Isolation and Purification of Genomic DNA and RNA from Jatropha curcas L

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Isolation and purification of genomic DNA and RNA

from Jatropha curcas L.

Vishakha Wadher

P.D. Patel Institute of Applied Science, Changa

Guided by

Bhavanath Jha

Marine Biotechnology and Ecology Division, CSIR- Central Salt and Marine Chemicals

Research Institute. Bhavnagar, Gujarat 364002, India

1. Introduction

Jatropha curcas belongs to the Euphorbiaceae family. It is believed to be native of South

America and Africa spread to other continents of the world. It can grow in arid, semiarid and

wasteland. It requires little water and fertilizer, and is not feed by cattles. It’s also pest-resistant

and has a high-seed yield that continues to be produced for 30–40 years. Oil content in the

Jatropha seeds is around 30–40%. Its oil has been used in India for several decades as biodiesel

of remote, rural and forest communities. It can be used directly after extraction (i.e. without

refining) in diesel generators and engines. Also, J. curcas oil is carbon-less, large-scale

production will improve the country's carbon emissions profile. Seed-oil of this plant is fast

emerging as an alternative to fossil fuel and has the desirable phytochemical characteristics. It

is also useful for medicines, insecticides, making candles and soap, as well as raw material for

biodiesel (Gubitz et al. 1999). The fruits, leaves, latex, and bark contain glycosides, tannins,

phytosterols, flavonoids and steroidal sapogenins that exhibit wide ranging medicinal

properties. By-products obtained while preparing biodiesel has industrial applications. India

has about 80–100 million hectare of wasteland, which can be used for the J.curcas cultivation.

In fact, implementation of biodiesel in India will lead to many advantages like providing green

cover to wasteland, support to agricultural and rural economy, and reduction in dependency on

imported crude oil and reduction in air pollution. Jatropha is tolerant to drought and heat stress,

and halophytes with their unique genetic makeup survive and complete their life cycle under

high saline conditions. It is also useful as an ornamental plant shrubs and herbs. Therefore

Jatropha is an important resource for stress-inducible genes. This plant is drought resistant and

is commonly planted as a garden fence.

Addition to this, the plant also contains secondary metabolites which are useful as protectant

for plants and as an ingredient for human medicine (Debnath and Bisen 2008). Some of the

obstacles encountered in developing castor oil, among others, lack of information about

varieties that have beneficial properties such as high production, fast multiplication, high oil

yield in seeds, as well as resistance to pests and diseases. This happens because so far the

Jatropha plant is only regarded as hedgerows that have low economic value so that research

and development of this plant is rarely done. To overcome this, plant breeding has a significant.

1.1. Deoxyribonucleic acid

Deoxyribonucleic acid (DNA) is a complex molecular structure that is found in all living

organisms. DNA codes genetic information for the transmission of inherited traits. James

Watson and Francis Crick determined that the structure of DNA is a double-helix polymer, a

spiral consisting of two DNA strands wound around each other. Each strand is composed of a

long chain of nucleotides. The nucleotide of DNA consists of a deoxyribosesugar molecule to

which is attached to a phosphate group and one of four nitrogenous bases: two purines (adenine

and guanine) and two pyrimidines (cytosine and thymine). The nucleotides are joined together

by covalent bonds between the phosphate of one nucleotide and the sugar of the next, forming

a phosphate-sugar backbone from which the nitrogenous bases protrude. One strand is held to

another by hydrogen bonds between the bases. This bonding between the bases is very specific-

i.e. adenine bonds only with thymine, and cytosine only with guanine. In double-helical DNA,

the number of A residues must be equal to the number of T residues, where as the number of

G and C residues must likewise be equal. As a result, the sequence of the bases of the two

chains of double helix has a complementary relationship.

DNA contains the genetic instructsions for the development and function of living things.

The DNA is important for inheritance, coding for proteins and it serve as the genetic blueprint

of life. The main role of DNA in the cell is the long-term storage of information.

The region of DNA that carries genetic information is called gene, but other DNA sequences

have structural purposes, or are involved in regulating the expression of genetic information.

In eukaryotes, DNA is stored inside the cell nucleus, while in prokaryotes the DNA is in the

cell's cytoplasm. The backbone of DNA carries four types of molecules called bases and it is

the sequence of these four bases that encodes information. The main function of DNA is to

encode the sequence of amino acid residues in proteins, using the genetic code. To read the

genetic code, cells make a copy of a stretch of DNA in the nucleic acid RNA. These RNA

copies are used to direct protein synthesis (Watson et al., 1953).

Fig. 1. Central dogma of molecular biology.

1.2. The central dogma

DNA functions as the template for RNA molecules, which subsequently move to the

cytoplasm, where they determine the arrangement of amino acids within proteins. In 1956

Francis Crick referred to this pathway as the central dogma for the ﬂow of genetic information

(Crick, 1970).

Here the arrows indicate the directions proposed for the transfer of genetic information. The

arrow encircling DNA indicates that DNA is the template for its self-replication. The arrow

between DNA and RNA represents that RNA synthesis is directed by a DNA template is called

transcription. Correspondingly, the synthesis of proteins is directed by an RNA template is

called translation. The last two arrows were presented as unidirectional; that is, RNA sequence

are never determined by protein templates nor was DNA then imagined ever to be made on

RNA templates. The idea that proteins never serve as templates for RNA has stood the test of

time. RNA chains sometimes do act as templates for DNA chains of complementary sequence.

Such reversals of the normal ﬂow of information are very rare events compared with the

enormous number of RNA molecules made on DNA templates. Thus, the central dogma as

originally proclaimed more than 50 years ago still remains essentially valid (Watson, 2008).

1.3. Ribonucleic acid

Ribonucleic acid (RNA) is typically single stranded and is made of ribonucleotides that are

linked by phosphodiester bonds. Structurally, RNA is quite similar to DNA. A ribonucleotide

Fig. 2. (a) DNA is double stranded, whereas RNA is single stranded. (b) RNA can fold upon

itself, with the folds stabilized by short areas of complementary base pairing within the

molecule, forming a three-dimensional structure.

in the RNA chain contains ribose sugar, one of the four nitrogenous bases (A, U, G, and C),

and a phosphate group. DNA is more suitable for storage of genetic information, whereas the

RNA is more suitable for its short-term functions. The pyrimidine uracil forms a

complementary base pair with adenine and is used instead of the thymine used in DNA. Even

though RNA is single stranded, most types of RNA molecules show extensive intra molecular

base pairing between complementary sequences within the RNA strand. It creates a predictable

three-dimensional structure essential for their function (Rich, 2009).

There are main three types of RNA which directly involved in protein synthesis, messenger

RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). The mRNA carries the

message from the DNA and controls all of the cellular activities in a cell. It is synthesized

through the process of transcription. The mRNA then interacts with ribosomes and other

cellular machinery to direct the synthesis of the protein. This process is known as

translation. rRNA is a major constituent of ribosome’s, comprise up to 60% of it. Ribosome

provides the location for binding of mRNA. rRNA of the ribosome has an enzymatic activity

and catalyzes the formation of the peptide bonds between two aligned amino acids during

protein synthesis. tRNA is the third main type of RNA and is the smallest one, usually 70–90

nucleotides long. It carries the correct amino acid at the site of protein synthesis in the

ribosome. The base pairing between the tRNA and mRNA allows for the correct amino acid to

be inserted in the polypeptide chain being synthesized. Any mutations in the tRNA or rRNA

can result in global problems for the cell because both are necessary for the synthesis of protein.

1.4. Problem with the isolation of DNA and RNA from J.curcus

Jatropha curcas contain latex, which is a true plant secondary metabolite. The presence of

secondary metabolites such as polyphenols, polysaccharides and tannins can inhibit the enzyme

activity (Pirtilla et al., 2001; Porebski, 1997). Isolation of DNA and RNA from this plant at a

distance often experienced problems due to high levels of secondary metabolites in the form of

polysaccharides and polyphenols. According to Sharma et al. (2002) the presence of

metabolites in several crops affect DNA isolation procedure, he used a modified CTAB to

isolate DNA from plant tissue containing high polysaccharide. According to Kiefer33 et al.

(2000), Pirtilla et al. (2001) and Sanchez-Hernandes, and Gaytan Oyarzun (2006), states that

isolation of DNA and RNA from plants containing polysaccharides and polyphenols is

difficult.

1.5. Polysaccharides

Polysaccharides are the prime interferers in the DNA isolation procedure as they are

undetectable and hard to remove. The presence of high amounts of polysaccharides, makes the

tissue homogenate very viscous, and gives a false indication of presence of high amounts of

DNA. Polysaccharides often co-precipitate with the DNA and impart a sticky, viscous

consistency to it. In this form, they also interfere with the activity of several biological enzymes

like polymerases, ligases and restriction endonucleases (Tel-Zur et al., 1999). Polysaccharide-

like contaminants can also cause anomalous re-association kinetics, and render the DNA

unsuitable for downstream processing. The contaminated DNA tends to stick in the well during

gel electrophoresis (Puchooa, 2004).

Use of high concentration of NaCl usually more than 0.5M is suitable for the removal of

polysaccharides from DNA solutions by increasing their solubility in ethanol. NaCl in

combination with the cationic detergent CTAB has also proved to be beneficial in DNA

isolation from polysaccharide rich plants (Murray et al., 1980; Syamkumar et al., 2003). CTAB

helps in precipitating DNA by forming a complex with it, and at high salt concentration, it

forms insoluble complexes with proteins and most acidic polysaccharides, leaving the nucleic

acids in the solution, which then can be easily isolated.

1.6. Polyphenols

The other problem in the plant DNA isolation protocols is the degradation of genomic DNA

due to presence of polyphenols. Polyphenols are extremely variable in their occurrence and

type. During cell lyses, polyphenols come out of the vacuoles and are easily oxidized by

cellular oxidases. The oxidized polyphenols undergo irreversible interactions with nucleic

acids and causes enzymatic browning of the DNA pellet, thereby rendering it useless for most

downstream processes. Polyphenols like flavonoids, terpenoids and tannins, which all occur

widely in plant kingdom, when bound to the nucleic acids cannot be removed by conventional

extraction procedures. To deal with the problem of polyphenolic contamination, antioxidants

are added to the extraction buffer to prevent the oxidation of phenols during cell lysis. Polyvinyl

pyrollidone (PVP, Sigmma) and polyvinyl polypyrollidon (PVPP, Sigma) are the most

commonly used chemicals to eliminate polyphenolics as they act as adsorbents of polyphenols,

especially at low pH. They form complexes with the polyphenolic compounds through

hydrogen bonding, allowing the polyphenolics to be separated from the DNA, thereby reducing

their levels in the product (Lodhi et al., 1994; John, 1992; Pich et al., 1993; Porebski et al.,

1997). Antioxidants such as β-mercaptoethanol, ascorbic acid, sodium azide, sodium sulfite,

sodium isoascorbate, DTT, etc., along with PVP are commonly used to solve the problems

related to phenolics (Chen et al., 1999; Dixit, 1998; Fang et al., 1992; Michiels et al., 2002;

Puchooa, 2004; Suzuki et al., 2003; Wang et al., 1996). β-Mercaptoethanol in particular is

widely used, and prevents the polymerization of tannins that makes it difficult the isolation

procedure.

Proper techniques of DNA isolation is needed in the plant breeding process to obtain DNA

with a high quality and quantity. To obtain pure DNA from plant tissue, generally carried out

repeated purification and modification of procedures (Kiefer et al., 2000), thus requiring

additional cost and effort. For that, you can use parts of plants that contain little secondary

metabolites. The content of secondary metabolites in plant tissues fluctuate in line with its

development. Secondary metabolites may vary because of differences in age and plant part

(Achakzai et al., 2009; Cirak et al., 2007). Therefore, to simplify the DNA extraction process

jatropha, have done research to learn the parts of plants containing secondary metabolites in

small amounts and produce DNA with high quality and quantity. This research aims to study

the jatropha plant leaves at different levels of development that have the potential to produce

the best quality and quantity of DNA and RNA in the extraction process.

2. Materials and method

2.1. Plant material

Experiments were performed using fresh leaves of Jatropha curcus collected from the plant

grown in the greenhouse of Central Salt and Marine Chemicals Research Institute, Bhavnagar,

Gujarat.

Fig. 3. Jatropha curcas.

2.2. Reagents required for DNA extraction

Sodium EDTA, Tris-HCl, NaCl,

Cyltrimethylammonium bromide (CTAB),

Polyvinylpolypyrrolidone (PVP), ß-mercaptoethanol,

Chloroform, Isoamylalcohol, Ethanol, RNase (20 mg/ml)

• CTAB buffer with pH 8.0

The extraction buffer contain 2% CTAB, 100 mM tris Cl, 3.5M NaCl, 20 mM EDTA, 0.2M β

mercaptoethanol, 2% PVP (β mercaptoethanol and PVP were added just before use.)

0.1M Tris HCl:

Dissolve 2.422 g of tris HCl in 200 ml of water and adjust the pH to 8.0

• 20 mM EDTA:

Take 1.488 g of EDTA and dissolve in 200 ml of water

• 3.5 M NaCl:

Take 40.90 g of NaCl and dissolve in 200 ml of water that will form 3.5 M solution of NaCl.

• Chloroform: Isoamyl alcohol 24:1 (v/v):

Take 24 ml of Chloroform and add with 1 ml of isoamylalcohol.

• TE buffer:

Dissolve 1 M Tris, 0.5 M EDTA in 100 ml of water and adjust pH to 8.0