Comparative Analysis for DNA Isolation from Jatropha curcas L.

INDEX

List of Tables

List of Figures

List of Abbreviations

1. Introduction

2. Review of literature

3. Materials and method

4. Result and discussion

5. Conclusion

6. References

Annexure – I
Reagent Preparation

Annexure – II
Reagent/Chemicals – Source

Annexure – II
Instrument Used - Company

LIST OF TABLES

1. Reaction component of RE digestion

2. Reaction component of RAPD analysis

3. PCR condition for RAPD analysis

4. Quantification of DNA

LIST OF FIGURES

1. Plant DNA isolation by protocol A, Protocol B, Protocol C.

2. Plant DNA isolation by protocol C2, Protocol D, Protocol E.

3. Plant DNA isolation by protocol E, Protocol F, Protocol G.

4. Plant DNA isolation by protocol G, Protocol H.

5. Restriction digestion of genomic DNA by 2 enzymes Eco R1 and Bam H1.

6. Electrophoretic pattern of DNA of Jatropha curcas accessions amplified with RAPD primer.

ABBREVIATION

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1. INTRODUCTION

Biotechnological approaches involve the exploitation of natural substances in production process. The utilization of various parts of Jatropha curcas potentially improving the economic situation of various tropical countries.

J. curcas is a drought-resistant shrub or tree belonging to the genus Euphorbiaceae, which is cultivated in Central and South America, South-east Asia, India and Africa. J. curcas, which can be easily propagated by cuttings, is widely planted as a hedge to protect field, as it is not browsed by cattle. Like many other Jatropha species J. curcas is a succulent that sheds its leaves during the dry season. Large green to pale-green leaves. Fruits are produced in winter, or there may be several crops during the year if soil moisture is good and temperatures are sufficiently high. It is well adapted to arid and semi-arid condition and often used for erosion control.

Cultivation is uncomplicated. Jatropha curcas grows in tropical and subtropical regions. The plant can grow in wastelands and grows on almost any terrain, even on gravelly, sandy and saline soils. Jatropha curcas has limited natural vegetative propagation and is usually propagated by seed. Propagation through seed (sexual propagation) leads to a lot of genetic variability in terms of growth, biomass, seed yield and oil content. Low seed viability and the recalcitrant nature of oil seeds also limit seed propagation. However, clonal techniques can help in overcoming these problems that hinder mass propagation of this tree-borne oilseed species. Vegetative propagation has been achieved by stem cuttings, grafting, and budding as well as by air layering techniques

Nuts of J.curcas Used as a contraceptive in South Sudan. Roots ashes are used as a salt substitute. The oil has been used for illumination, soap, candles, and the adulteration of olive oil. Bark Used as a fish poison. The latex of J. curcas has been found to be strongly inhibitory to watermelon mosaic virus. The latex has been also used to promote healing to wounds.

The first commercial application of J. curcas was reported from Lisbon, where the oil imported from Cape Verde was used for soap production and for lamps. The press cake was used as a fertilizer for potatoes. Even today, J. curcas is mainly cultivated for the production of oil as a fuel substitute. However, trans-esterification of the oil for use in standard diesel engines is gaining more importance then direct utilization of the oil in adapted engines. New techniques such as the enzyme-supported oil extraction and more efficient trans-esterification processes have been evaluated.

All parts of J. curcas have been used in traditional medicine and for Veterinary purposes for a long time. The oil has been used as a purgative, to treat skin diseases and to soothe pain such as that caused by rheumatism. Detection of the leaves has been used against coughs or as antiseptics after birth, and branches as chewing sticks. Recently the substances responsible for wound healing and anti-inflammatory effects have been isolated and characterized.

Various extracts from J. curcas seeds and leaves showed molluscicidal, insecticidal and fungicidal properties. Biotechnological process related to the exploitation of J. curcas include genetic improvement of the plant, biological pest control, enzyme supported oil extraction, anaerobic fermentation of the press cake and the isolation of anti-inflammatory substance and wound healing enzymes.

In India and Africa still various parts of J. curcas have been used in tradition medicine. In Mali the leaves are known as a treatment for malaria. The leaf decoction is applied externally for inflammation. The root decoction is drunk against pneumonia and syphilis. In Mexico the latex is used for fungal infection in the mouth and digestive problems of children.

J. curcas are, in general, toxic to humans and animals. Numerous feeding experiments with different animal species have demonstrated the toxicity of the seeds as well as of the oil and the press cake. The latex of J. curcas has been found to be strongly inhibitory to watermelon mosaic virus. The latex has been also used to promote healing to wounds. The use of J. curcas oil for the control of cotton insect pests seemed to be a promising alternative to hazardous chemicals. The lowest gas exhaust was obtained with J. curcas.

Limitation of Jatropha curcas

Seeds of Jatropha curcas are toxic and its cake cannot be used as a fodder. The low yields may have been caused by the fact that un-adapted provenances have been used. Being highly cross-pollinated crop, Ratanjyot within its species is genetically different. Large variation for oil percentage in the seed, from 25-40% has been observed in it.

2. REVIEW OF LITERATURE

1. Jatropha curcas:-

J. curcas is a drought-resistant shrub or tree belonging to the genus Euphorbiaceae, which is cultivated in Central and South America, south-east Asia, India and Africa. Large green to pale-green leaves. Fruits are produced in winter, or there may be several crops during the year if soil moisture is good and temperatures are sufficiently high.

Cultivation is uncomplicated. Jatropha curcas grows in tropical and subtropical regions. The plant can grow in wastelands and grows on almost any terrain, even on gravelly, sandy and saline soils. Currently the oil from Jatropha curcas seeds is used for making biodiesel fuel in Philippines and in Brazil.

Nuts of J. curcas Used as a contraceptive in South Sudan. Roots ashes are used as a salt substitute. The oil has been used for illumination, soap, candles, and the adulteration of olive oil. Bark Used as a fish poison. The latex of J. curcas has been found to be strongly inhibitory to watermelon mosaic virus. The latex has been also used to promote healing to wounds.

2. DNA isolation from J. curcas plant:-

Non-renewable hydrocarbons are being used as the major energy source, which are mainly responsible for global warming. Biofuels and bioenergy encompass a wide range of alternative source of energy of biological origin. Plant based fuels create a better balance between the formation and consumption of CO2; decrease particulate matter, co, unburnt hydrocarbon and so2 emission into the atmosphere. In this regard, biodiesel derived from the seed-oil of J. curcas is fast emerging as an available alternative to fossil fuels and has a desirable physiochemical activity, even superior to diesel. The by-products, obtain while preparing biodiesel, have industrial application.

The deoiled cake is used as fertilizer and in biogas production. Almost all parts of the plant are used in medicine. Hence, there is a need to identify high yielding clones of J. curcas for its further improvement and large scale cultivation.

DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and one optional step in a DNA extraction:

1. Breaking the cells open commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by grinding or sonicating the sample.
2. Removing membrane lipids by adding a detergent.
3. Removing proteins by adding a protease (optional but almost always done).
4. Precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt.
3. Detecting DNA

Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280 nm is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nanometers, and aromatic proteins absorb UV light at 280 nm; a pure sample of DNA has the 260/280 ratio at 1.8 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.

A260 = measure concentration of the DNA (1.0 A260 = 50μg/ml)

A260/A280 = Estimate DNA purity

4. Restriction digestion of DNA:-

Recombinant DNA technology is a modern methodology in the field of molecular biology to manipulate genes. It includes enzymes to cut DNA. The restriction endonuclease cut at the interior part of the DNA. These enzymes found in bacteria and in vivo are involved in recognition and destruction of foreign DNA. Invading phage DNA for instance will be restricted by such enzymes. The bacteria protect their own DNA by modification process.

The essential feature of restriction endonuclease action is that the enzyme recognizes a particular sequence of bases. Bacteria are constantly attacked by bacteriophages to protect themselves; bacteria have developed defense mechanism in the form of enzyme known as restriction endonuclease.

5. RAPD analysis:-

RAPD stands for Random Amplification of Polymorphic DNA. It is a type of PCR reaction, but the segments of DNA that are amplified are random. The scientist performing RAPD creates several arbitrary, short primers (8-12 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify.

RAPD analysis requires only a small amount of genomic DNA and can produce high levels of polymorphism and may facilitate more effective diversity analysis in plants (Williams et al., 1990). RAPD analysis provides information that can help to define the distinctiveness of species and phylogenetic relationships at molecular level (Subramanyam et al. 1901).

RAPD is an inexpensive and rapid method not requiring any information regarding the genome of the plant and has been widely used to ascertain genetic diversity in several plants

How It Works:-

Unlike traditional PCR analysis, RAPD does not require any specific knowledge of the DNA sequence of the target organism: the identical 10-mer primers will or will not amplify a segment of DNA, depending on positions that are complementary to the primers' sequence. For example, no fragment is produced if primers annealed too far apart or 3' ends of the primers are not facing each other. Therefore, if a mutation has occurred in the template DNA at the site that was previously complementary to the primer, a PCR product will not be produced, resulting in a different pattern of amplified DNA segments on the gel.

Limitations of RAPD:-

- Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish whether a DNA segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2 copies). Co dominant RAPD markers, observed as different-sized DNA segments amplified from the same locus, are detected only rarely.
- PCR is an enzymatic reaction, therefore the quality and concentration of template DNA, concentrations of PCR components, and the PCR cycling conditions may greatly influence the outcome. Thus, the RAPD technique is notoriously laboratory dependent and needs carefully developed laboratory protocols to be reproducible.
- Mismatches between the primer and the template may result in the total absence of PCR products well as in a merely decreased amount of the product. Thus, the RAPD results can be difficult to interpret.

3. MATERIALS AND METHODS

Plant material

Nine DNA extraction methods are examined on young and mature leaf samples of Jatropha curcas. Fresh leaves of J. curcas were collected from our experimental field located at Shree M. & N. Virani Science College, Rajkot, Gujarat, India, and brought to the laboratory. Leaves were harvested fresh before DNA isolation. The DNA was extracted from fresh leaves on the same day using different method to obtain high quality intact DNA.

For DNA isolation different nine protocols were examined, but from those only six protocols had given positive result. These nine methods and their chemical requirements are given below.

DNA extraction protocol A (modified - Doyle and Doyle, 1990)

Chemicals

- Extraction buffer: 2% CTAB, 100 mM Tris/HCl, pH 7.5, 1.4 M NaCl, 2% polyvinylpyrrolidone (PVP)-40, 20 mM ethylene diamine tetra acetic acid (EDTA), pH 8.0. Add 20 µL/mL b-mercaptoethanol immediately prior to use.
- Chloroform: isoamylalcohol 24:1 (CIA).
- Isopropanol, 70% ethanol.
- TE-RNase solution: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0, 10 mg/mL RNase.

Protocol

1. 500 mg of leaf sample was crushed in mortal and pestle with the help of extraction buffer A and the crushed samples were equally distributed in the eppendroff tubes.
2. To ground sample, add 1 mL extraction buffer and incubate samples for 1 h at 60°C in boiling water bath with occasional swirling.
3. Cool samples at room temperature, and centrifuge at 14000 rpm for 10 minutes.
4. Supernatant was collected in fresh vial and add 600 µL CIA and mix gently for 5 min.
5. Then the sample was centrifuged for 15 min at 12000 rpm at 4°C. Transfer the supernatant to a new tube and add equal volume of Isopropanol. Mix gently and incubate at -20°C overnight.
6. Centrifuge for 15 min at 14000 rpm. The tubes were allowed to drain and Wash the pellet with 70% ethanol and allowed to dry.
7. Dry the DNA and dissolve the pellet in 20-50 µL (according to your quantity) TE-RNase solution. Incubate for 1 h at 37°C in dry bath
8. Equal amount of Isopropanol were added and samples were incubated at -20°C for 2 hours.
9. Samples were centrifuged at 14000 rpm for 15 minutes at 4°C in cooling centrifuge.
10. The tubes were allowed to drain and dried at room temperature for 20-30 minutes, and then DNA was resuspended in 50µl of Tris-EDTA buffer.

DNA extraction protocol B (Jobes et al., 1995)

Chemicals

- Extraction buffer: 100 mM sodium acetate, pH 4.8, 100 mM EDTA, pH 8.0, 500 mM NaCl, 10 mM dithiothreitol (DTT), 2% PVP, pH 5.5. Add 100 µg/mL proteinase K immediately prior to use.
- 20% sodium dodecyl sulfate (SDS) solution.
- 5 M potassium acetate, 8 M LiCl, 5 M NaCl.
- Isopropanol, absolute and 70% ethanol.
- TE buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.

Protocol

1. 500 mg of leaf sample was crushed in mortal and pestle with the help of extraction buffer B and the crushed samples were equally distributed in the eppendroff tubes.
2. To each ground leaf sample, add 1 mL extraction buffer and incubate sample for 1 h at 60°C with occasional swirling.
3. Samples were cooled at room temperature, and centrifuged at 12000 rpm for 10 minutes at 4°C
4. Transfer the supernatant to a new tube and add 1/3 volume of 5 M potassium acetate. Mix gently and incubate for 30 min at -20°C.
5. Centrifuge for 10 min at 14000 rpm. Transfer the supernatant to new tube and add 0.6 volume of Isopropanol. Mix gently and incubate at -20°C overnight.
6. Centrifuge for 15 min at 14000 rpm. The tubes were allowed to drain and Wash the pellet with 70% ethanol and allowed to dry.
7. Dissolve the pellet in autoclaved Deionized water. Add 0.5 volume of 5 M NaCl and mix well. Add two volumes of cold absolute ethanol and incubate for 30 min at -20°C.
8. Centrifuge for 10 min at 14000 rpm. Dissolve the pellet in autoclaved deionized water. Precipitate RNA with 1/3 volume of cold 8 M LiCl (final concentration 2 M) and incubate at -20°C for at least 1 h.
9. Recover RNA by centrifugation for 15 min at 14000 rpm and carefully transfer supernatant to a new tube. Precipitate DNA by adding 0.6 volume of Isopropanol and incubate for 1 h at -20°C.
10. Centrifuge for 10 min at 14000 rpm. Wash the pellet with 70% ethanol and was allowed to dry.
11. Dried DNA was dissolved in 30µl of TE buffer.

DNA extraction protocol C (Dellaporta et al., 1983)

Chemicals

- Extraction buffer: 10% SDS, 50 mM Tris/HCl, 100 mM NaCl, 10 mM EDTA, pH 8.0. Add 20 µL/mL b-mercaptoethanol and approximately 0.01 g PVP-40 immediately prior to use.
- 5 M potassium acetate, 3 M sodium acetate.
- Isopropanol, absolute and 70% ethanol.
- TE-RNase solution, TE buffer.

Protocol

1. 500 mg of leaf sample was crushed in mortal and pestle with the help of extraction buffer C and the crushed samples were equally distributed in the eppendroff tubes.
2. To each ground leaf sample, add 1 mL extraction buffer and incubate sample for 1 hour at 60°C with occasional swirling.
3. Add 300 µL 5 M potassium acetate and mix gently. Incubate sample for 20 min on ice.
4. Centrifuge for 10 min at 14000 rpm. Transfer the supernatant to a new tube and add equal volume of Isopropanol. Mix gently and incubate for 1 h at -20°C.
5. Centrifuged at 14000 rpm for 15 min. Wash the pellet with 70% ethanol.
6. Dry the DNA and dissolve the pellet in 20-50 µL TE-RNase solution. Incubate for 1 h at 37°C in dry bath.
7. Precipitate the DNA by adding 10% volume of 3 M sodium acetate and two volumes of absolute ethanol. Incubate at -20°C overnight.
8. Samples were Centrifuged at 14000 rpm for 15 min at 4°C and tubes were allowed to drain and dry at room temperature for 20-30 minutes.
9. Dry the DNA and dissolve the pellet in 20-50 µL TE buffer.

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