Indian Journal of Biotechnology

Vol 8, April 2009, pp 187-192

A simplified method for extraction of high quality genomic DNA from

Jatropha curcas for genetic diversity and molecular marker studies

D V N Sudheer Pamidimarri, Meenakshi, Ritam Sarkar, Girish Boricha and Muppala P Reddy*

Discipline of Wasteland Research, Central Salt and Marine Chemicals Research Institute, Bhavnagar, 364 002, India

Received 8 June 2007; revised 1 April 2008; accepted 20 August 2008

A simple and efficient protocol for the extraction of high quality genomic DNA from different tissues, including callus

generated from leaves of Jatropha curcas has been developed. The important steps in this protocol include (a) use of 3.5 M

NaCl in extraction buffer; (b) 2.0 M NaCl (final concentration) during precipitation; (c) Tris saturated phenol in place of

phenol:chloroform:isoamyl alcohol at purification phase; (d) 80% ethanol for DNA precipitation, and (e) performing all the

steps at RT. The DNA thus extracted from the leaves had 1.81±0.063, OD at A260/280 and the yield was 120 to 140 µg/g of

material. The extracted DNA was found suitable for restriction digestion, ligation and PCR amplification. It was also used

for DNA fingerprinting techniques, RAPD and AFLP, for development of molecular markers and studies on genetic

diversity.

Keywords: CTAB, Jatropha curcas L, molecular markers, RAPD, AFLP

Introduction Non-renewable hydrocarbons are being used as the

major energy source the worldover. Their fast

depletion has forced the scientific community to

search for renewable sources, which could be used as

fuel. Besides the threat of depleted reserves, their

excessive use can aggravate greenhouse gases, which

are now held responsible for global warming.

Biofuels and bioenergy encompass a wide range of

alternative sources of energy of biological origin.

These renewable energy sources have the potential of

increasing energy supplies in a self-reliant way in

developing countries. Plant-based fuels create a better

balance between the formation and consumption of

CO2; decrease particulate matter, CO, unburnt

hydrocarbons and SO2 emissions into the

atmosphere1. In this regard, biodiesel derived from the

seed-oil of Jatropha curcas is fast emerging as an

available alternative to fossil fuels2,3

and has the

desirable physio-chemical characteristics and

performance, even superior to diesel4. J. curcas is not

browsed by animals, has the potential to grow on

eroded soils and is tolerant to stresses including

drought. The by-products, obtained while preparing

biodiesel, have industrial applications5. The deoiled

cake is used as fertilizer and in biogas production6,7

.

Almost all parts of the plant are used in medicine6.

Despite its potential as an alternative to fossil fuel,

jatropha is not being fully exploited due to lack of

quality planting material for cultivation. Hence, there

is a need to identify high yielding clones of J. curcas

for its further improvement and large-scale cultivation.

Molecular marker analysis in genome studies

greatly enhances the speed and efficiency of crop

improvement. Molecular markers closely linked to

traits of economic importance have been developed in

several crops8. These have allowed the selection of

desirable traits in a genotype. DNA fingerprinting

techniques like, Restriction fragment length

polymorphism (RFLP), Amplified fragment length

polymorphism (AFLP), Random amplified

polymorphic DNA (RAPD), microsatellite markers

like SSR’s, Sequence characterized amplified regions

(SCAR), Sequence tagged sites (STS), and rDNA-ITS

have been used for the generation of molecular

markers and their efficient use in breeding9-12

. For

these PCR based marker-assisted selection, suitably

modified extraction method for genomic DNA in

terms of quality and quantity is essential for a

particular crop.

Protocols are available for the extraction of pure

DNA from many plant species, particularly the latex

containing plants13

and those producing secondary

__________________________ *Author for correspondence:

Tel: 91-278-2567760; Fax: 91-278-2566970/2567562

E-mail: mpr@csmcri.org.

INDIAN J BIOTECHNOL, APRIL 2009

188

metabolites14

. However, published protocols available

in the literature could not be successfully used for the

isolation of high quality DNA from J. curcas.

Therefore, we developed a reliable and cost-effective

method by modifying the CTAB protocol15

. The

important modifications included in this protocol are

the use of (i) 3.5 M NaCl in extraction buffer; (ii) Tris

saturated phenol during purification (pH-8.0); (iii)

80% ethanol and 2.0 M NaCl (final concentration)

during precipitation; and (iv) conducting all the steps

at room temperature (RT). The DNA thus extracted

from the leaves had 1.81±0.063 OD at A260/280

(Table 1) could be efficiently digested by restriction

endonucleases, subjected to ligation, and was found

suitable for PCR amplification as well as other down-

stream processes in genetic diversity and molecular

marker studies.

Materials and Methods Several experiments based on the available

protocols15-17

were performed using fresh plant

material collected from one-year-old plants grown in

the genetic garden of Central Salt and Marine

Chemicals Research Institute, Bhavnagar for (i)

Determining the type of plant material to be used for

the extraction, (ii) Incubation time of buffer and tissue

mixture at 65°C, (iii) Buffer to tissue ratio and (iv)

Extraction with phenol:chloroform:isoamyl alcohol

vs. Tris saturated phenol followed by chloroform:

isoamyl alcohol extraction in extraction and

purification phases. All the experiments were repeated

3-4 times to check reproducibility.

Extraction Buffer

The extraction buffer (pH 8.0) contained 2%

CTAB, 100 mM Tris-HCl, 3.5 M NaCl, 20 mM

EDTA, 0.2 M β-Mercaptoethanol and 2% PVP.

Chemicals

Tris saturated phenol, phenol:chloroform:isoamyl

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

70% and 80% ethanol, 5 M NaCl, 3 M sodium acetate

(pH 5.2) and TE buffer (10 mM Tris-HCl, 1 mM

EDTA, pH 8.0). Solutions and buffers were

autoclaved at 121°C at 15 psi pressure (Tommy

autoclave, Japan). The stock solution 10 mg/mL of

RNase was prepared as per the user’s manual (Sigma

USA).

DNA Extraction Extraction Phase

Fresh young plant tissues plucked from genetic

garden were rinsed with distilled water and blotted

gently with soft tissue paper; 0.1 g of this tissue,

precooled using liquid nitrogen, was ground to a fine

powder with a mortar and pestle along with 10 mg (2%

of extraction buffer) of PVP (Sigma). The powdered

tissue was scraped into a 2.0 mL microcentrifuge tubes

containing preheated (65°C) extraction buffer in a 1:5

ratio (0.5 mL). β-Mercaptoethanol was then added to

the final concentration of 0.2 M and mixed well. The

mixture was incubated in water bath at 65°C for 90 min

and cooled for 5 min. An equal volume of

chloroform:isoamyl alcohol mixture (24:1) was added

to the extract and mixed by gentle inversion for 5 to 10

min to form an uniform emulsion. The mixture was

centrifuged at 8000 rpm for 8 min at RT.

Chloroform:isoamyl alcohol extraction step was

repeated again. The aqueous phase was pipetted out

gently, avoiding the interface. To the above solution, 5

M NaCl (to final concentration 2M) and 0.6 volume of

isopropanol of the total solution was added and

incubated at RT for 1 h. To the above solution, two

volumes of 80% ethanol was added and incubated

again for 10 min at room temperature for DNA

precipitation. After incubation, the mixture was

centrifuged at 10,000 rpm for 15 min. The

white/translucent pellet was washed with 70% ethanol,

dried and resuspended in 200 µL of TE buffer.

Purification Phase

The sample was then treated with 20 µL of 10

mg/mL of RNase and incubated at 37°C for 60 min.

After incubation with RNase, one volume of Tris

saturated phenol (pH 8.0) was added and mixed

gently by inverting the microcentrifuge tube till it

formed a milky white emulsion. The emulsion was

centrifuged at 10,000 rpm for 5 min at RT. The

supernatant was pipetted out into a fresh tube. The

sample was then extracted with equal volumes of

chloroform:isoamyl alcohol (24:1) twice. The DNA

was reprecipitated with 0.6 volumes of isopropanol,

Table 1Effect of tissue: buffer ratio on quality and quantity of

genomic DNA extracted from J. curcas leaves

Tissue–buffer ratio A260/280 Concentration (µg/g tissue)

1:3 1.67±0.063 66.00±2.3

1:4 1.72±0.11 72.50±8.6

1:5 1.81±0.069 142.00±15.8

1:6 1.84±0.12 112.50±11.3

1:7 1.77±0.059 95.50±6.9

Mean±SD of 4 independent experiments.

PAMIDIMARRI et al: ISOLATION OF GENOMIC DNA FROM J. CURCAS

189

2.0 M NaCl (final concentration) and incubated for 10

min. To the above, 20 µL of sodium acetate and 1

volume of 80% ethanol were added, incubated for 30

min, and centrifuged at 10,000 rpm for 15 min to

pellet the DNA. The pellet was then washed with 70%

ethanol twice; air-dried and finally suspended in 40-

50 µL of TE buffer.

Quantification of DNA

The yield of the extracted DNA was quantified by

spectrophotometer (UV-160A, Shimadzu Corpor-

ation, Japan) at 260 nm and the purity of DNA was

determined by calculating the ratio of absorbance at

260 nm to that of 280 nm according to the procedure

of Sambrook et al18

. DNA concentration and purity

was also checked by running the samples on 0.8%

agarose gels along with standard 1 Kb marker

(Biogene, USA).

Restriction Digestion

The extracted genomic DNA was digested by

incubating with EcoRI, HindIII and SauIII A

restriction endonucleases (Bioenzyme, USA) along

with control (with out adding enzyme) in the

corresponding buffers at 37°C for 3 h according to the

user’s manual. Digested DNA along with control was

analyzed by running the samples on 1.2% agarose gel

at 50 V and stained with ethidium bromide.

RAPD Analysis

Amplification of RAPD fragments was performed

according to Williams et al19

using decamer arbitrary

primer OPL-5 (KIT-L, Operon Technologies Inc,

USA) with 25 µL of reaction mixture. Amplification

mix consisted of 10 mM Tris-HCl (pH 9.0), 50 mM

KCL, 0.1 Triton X-100, 0.2 mM each dNTP, 3.0 mM

MgCl2, 0.4 µM primer, 25 ng template, 1unit Taq

DNA polymerase (Promega USA). Amplification was

done using thermal cycler (master cycle epgradient S,

Eppendorf, Germany) with following program: initial

denaturation at 94°C for 3 min, 42 cycles were run

with denaturation at 94°C for 30 sec, primer annealing

at 32°C for 1 min, extension at 72°C for 2.5 min, and

final extension at 72°C for 4 min. PCR products were

run on 1.5% agarose gel in 1X TBE buffer. The gels

were stained with ethidium bromide and

photographed using Gel documentation system

(Syngene, USA).

AFLP Analysis

Extracted genomic DNA was analyzed by AFLP

analysis system-II kit (Invitrogen Life Science Ltd,

USA). The genomic DNA (300 ng) was digested with

EcoRI and MseI. After complete digestion, aliquot

was ligated to EcoRI and MseI specific adopters at

20°C for 3 h. The adopter ligated DNA was

preamplified using EcoRI + ‘1 selective nucleotide’

and MseI + ‘1 selective nucleotide’ primer. The pre-

amplified product was diluted 1:20 with sterile TE

buffer. With the diluted product, selective

amplification was carried out using primers with three

selective nucleotides for EcoRI primer and three

selective nucleotides for MseI primer at the 3′. PCR

was conducted using 65°C as the initial annealing

temperature for the first cycle. For the subsequent 11

cycles, the annealing temperature was successively

reduced by 0.7°C. Twenty-three cycles were run at

56°C annealing temperature. To the PCR product an

equal amount of formamide dye was added and

subjected to electrophoretic separation on 6%

denaturing polyacrylamide gel in 1X TBE buffer in a

sequencing gel system (LKB, Sweden). The gels were

stained with silver nitrate using silver staining kit

(Sigma, USA).

Results and Discussion The unsuccessful attempts for extraction of good

quality DNA from J. curcas with the available

protocols made us to search for the new extraction

protocol. We developed a simple and efficient method

of genomic DNA extraction from different tissues of

J. curcas including callus by modifying CTAB

protocol14

.

Different CTAB extraction methods13,14

gave very

less quantity of DNA with loads of polysaccharide

and protein contamination, samples were very viscous

and took a long time to get dissolved in TE buffer.

The A260/280 OD ratio from 1.34 to 1.65 indicated

heavy contamination of the DNA with proteins. The

extracted sample, upon electrophoresis, gave fire-type

bands, uneven migration, and often remained in the

wells during electrophoresis as reported in the

literature20,21

confirmed the presence of

polysaccharides and protein (Fig. 1, lanes II-V).

Polysaccharides can co-precipitate with DNA after

adding alcohol during DNA precipitation to form

highly viscous solutions19

. By using 3.5 M NaCl in

extraction buffer and 80% ethanol with 2.0 M NaCl

(final concentration) during precipitation and further

purification with Tris saturated phenol during

purification phase the quality (A260/280 1.81±0.063,

Table 2) and quantity (120 to 140 µg/g) of DNA was

improved significantly without contamination of

INDIAN J BIOTECHNOL, APRIL 2009

190

proteins and polysaccharides. On agarose gel

electrophoresis, DNA gave sharp bands (Fig. 2, lane

I). When 100% ethanol was used along with 2.0 M

NaCl (final concentration) in extraction and

purification phases, quantity of DNA decreased, and

salts got precipitated along with DNA. On

electrophoresis, the DNA gave fire type bands. Use of

high concentration of NaCl in the extraction buffer

decreased contamination of polysaccharides22

. In the

present protocol, the use of 3.5 M NaCl in the

extraction buffer reduced 90% of polysaccharides

contamination and very little or no jelly like

precipitate was found during precipitation of DNA.

One of the most significant steps of our protocol was

the use of only Tris saturated phenol (pH 8.0),

followed by chloroform: isoamyl alcohol extraction.

Most of the protocols in the literature used phenol:

chloroform: isoamyl alcohol (25:24:1) or chloroform:

isoamyl alcohol (24:1) for protein removal15,23

,

whereas in our experiments use of either phenol:

chloroform: isoamyl alcohol (25:24:1) or chloroform:

isoamyl alcohol (24:1) gave yellowish pellet and OD

at A260/280 was 1.60±0.75, which confirmed the

presence of protein contamination. By using Tris

saturated phenol (pH 8.0) followed by

chloroform:soamyl alcohol (24:1) extraction, protein

impurities could be successfully removed, without

affecting DNA yield (Fig. 1, lane 1, Fig. 2; lanes 2, 4).

It was also observed that buffer to tissue ratio, and

incubation time were also important factors for

obtaining higher yields of DNA and in case of J.

curcas 5:1 buffer to tissue ratio (Table 1) and 90 min

incubation at 65°C (Table 3) gave the best results.

Compared to precipitation at −20°C14

, we could

Fig. 2Electrophoretic separation of genomic DNA extracted from

J. curcas leaves using phenol:chloroform:isoamyl alcohol

combinations. Lane I-DNA extracted with Tris saturated phenol;

lane II-DNA extracted with phenol:chloroform:isoamyl alcohol

(25:24:1); lane III-1 Kbs marker; lane IV-DNA extracted with

chloroform: isoamyl alcohol (24:1).

Table 3Effect of incubation time on quality and quantity of

genomic DNA extracted from J. curcas leaves

Time of incubation at

65°C (min)

A260/280 Concentration (µg/g

of tissue)

30 1.74±0.062 57.50±5.3

60 1.75±0.073 77.50±5.8

90 1.80±0.059 132.50±7.8

120 1.95±0.063 122.50±11.3

150 1.82±0.071 56.25±8.6

180 1.84±0.079 59.75±8.2

Mean±SD of 4 independent experiments.

Fig. 1Electrophorotic separation of genomic DNA extracted

from J. curcas leaves using different protocols. Lane I-1Kbs

marker; lane II-V DNA extracted using published protocols.

Table 2Qualitative and quantitative differences in genomic

DNA extracted from different tissues of J. curcas

Plant parts A260/280 Concentration

(µg/g of tissue)

Germinated seedling 1.64±0.061 78.60±6.8

Petiole 1.84±0.042 43.75±7.2

Root 1.29±0.23 45.00±5.3

Stem 1.75±0.093 46.25±4.1

Callus (leaves) 1.82±0.079 88.30±3.4

Leaves 1.81±0.063 126.40±8.9

Mean ± SD of 4 independent experiments.

PAMIDIMARRI et al: ISOLATION OF GENOMIC DNA FROM J. CURCAS

191

achieve the same at RT without compromising the

quality and quantity of DNA suitable for restriction

digestion, ligation, PCR amplification and other

downstream processes necessary for DNA

fingerprinting (Figs 4 & 5). This protocol was also

found suitable for the extraction of genomic DNA

from root, petiole, stem, germinated seedling and also

from the callus (Table 3, Fig. 6).

Acknowledgement

The authors wish to thank Director Dr P K Ghosh,

Central Salt & Marine Chemicals Research Institute,

Bhavnagar for providing facilities and Dr T

Radhakrishnan, Senior Scientist. National Research

Center for Groundnut, Junagadh for technical support

in AFLP analysis.

Fig. 3Restriction digested J. curcas genomic DNA on 1.2%

agarose gel. Lane I-DNA digested with Eco RI; lane II-DNA

digested with Bam H I; and lane III-DNA digested with Saw III A,

lane IV-undigested genomic DNA.

Fig. 4Electrophoretic separation of genomic DNA extracted

from different parts of J. curcas. Lane I-1 Kbs DNA marker; lane

II-from germinated seedlings; lane III-from roots, lane IV-from

stem of 2 wk plant, lane V-from stem of mature plant, lane VI-

from petiole, lane VII-from leaves, lane VIII-from callus.

Fig. 5AFLP fingerprint of J. curcas genomic DNA. Lane I, 1

Kbs+100 bps mixture; lane II-XVI Selective amplified with

primer set E-ACC/M-CAC.

Fig. 6RAPD fingerprint analysis of J. curcas genomic DNA.

Lane I, 1 Kb standard marker (Biogene, USA); lane II-XV, RAPD

profile of J. curcas with primer OPL5 from KIT-L (Operon

Technologies Inc, USA).

INDIAN J BIOTECHNOL, APRIL 2009

192

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