Standardization of DNA extraction from invasive alien weed Parthenium hysterophorus

DNA isolation from the weed, Parthenium hysterophorus is complicated due to the presence of high amount of allelochemicals in the form of secondary metabolites that causes hindrance in extraction and enzymatic reactions. A modified and efficient DNA extraction from P. hysterophorus leaf has been developed. The present protocol is a modified version of cetyltrimethylammonium bromide (CTAB) method constituting high salt concentration to remove polysaccharides. The increased concentration of β-mercaptoethanol, polyvinylpolypyrrolidone (PVPP), and phenol/chloroform/isoamyl alcohol extractions eliminated protein and phenolic compounds well. Good amount and quality DNA was obtained by this method. The resulted genomic DNA showed fine random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) banding pattern, polymerase chain reaction (PCR) amplification of actin gene and restriction digestion confirm the efficiency of modified procedure.


INTRODUCTION
Parthenium hysterophorus is an annual invasive weed of asteraceae.It spreads rapidly worldwide in a short period due to its great structural and physio-chemical flexibility, vegetative multiplication, effective dispersal of seeds, longer seed dormancy, stress tolerance and presence of secondary metabolites (Patel, 2011).P. hysterophorus exhibited wide adaptability towards various environmental conditions which enabled this plant to grow successfully in drought and heat prone areas (Hedge and Patil, 1982;Shubneet et al., 2014;Kumar, 2014).Apart from these properties, P. hysterophorus also displayed antibacterial, antioxidant, and cytotoxic activities (Kumar et al., 2013).The occurrence of diverse alkaloids, terpenoids, flavonoids, hydrocarbons and fatty acids in its tissues may execute these properties (Panwar et al., 2015).However, no molecular information is available on P. hysterophorus.Keeping the above facts in mind, P. hysterophorus can be used as a good source for identification of valuable genes, proteins and metabolites that is responsible for diverse properties of this weed.Therefore, molecular characterization of this weed is necessary for further use in transgenic approach.The quality as well as quantity of DNA is a matter of great concern in functional genomics.Fine quality DNA is an *Corresponding author.E-mail: mirfanq@gmail.com.Tel: +91 9911491949.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License essential requirement in all the manipulations related to recombinant DNA technology.A fast, simple and reliable DNA isolation method is immensely required for this purpose since similar DNA isolation protocols may not be suitable for all plants (Akhtar et al., 2013), because size, content and organization of genome and metabolic setup of different plant systems differ from each other (Sangwan et al., 1998).Many problems were faced in the isolation and purification of DNA which include degradation of DNA, contamination with highly viscous polysaccharides, polyphenols, tannins, terpenoids, flavonoids, quinines and other allelochemicals which affects the downstream application (Kotchoni et al., 2011).Therefore, a genomic DNA isolation protocol was modified from the earlier method (Doyle and Doyle, 1990) to simplify and optimize the procedure for P. hysterophorus.

Collection of plant sample
Seeds from authenticated P. hysterophorus were collected from Jamia Millia Islamia University campus, New Delhi, India (Latitude 28.6° 4'N and Longitude 77.2°) growing in the alluvial soil.The plant was further identified with authentic specimen and deposited with accession no.115597 at Botanical Survey of India, Dehradun, India.Seed sterilization was done by soaking of seeds in 0.3% KMnO4 for 10 min, and then rinsed ten times with sterilized distilled water.Germinated seed (five days old seedlings) were transferred to Soilrite™ (Keltech Energies Ltd., India) containing pots (6"× 6", 300 g/pot) and cultured in 14 h/10 h light/dark at 25°C with 250 µmol photons m-2 s -1 for 55 days.Thus, two months old plants were used in the present study.

DNA extraction procedure
(1) 1.0 g of plant material (P.hysterophorus leaves) was immediately placed in pre-chilled (-80°C) mortar and pestle and ground thoroughly.The ground sample was transferred to 30 ml sterile centrifuge tube containing 5 ml preheated extraction buffer and mixed by inversion.The slurry was formed and tube incubated at 65°C for 60 min in water bath with occasional gentle swirling.
(2) Next, incubated sample was extracted with equal volume of chloroform: isoamyl alcohol (24:1) and mixed gently by inversion thoroughly for about 30 min at room temperature.The mixture was centrifuged at 12,000 rpm (10 min, 25°C) for phase separation.
(3) After the two phases were separated visibly of which top clear aqueous phase was carefully transferred to a new sterile centrifuge tube and equal volume of chilled isopropanol was added to precipitated nucleic acid.The whole preparation was mixed gently and kept at -20°C for 1 h.
(4) The mixture was centrifuged at 10,000 rpm for 15 min.The supernatant was removed carefully and pellet was washed with 70% ethanol, air dried at room temperature for 1 h and dissolved in 400 µl TE buffer.Next, 5 µl RNase A (1 μg/μl) was added and incubated at 37°C for 1 h.
(5) For further purification, mixture was extracted with an equal volume of phenol: chloroform: isoamyl alcohol.(6) The upper aqueous layer was transferred to another sterile 2.0 ml micro-centrifuge tube and further extracted with equal volume of chloroform: isoamyl alcohol.It was then centrifuged at 10,000 rpm for 10 min (7) In the next step, the top aqueous layer was picked carefully and transferred to fresh sterile 2.0 ml micro-centrifuge tube and precipitated the DNA by addition of 1/10th volume of sodium acetate (3 M pH 5.2) with equal volume of chilled isopropanol.The tube was inverted thoroughly and kept at -20°C for at least 1 h.Later, the mixture was centrifuged at 10,000 rpm for 15 min.(8) The supernatant was decanted and pellet was washed with 70% (v/v) ethanol.Resulted air dried pellet was dissolved in 100 µl TE buffer.

Quantification of DNA
The DNA (5 µl) was added to 495 µl TE, and mixed by vortexing.The absorbance was read in spectrophotometer (SICAN 2301, India) by setting the blank against TE.The observations were taken at 260 and 280 nm.The ratio of OD260 nm/OD280 nm provides an estimate of purity of nucleic acid.Pure preparation of DNA have OD260/OD280 ratio of 1.8 to 2.0 (Sambrook et al., 1989).The DNA concentration (µg/µl) was calculated by using the following formula:

Qualitative analysis of DNA
Qualitative analysis of DNA isolated from fresh leaves was done by 0.8% agarose gel electrophoresis.Gels were stained with ethidium bromide and documented using gel documentation system (Bio-Rad, USA).

Restriction digestion
The composition of reaction mixture and procedure used for the restriction digestion is shown in Table 1.Genomic DNA (4 µg) was used in restriction digestion mediated by Eco R I (Figure 2).

RAPD amplification
Random decamer primers synthesized from Life Technologies (India) were used in random amplified polymorphic DNA (RAPD) amplification.The amplification of template DNA was carried out by using a total volume of 25 μl containing 2.5 μl reaction buffer (10X), 2.0 μl dNTPs mix (200 μM each), 30 ng of decamer primer, 50 ng of genomic DNA and 0.5 μl (3U μl -1 ) Taq DNA polymerase.Thermal cycler (VeritiR, Applied Biosystem, USA) was used to perform PCR reactions having an initial denaturation at 95°C for 5 min, denaturing at 94°C for 1 min, annealing at 40°C for 30 s and polymerization at 72°C for 2 min and final extension 72°C for 5 min.After amplification, the PCR product was resolved on 1.5% agarose gel in 1X TAE buffer.DNA was stained with ethidium bromide and visualized under UV light using gel documentation system (Bio-Rad, USA) (Figure 3).

Procedure
The reaction mixture was prepared as above in an Eppendorf tube The mixture was mixed gently Then it was incubated at 37 º C for 2 h The mixture was taken out after 2 h and the digestion was monitored on 1.5% agarose gel running at 50 V for 45 min

ISSR amplification
Six inter simple sequence repeat (ISSR) primers were employed with genomic DNA of P. hysterophorus for the amplification.The PCR reaction was performed by using mixture that consisted of 50 ng genomic DNA, 2.0 mM MgCl2, 1X PCR buffer, 100 mM of dNTP, 25 ng primer and 1.5 U Taq polymerase in 25 μl volumes.PCR programme had an initial denaturation for 5 min at 95°C, followed by 40 cycles for 1 min at 94°C denaturation, annealing at 50°C for 1 min, extension at 72°C for 2 min and final extension at 72°C for 7 min.The amplification was conducted in a thermal PCR (VeritiR, Applied Biosystems, USA).After amplification, 10 μl of PCR products were loaded on the 1.5% agarose gel and electrophoresed in 1X TAE buffer.The image was taken under UV-light using gel documentation system (Bio-Rad, USA).

PCR amplification of actin gene
The gene specific primer for actin gene was used in PCR amplification.A reaction tube mixture contained 40 ng of DNA, dNTP mixture (100 mM each), Taq DNA Polymerase (1.0 U), 10X reaction buffer and gene specific primers (30 ng).The thermal cycling of DNA was performed with program as follows 95°C for 5 min (Initial denaturation), 35 cycles at 95°C for 1 min (denaturation), 52°C for 1 min (annealing), 72°C for 1 min (extension) and final extension at 72°C for 5 min.The amplified PCR product was resolved on 1.2% agarose gel in 1X TAE buffer.The gels were examined under UV light and photographed by documentation system (Bio-Rad, USA).

RESULTS AND DISCUSSION
In the present study, optimized CTAB method was used for isolation of DNA from parthenium leaf.The main complications of plant DNA isolation were associated with the presence of polyphenolic compounds, terpenoids and polysaccharides (Kotchoni and Gachomo, 2009).Weed plant such as P. hysterophorus showed strong allelopathic character which is associated with secondary metabolites (Bhadoria, 2011;Mawal et al., 2015).Thus, quality and amount of DNA were greatly affected by these interfering compounds which co-precipitate during the DNA isolation from P. hysterophorus leaf.Extraction using high salt CTAB buffer was done to remove polysaccharides.CTAB also acts as a good detergent that solubilizes cell wall, lipid membrane and denatures protein (Clarke, 2009).Polyphenols associated with DNA cause hindrance in polymerase chain reaction and restriction digestion.DNA molecules are damaged by reactive oxygen species produced by oxidized product of phenolics (Li and Trush, 1994).Plant cells were effectively lysed on increasing the temperature (65°C) of heat treatment.Increased amount of PVPP (3%) was used to support elimination of phenolic compounds mediated by hydrogen bond formation with them.Oxidation of polyphenol as well as browning of the DNA is effectively prevented by PVPP (Guillemaut and Maréchal-Drouard, 1992;Borse et al., 2011).On the other hand, increased concentration of βmercaptoethanol (0.4%) in modified protocol also improves the quality of DNA because βmercaptoethanol works as a strong reducing agent which contributes in removal of polyphenols from the crude extract of plant and facilitates the linearization of proteins by disruption of disulphide bonds between cysteine residues (Nalini et al., 2004).Elimination of protein was carried out by phenol: chloroform mixture followed by centrifugation steps.Long term treatment using chloroform: isoamyl alcohol was done to get rid of pigments.Isolation of RNA free DNA was achieved by 1 h RNase treatment at 37°C.The use of liquid nitrogen was substituted by prechilled mortar and pestle stored overnight at −80°C.The shearing of DNA was not visible in the present method (Figure 1), which proved the good quality of extracted DNA.
Finally, white pellet of DNA was obtained.The value of A260/280 ratio was 1.79±0.005(Table 2) showing the absence of contaminants.Suitability of EcoRI mediated restriction digestion (Figure 2) of DNA isolated by modified method indicating the efficiency of this method.Clear, reproducible and consistent banding patterns yield demonstrated its compatibility for polymerase chain reaction using RAPD (Figure 3) and ISSR primers (Figure 4).A distinct PCR amplification of actin gene (Figure 5) was also obtained, which further proved that a good  quality of DNA was obtained.Several independent extractions and replicates proved the reliability of the modified method over Doyle and Doyle's (1990) (Table 3).

Conclusion
Conclusively, a modified CTAB method for DNA isolation from weed plant P. hysterophorus was described.It is reliable and cost-effective and does not require liquid  nitrogen.The quality as well as yield of extracted DNA is also not compromised.Absorbance ratio in purity range and sharp band intensity on agarose gel electrophoresis indicated the effectiveness of the optimized method for DNA isolation.The extracted DNA product is suitable for molecular studies such as RAPD, ISSR, PCR amplification and restriction digestion analysis.

Figure
Figure 1.Total DNA (Lanes 1, 2, 3 and 4) extracted from P. hysterophorus leaf using present modified method visible on 1% agarose gel stained with ethidium bromide.Lane M is 1Kb ladder.

Table 1 .
Composition of reaction mixture and procedure used in restriction digestion.

Table 2 .
Quantification of DNA extracted by Doyle and Doyle's (1990) and modified methods from leaf of Parthenium hysterophorus.

Table 3 .
Comparison of components of two DNA extraction methods.