etc

(Kasahara and Mangunkawatjia, 1986). It has

pharmacological specialty for various health problems,

such as: to release anuria, constipation, hemorrhoids,

maturing boil process, weak laxative for purgation, skin

softener and to enhance menstrual blood flow

(Dalimarta, 1999). The aqueous extract of the leaves of

G. pictum possess hypoglycemic effect which is

comparable to metformin and can be safely administered

orally without any immediate unwanted effect

(Olagbende-Dada et al., 2010), whereas the infusion of

the plant leaves has anti-diabetic activities (Lavergne

and Vera, 1989). Alcoholic extract of the leaf possess

analgesic, and anti-inflammatory effect (Ozaki et al.,

1989).

A reliable plant material used as medicinal products

such as herbal medicine s must be employed by

w e l l - a u t h e n t i c a t e d o f t h e p l a n t f o r e n s u r i n g

its pharmacological efficacy and safety. There is

no single authentication method that can be applied to

every med icinal pla nt (Da -Cheng et al., 2010).

Morphological and histological authentication is now

commonly practiced but they are not precise enough to

authenticate those herbs which are possibly substituted

or adulterated by plants with similar shapes and tissue

constructs. Ordinary chemical authentication is often not

reliable enough to produce easy-to-interpret results.

Moreover, it is difficult to distinguish closely related

species due to similar chemical compounds (Zhang et al.,

2007).

DNA-based analysis is one of the techniques that have

been widely used for authentication of plant species of

medicinal importance. It has the advantages that they

are least affected by age, environmental factors, and

physiological conditions of the samples (Zhang et al.,

2007). However isolation of superior quality, high

molecular mass genomic DNA, becomes difficult for

many medicinally important plants (Haque et al., 2008).

Although a number of methods for DNA isolation from

plants containing polyphenolics, polysaccharides

compound and high level of secondary metabolites and

have been developed (Pirttilä, 2001; Qiang et al., 2004)

none of these are universally applicable to all plants

(Varma et al., 2007). The published protocols are also

limited because of degradation of DNA by DNases and

other nucleases (Sharma and Sharma, 1980).

This study presents a method for extracting genomic

DNA from three varieties of G. pictum collected by

Medicinal Plant and Traditional Medicine Research and

Development Office (B2P2TO-OT) to lay down further

genetic-based analyses. Random amplified polymorphic

DNA (RAPD) and inter-simple sequence repeat (ISSR)

primers were utilized to evaluate those extracted

genomic DNA.

ISBN No. 978-979-25-1209-0


MATERIALS AND METHODS

Plant material

Leaves from three varieties of G. pictum which can be

distinguished by their leave’s color i.e.: green with pale

(white) blotches along the midrib (HL1), purple with pale

(white) blotches along the midrib (UL) and dark purple

(UP) collection of B2P2TO-OT.

Methods

DNA isolation from G. pictum leaves:

The leaves were weighed to about 100 mg per tube and

freezed at a temperature of -80°C for at least an hour.

Pre-warmed the buffer solution (2% CTAB extraction

buffer containing 100 mM Tris-HCl pH 8.0, 20 mM EDTA

pH 8.0, 1.4 M NaCl, 2% CTAB and 1% 2-

- 328 -

o C for 30 min. The leaves were

then ground using pre-freezed mortar and pestle (-80 o C)

and added with 1% (0.007 g) polyvinylpyrrolidone (PVP)

powder into fine powder, and add 700 µL buffer

solution, stired thoroughly. Transferred into micro tube,

preheated to 65°C for 45 min in incubator (Provocell-

Escol) and inverted every 10 min. Centrifuged the tube

at 12,000 rpm for 10 min at room temperature,

transferred the upper phase into a new tube (notice the

volume of upper phase). Added the same volume with

phenol : chloroform : isoamyl alcohol (25 : 24 : 1),

carefully inverted the tube (± 30 times). Centrifuged the

tube at 12,000 rpm for 10 min at room temperature,

transferred the upper phase into a new tube (notice the

volume of upper phase). Added the same volume with

chloroform : isoamyl alcohol (24 : 1), carefully inverted

the tube (± 30 times). Centrifuged the tube at 12,000

rpm for 10 min at room temperature, transferred the

upper phase into a new tube (notice the volume of upper

phase). Added 0,1 and 0,7 of volume supernatant with 3

M sodium acetate and isopropanol respectively. Placed

the tube at -20 o C for 60 min and centrifuged the tube at

12,000 rpm for 10 min at 4 o C. Discarded the

supernatant, added 500 µL 70% cold ethanol,

centrifuged at 12,000 rpm for 10 min at 4 o C (this step

was done twice). Discarded the supernatant and let the

tube open and the remaining ethanol evaporate.

Dissolved the pellet 100 µL Tris EDTA, and added 2 µL

RNase and incubated at 37 o C for ten hours, Added 10 µL

sodium acetate and 250 µL absolute ethanol, inverted for

about 6 times. Placed at -20 o C for 60 min, centrifuged at

13,000 rpm for 10 min at 4 o C.

Discarded the supernatant, added 500 µL cold ethanol,

centrifuged at 12,000 rpm for 10 min at 4 o C (this step

was done twice). Discarded the supernatant and let the

tube open and the remaining ethanol evaporate.

Dissolved the pellet in 50 µL Tris EDTA, stored at -20 o C

till further usage.

The purity of DNA was determined by obtaining the

mean of the absorbance ratios A260/A280 by

Spectrophotometer (uv-vis Spectrophotometer,

Shimadzu) and calculated the DNA concentration from

A260 (Sambrook and Russell, 2001).

PCR amplification of the DNA with RAPD primers:

PCR amplification was performed with a random decamer

primer of OPA1. Amplification was performed in a 25 μl

reaction volume and containing 25 ng/µL DNA, 50 μM

primers, 12.6 µL PCR master mixtures and 9.6 µL

nuclease free water (Promega). DNA amplification was

performed in thermal cycler (Bio Rad) under the

following condition: an initial denaturation at 94°C for 4

min and 44 cycles of 94°C for 45 sec (denaturation),

36°C for 1 min (annealing), and 72°C for 2 min

(elongation), followed one additional cycle by 8 min at

72°C.

PCR amplification of the DNA with ISSR primers:

An ISSR primer: HB-15 was used in this study. Genomic

DNA was used for amplification reactions by preparing 25

µL reaction mixture containing 25 ng/ µL DNA, 50 μM

primers, 12.6 μl PCR master mixtures and 9.6 µL

nuclease free water (Promega). DNA amplification was

performed in thermal cycler (BioRAD) under the following

condition, an initial denaturation at 95°C for 5 min

followed by 39 cycles of denaturation at 94°C for 1 min,

annealing at 52°C for 45 sec and elongation at 72°C for

2 min. One additional cycle of 8 min at 72°C was used

for final elongation.

Amplified products from both RAPD and ISSR were

electrophoresed in 2% agarose gel containing DNA

staining [(13.3: 1 v/v) (Good View – SBS Genetech)] for

about 5 hrs at 60 Volts in 1X TBE buffer solution and

visualized under UV light (GelDoc-Bio Rad).

ISBN No. 978-979-25-1209-0


RESULTS AND DISCUSSION

Sample was collected from young leaves, as secondary

metabolic compounds generally get accumulated in the

tissue, particularly those with medicinal properties, the

problem that becomes severe as the material gets older.

Instead of lyophilizing with liquid nitrogen, the sample

was ground using a pre-chilled mortar and pestle (-20 o C

or -80 o C) which allowed harmful cellular enzymes and

chemicals inactive. It will be suitable for either a sample

from alcohol fixed tissue or a condition which no liquid

nitrogen available.

The chemicals and steps used in this protocol were

suitable for isolation genomic DNA from all of three

varieties of G. pictum. Addition of PVP and buffer-

containing β-marcaptoethanol could prevent oxidation of

secondary metabolites in the disrupted plant sample.

CTAB is a cationic detergent, which solubilises

membranes and forms a complex with DNA (Sghaier and

Mohammed, 2005). High ionic strength of CTAB forms

complexes with proteins and most of the acidic

polysaccharides.

Polysaccharide contaminations are particularly

problematic, as they can inhibit the activity of many

commonly used molecular biology enzymes, such as

polymerases (Fang et al., 1992), ligases and restriction

endonucleases. This is because nucleic acids form tight

complexes with polysaccharides creating a gelatinous

pellet which makes it difficult for the embedded DNA to

be inaccessible to the commonly used molecular biology

enzymes (Sharma et al., 2002). Combination of Natrium

acetat and isopropanol were efficient in removing most

of secondary metabolites and polysaccharides from DNA

(Aljanabi et al., 1999). Whereas, PVP was used to

remove phenolics compounds. Polyphenol contamination

of DNA also makes it resistant to restriction enzymes and

it is co-purify with DNA (Katterman & Shattuck, 1983)

and interact irreversibly with proteins and nucleic acids

(Loomis, 1974).

Table 1. The average of concentration and purity of genomic DNA

isolation from three varieties of Graptophyllum pictum

No Sample

1.

2.

3.

Green with

pale (white)

blotches along

the midrib

leaves (HL1)

Purple with

pale (white)

blotches

along the

midrib

leaves (UL)

Dark purple

leaves (UP)

- 329 -

A 260

OD Concentration

A 280

µg/ mL

Purity

0.0445 0.0235 222.5 1.89

0.1490 0.0775 745 1.92

0.2405 0.1220 1202.5 1.97

The spectrophotometric determination results were as

the expected (1.8 – 2.0) (Table 1.) There was neither

RNA contamination after incubation for about ten hours

nor any sign of degraded DNA in all three samples (Fig.

1). The DNA obtained worked well for the subsequent

studies in PCR-based analysis. This genomic DNA obtain

could be amplified using RAPD-PCR and ISSR and good

amplification was observed (Fig. 2)

Figure 1. Genomic DNA isolated from three varieties of

Graptophyllum pictum

M: weight marker, Lane 1: HL, Lane 2: PL, and Lane 3: UP

Based on these findings, it can be concluded that this

protocol provides nuclear DNA that has little or no visible

coloration; possesses a µspectrophotometric A 260 / A 280

value > 1.8; has an intact DNA or at least the mean

fragment length more than 10 kb. Moreover, the protocol

can be used to isolate DNA from young plant leaves as

well as younger tissues including seedlings, and it works

well with frozen tissue which is suitable at condition that

liquid nitrogen is not available.

Figure 2. PCR amplification of genomic DNA from three

varieties of Graptophyllum pictum with RAPD primer:

OPA1 (A), and ISSR primer: HB-15 (B)

M: weight marker, Lane 1: HL, Lane 2: PL, and Lane 3: UP

ISBN No. 978-979-25-1209-0


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ISBN No. 978-979-25-1209-0

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