Genomic DNA Extraction Protocol for Artemisia Annua L. Without Using Liquid Nitrogen and Phenol

Artemisia annua is an important medicinal plant, used for curing various diseases especially malaria. It secretes verities of secondary metabolites which hinders in the DNA extraction. This investigation describes an efficient DNA extraction protocol for A. annua based upon the Cetyl Trimethyl Ammonium Bromide (CTAB) extraction method without using hazardous chemicals i.e. liquid nitrogen and phenol. The developed protocol is simple, reliable and operative in normal laboratory condition yielding intact DNA having good quantity (502.7 to 1288.5 ng/μl) and quality (A260/280 ratio 1.82 to 1.85) in two working days. Proper amplification of extracted DNA indicates its suitability for the various molecular biological applications.


Introduction
Artemisia anuua L. is a traditional Chinese medicinal plant well known for its bio-active compound artemisinin-an antimalarial drug.Artemisinin based Combination Therapy (ACT) is most commonly used for the curing of malaria including cerebral malaria (WHO, 2015), but low and fickle production of artemisinin does not justify the total requirement of ACT worldwide (WHO, 2011).Emphases are being made to enhance the economic commercial production of artemisinin and the molecular techniques will play a major role.Good quality, quantity and intact DNA extraction is very crucial for PCR based applications, cloning, sequencing and other molecular research.Plants which synthesize and accumulate a large numbers of bioactive compounds like A. annua (Bhakuni et al., 2001) their DNA extraction became a tedious process for the molecular biologist.Presence of polysaccharides in the extracted DNA shown to inhibit the Taq polymerase activity while the oxidized form of polyphenols covalently binds to DNA giving a brown colour and reduces maintenance time, making it useless for molecular studies (Katterman and Shattuck, 1983;Fang et al., 1992).
Although there are many DNA extraction protocol for A. annua has been developed by using liquid nitrogen and/or phenol (Kumar et al., 2011;Lakshmi et al., 2012).Sangwan et al., (1998) isolated The DNA form A. annua by using CTAB method that includes a rapid micro-column chromatography through DE-52 ion-exchange resin.Many researchers also used DNA extraction kits for the DNA extraction which is less time consuming and eliminate the use of toxic chemicals, however they used liquid nitrogen.But there is a need for development of DNA extraction method for normal laboratory conditions, which avoid the use of liquid nitrogen and phenols.Keeping these views, the present investigation was intent to develop a simple, efficient genomic DNA extraction protocol for A. annua without using liquid nitrogen and phenol.

Material and Methods
Fresh, young and disease free leaves of A. annua variety Jeevanraksha (Kumar et al., 1999) was taken from the medicinal plants collection of Prof. T. S. Murthy Science and Technology Station, MP Council of Science and Technology, Obaidullaganj, Raisen, Madhya Pradesh, India.Leaves were washed in a series of Millipore water, 70% ethanol and again two times in Millipore water.Fresh weight of the leaves was taken and they were chopped into fine pieces and subjected to genomic DNA isolation with following steps.

Research Article
1.The chopped leaves (500 mg) were ground into pre-chilled mortar and pestle (at refrigerator for 1 hour) and homogenized with 700 µl of pre warmed (at 65 o C) extraction buffer (Haque et al., 2008) consisting, 150 mM Tris Buffer (pH 8.0), 25 mM EDTA (pH 8.0), 1.5 M NaCl, 3.5% CTAB, 3% PVP and 0.3% β-mercaptoethanol.2. The suspension was collected in 1.5 ml Eppendorf tubes and mixed with 5 µl of RNAse (stock 20 mg/ml).3. The Eppendorf tubes were incubated for 1 hour at 65°C in water bath with 2-3 times gentle inversions.4. Equal amount of chloroform: isoamyl alcohol (24:1) was added into the room temperature (RT) cooled eppendorf tube, missed gently mixed to form a emulsion and centrifuged at 10000 rpm for 10 minutes at RT. 5.The upper aqueous layer was taken and the step 4 (treatment of chloroform: isoamyl alcohol) was repeated again.6. Isopropyl alcohol (0.6 volumes) was added into the upper aqueous layer, mixed well by gentle inversion and incubated at -20 °C for 30 minutes.7. The tubes were centrifuged at 10000 rpm for 10 minutes at 5 0 C and pellet was washed with 70% ethanol.8.The pellet was air dried, dissolved in 500 µl of TE buffer (10 mM Tris buffer, pH 8.0 and 0.1mM EDTA, pH 8.0) and stored at 4 °C for overnight.(First day extracted DNA) 9. On the subsequent day, DNA dissolved in TE buffer was treated with equal volume of chloroform: isoamyl alcohol (24:1) added into the Eppendorf tube and centrifuged at 10000 rpm for 10 minutes at RT. 10.Isopropyl alcohol (0.6 volumes) was added into the supernatant aqueous layer, incubated at -20 °C for 30 minutes and centrifuged at 10000 rpm for 10 minutes at 5 °C.11.The pellets were washed with 70% alcohol, air dried pellet was dissolved in 50 µl of TE buffer and stored at 4 °C for overnight (Second day extracted DNA).
Quantification of first and second day extracted DNA was performed in a Nanodrop spectrophotometer at 260 nm and the purity was checked from A260/A280 ratio.The DNA quality was also tested for PCR amplification with using

Results and Discussion
In the present study, genomic DNA of A. annua was isolated without using liquid nitrogen and phenol in two subsequent days.The first day extracted DNA was brown in colour having DNA concentration from 742.6 to 1556.9 ng/µl with A260/A280 ratio of 0.83 to 0.95, while the DNA concentration of second day extracted DNA was varied from 502.7 ng/µl to 1288.5 ng/µl with A260/A280 ratio of 1.82 to 1.85 (Table 1).A ratio of A260/A280 is used to assess the purity of nucleic acid.A260/A280 ratios of good quality DNA are commonly ~1.8 and ratio below this indicates the contamination of chemical constituents (Thermo Scientific).Thus the first day extracted DNA had contaminants of proteins, polysaccharides and other secondary metabolites, which was eliminated in second day by the another treatment of chloroform: isoamyl alcohol (24:1).Although The DNA concentration of first day extracted DNA was higher than second day extracted DNA, might because of residual chemical contamination present in the first day extracted DNA which result in the overestimation of DNA concentration (Thermo Scientific) or loss of DNA along with chemical contamination during the treatment of chloroform: isoamyl alcohol (24:1) on second day.Use of liquid nitrogen and phenol is very crucial in the DNA extraction procedure.Liquid nitrogen is used for grinding plant tissue to break down the cell wall of plants, while phenol used for the elimination of protein contaminants from the genomic DNA.However both the chemicals are highly hazardous in nature and thus require proper handling during the procedure, which may be not possible in the normal laboratory conditions.Therefore, efforts have been made for the isolation of genomic DNA without using these chemicals, which leads into the development of DNA extraction protocols for plants avoiding the use of liquid nitrogen and/or phenol (Sharma et al., 2003(Sharma et al., , 2010;;Biswas and Biswas, 2011;Ferdous et al., 2012;Sahu et al., 2012;Sairkar el al., 2013).As for as A. annua plant, there are many DNA extraction protocols had been developed, although they use either liquid nitrogen or phenol or both (Kumar et al., 2011;Lakshmi et al., 2012).In the present investigation, genomic DNA of A. annua was isolated without using liquid nitrogen and phenol and yielded good quantity and quality DNA.
The extracted DNA was further checked for its proper PCR amplification through EST-SSR primer (AAESP19).The first day extracted DNA did not amplified, while the second day extracted DNA give proper amplification (Fig. 1).This confirms that the second day extracted DNA was good enough to be used for the molecular biological experiments of A. annua.

Table 1 :
Quantity and quality of isolated genomic DNA of A. annua AAD = Artemisia annua DNA