Extraction of Genomic DNA from Lantana camara Leaf: Simple Protocol for Freshers in Molecular Biology and Biotechnology

This research note has been accepted for publication by Harmony Newsletter published online from Patna, Bihar, INDIA

***************************

Extraction of Genomic DNA from Lantana camara Leaf: Simple Protocol for Freshers in Molecular Biology and Biotechnology

Sourav Datta¹, Suman Roy², Sourav Kumar Das³, Palash Mukherjee⁴ and SKT Nasar*

Department of Biotechnology, Bengal College of Engineering and Technology

(Affiliated to West Bengal University of Technology), Durgapur-713212, West Bengal

Abstract

We present here an ultrasimple, effective and inexpensive procedure to extract genomic DNA from plants and other organisms with special focus on a common weed Lantana camara L. This report also presents a brief discussion on some selected downstream applications of genomic DNA extracted by this protocol. 

Introduction

Genomic DNA comprising nuclear, mitochondrial and chloroplast DNA is basic to molecular biology and biotechnology and application of genetic technologies. Extraction of DNA from organisms is the first step for the application of molecular biology. Shirazu et al. (2009), Nasar and Nasar (2009, 2010) and others demonstrated that extraction of DNA from different organisms is easy, inexpensive and fun.

Lantana camara (L.) is a common invasive weed plant showing wide diversity. Lantana biodiversity has been used to produce commercially available ornamental varieties for hedges and gardens. We have selected Lantana camara for the present work for two main reasons: Durgapur shows wide variability and DNA extraction is rendered difficult due to high content of pentacyclic terpenoids and volatile oil in leaves.

We show here that gDNA extraction from Lantana leaf is effectively as easy as for other plant species. We also show that its applicability in selected downstream procedures is feasible.

Materials and Methods

Planting selected materials in pots

Selected disease-free plants of Lantana camera L. were maintained live in earthen pots at the departmental garden. Healthy fresh leaves were collected for DNA extraction experiments.

Extraction of genomic DNA

The procedure standardised by Nasar & Nasar (2010) and Nasar, Mukherjee & Trivedi (2010, unpublished; personal comm.) has been used here. This protocol requires household items such as refrigerator, mortar-pestle, table salt, dish washing liquid soap and alcohol.

The method of gDNA extraction from leaf of Lantana camera L. constituted the following steps:

(i) All items – leaves, glassware, and chemicals – were pre-cooled in the freezing chamber of a refrigerator and all steps were carried out on a bed of ice cubes or in ice box; (ii) Only   healthy and young leaves were collected and washed with clean water (Fig 1 A); (iii) leaves were cut into small pieces and surface dirt of the material removed by washing  repeatedly with distilled or drinkable water (Fig 1 B); (iv) A tablespoonful of clean water, a pinch each of ethylenediaminetetraacetic acid (EDTA) and Tata® salt (NaCl) were added to the material and it was pulverised with mortar-pestle to make a paste of tissue; (v) The paste was filtered through fibre-free cloth and the filtrate was collected in a test tube or small glass container; the residue was discarded; (vi) 2-4 drops of Vim® liquid dish washing soap were carefully added to the suspension of pulverised tissue; very gentle stirring with a pre-cooled glass rod avoided lathering; (vii) Finally, pre-cooled ethanol was poured slowly along the wall of test tube/container without disturbing the tissue suspension; (viii) The test tube was kept in the refrigerator at 14-15^O C for 20-30 minutes and progress was watched intermittently; (ix) Bubbles began rising through alcohol layer raising DNA threads that aggregated as a white cloud floating in the ethanol layer (Fig 1 C & D); (x) DNA, at his stage, was carefully sucked out with a long-nozzle glass dropper and was then expelled from the dropper in pre-cooled 70% ethanol in a glass container for storing in a refrigerator (Fig 1 E).

Fig. 1. Steps of gDNA extraction from leaves of Lantana camara L. from preparing the material to DNA isolation to storing

Sufficient amounts of DNA were collected in about 30 minutes as can be seen in Fig. 1 D and E.

Confirmation of the extracted substance being DNA

Several protocols exist for confirmation of the extracted substance being DNA. We used ethidium bromide [EtBr] in a simple experiment. The extracted DNA stored in 70% ethanol was poured on a clean watch glass (#1) and kept in a refrigerator for four-five days till the alcohol evaporated. Air-dried extracted DNA clung to glass surface at the centre. A drop of EtBr was placed over the substance. Another drop of EtBr was placed on a clean watch glass (#2) to act as experimental control. Watch glasses #1 and #2 were exposed to UV light in a transilluminator. The fluorescence patterns were photographed [Fig. 2 (a)]. A teaspoonful of water each was added to another set of #1 and #2 and similarly fluoresced [Fig. 2 (b)].

Fig. 2. Ethidium bromide (EtBr) fluorescence and EtBr-stained air-dried DNA and DNA dissolved in water

It was observed that EtBr alone fluoresced with lesser intensity than in conjunction with the extracted substance [Fig. 2(a) #2 and (b) #2]. EtBr is known to intercalate with dsDNA and produce intense fluorescence. On the other hand, ssDNA, RNA and stretches of dsRNA fluoresce with much lower intensity.  This mini-experiment confirmed that the extracted substance is indeed DNA.

Confirmation of gDNA

Leaf cell contains nuclear, chloroplast and mitochondrial DNA. It was important to verify if the extracted DNA was a mixture of nuclear and organellar DNA since downstream applications would depend upon this information.  To this end, the extracted DNA was subjected to agarose gel electrophoresis in the laboratory.

Agarose gel electrophoresis for analysis of gDNA

The basic information utilised for this section was based on Ogden and Adams (1987) and Brody and Kern (2004). The extracted DNA was placed in pre-cooled water in eppedorf tube and rinsed by centrifugation several times before use. Washing with water would remove salt, ethanol and lighter segments of sheared DNA, 

The extracted DNA samples were electrophoresed in 1% agarose gel with EtBr at 75 v and, 60 mA for 1 hr.  The EtBr-DNA fluorescent bands under transilluminator were captured in digital pictures.

Fig. 3. DNA bands of Lantana camara (L.) after agarose gel electrophoresis. Heavy band likely nuclear DNA, Light band likely chloroplast band and Lightest band likely mitochondrial DNA

The electrophoresis yielded three distinct bands. A comparison of the results (Fig. 3) with other results showed that Lantana leaf DNA bands were similar to those of DNA extracted from different plant species. The heaviest band represents nuclear DNA (nDNA), the light band is chloroplast DNA (ctDNA) and the lightest band comprises mitochondrial DNA (mtDNA).

The result shows that extraction of DNA from Lantana camara (L.) leaf is easy, quick and inexpensive. It also confirms that the extracted ‘white cloud’ in ethanol is indeed DNA as further confirmed by the electrophoresis result.

Discussion

Experiments conducted at Department of Biotechnology, Bengal College of Engineering and Technology, Durgapur, confirmed that the ultrasimple procedure of gDNA  extraction from Aloe vera leaf (Mala 2010), Oryza sativa leaf (Ganguly 2010; Tewari 2011), giant tiger prawn (Penaeus monodon Fab.) muscle tissue (Nasar and Trivedi, pers. comm.),  goat liver and Lactobacillus (Nasar and Mukherjee, pers. comm.) are uniformly effective.

Four basic steps are essential for all DNA extraction protocols: (a) breaking cell membranes by grinding to expose cellular contents and DNA, (b) getting rid of intracellular boundaries by disrupting lipo-protein membranes by a detergent containing sodium dodecyl sulphate, (c) removing proteins by protease such as papaya juice and (d) precipitating DNA with alcohol, preferably ethanol. The experiment must be conducted in cool conditions to decrease endonuclease activity. Procedures must be carried out gently to avoid shearing of DNA.

An inexpensive gDNA extraction protocol (Shirazu et al. 2009; Nasar & Nasar 2010) that consumes less time without compromising with quality has been considered handy for this work. This report has shown that the ultrasimple protocol of gDNA extraction is inexpensive, quick and effective as compared with standard procedures such as the hexadecyltrimethylammonium bromide (CTAB) method (Ausubel et al. 1994, Tewari 2011) are elaborate, costly and time consuming. This report corroborates our other experiments. The total time duration for the experiment from harvesting plant tissues to harvesting gDNA has been found to be typically less than an hour.

Extraction of DNA from plant tissue varies with experimental materials. Required modifications adopted for DNA extraction from Lantana camara without changing the fundamentals of different steps yields sufficient amounts of gDNA amenable to downstream analysis.

Addition of table salt in lieu of lab-grade NaCl is important. The negative charge of one of the oxygen atoms linked to the phosphorous provides high polarity to DNA that dissolves in water at neutral pH.  The negative charge of phosphodiester group of DNA is neutralised on addition of salt. DNA then becomes much less soluble in water. Iodised Tata® table salt contains enough NaCl for the purpose of the protocol under report. Table salt worked well in all our experiments.

Ethylenediaminetetraacetic acid (EDTA) provided better results when used in our experiments. EDTA is a chelating agent for metal ions such as Ca²⁺, Mg²⁺ and Fe³⁺. Nucleases need divalent cations such as Mg²⁺ which when depleted deactivates the enzymes.

Experiments reported here used Vim® dishwashing liquid soap containing sodium dodecyl/lauryl sulphate (SDS/SLS). SDS in the soap is a strong anionic detergent that solubilises and breaks lipo-protein membranes and nuclear envelope. In addition to breaking down membranes, SDS emulsifies lipid bi-layer structure of cell and nuclear membranes. SDS also helps the release of chromosomal DNA from histones and other DNA binding proteins by denaturing them.

After careful examination of DNA precipitation in isopropyl alcohol, denatured alcohol or ethanol in pilot experiments, cold ethanol was found to yield the best results.  Addition of alcohol and salt causes DNA to precipitate while other soluble cell components remain in solution in the aqueous phase. Alcohol also removes alcohol-soluble-salt (see Kurabo PI-80X 2010; CTAB, CIMMYT 2005).

Experiments in our laboratory revealed three bands of DNA extracted from leaf of Rice (Oryza sativa L. cv. MTU 7029 ‘Swarna’; Ganguly 2010 and Tewari 2011), Aloe vera L.  (Mala 2010) and Lantana camara L. (present report) as can be seen in the collage presented at Fig. 4.

Ganguly (2010), in another experiment on the DNA of fresh and senesced leaf and fresh root of rice (Oryza sativa L. cv. MTU 7029) successfully showed that the three bands as seen in Fig. 4 represent nuclear DNA (nDNA; heavy band), chloroplast DNA (ctDNA; light band) and mitochondrial DNA (mtDNA; lightest band). The parallel illustrated in Fig. 4 confirms that the ultrasimple simple protocol is undeniably effective for gDNA extraction from Lantana leaf.

The gDNA extracted by the ultrasimple procedure is fit for downstream application. Tewari (2011) subjected gDNA extracted by standard (CTAB) and ultrasimple protocols to digestion with two restriction enzymes i.e. Hind 111 and EcoR1 for RFLP studies on rice (Oryza sativa L. cv. MTU 7029). He observed that gDNA extracted by these procedures yielded the same results.  Similarly, our PCR studies on gDNA extracted by standard and ultrasimple protocols from giant tiger prawn (Penaeus monodon Fab.) muscle tissue (Nasar and Trivedi, pers. comm.) showed equally acceptable result.

Fig. 4 (A, B, C & D) Collage of leaf DNA from (A & B) Oryza sativa L. (C) Lantana camara L. and (D) Aloe vera L. after agarose gel electrophoresis showing three bands each. (A & B) DNA bands of Oryza sativa L. cv. MTU 7029 leaf; (A) DNA extracted by the ultrasimple protocol as in the present report; (B) DNA extracted by standard (CTAB) protocol; (C) DNA bands of Lantana camara L.; Present work, DNA extracted by ultrasimple protocol under this report; (D) DNA bands of Aloe vera L.; DNA extracted by the ultrasimple protocol as in the present report;

(1% Agarose gel, 20µl EtBr; electrophoresis running time 3 hr 30 minutes at 50 volts)

Although gDNA extracted by ultrasimple protocol has an OD of 1.4-1.5 at 260nm/280nm UV absorbance (Tewari 2011), our conclusion that the ultrasimple protocol for extraction of gDNA good for downstream applications is inescapable. This is corroborated by Chum et al. (2012) who showed that DNA extracted directly from tissues can be used for PCR analysis.

A major criticism of teaching in molecular biology and biotechnology is that practicals are not conducted appropriately (Lakhotia 2008). Such inadequacies are due to the prohibitive costs of infrastructure coupled with fund crunch. Experiments in selected areas with innovative and cost cutting measures (Nasar 2009) can lessen the burden. The present work has been undertaken to address this issue.

The present work shows that, with a good understanding of fundamental principles of protocols, innovative experiments in molecular biology and biotechnology can be cost-effective and quick. Students can, for example, extract DNA at residences and bring samples to laboratory for further analysis.

Acknowledgement

Authors are indebted to Bengal College of Engineering and Biotechnology, Durgapur for providing facilities and to Mr. Prasenjit Tewari, Mrs. Kanchan Mala and Mr. Joydev Ganguly, all former students of SKTN, for allowing us to use data from their dissertations.

References

1.     Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. 1994 Current Protocols in Molecular Biology. New York City, NY: John Wiley & Sons Inc.

2.     Brody, JR and Kern SE (2004) History and principles of conductive media for standard DNA electrophoresis. Anal Biochem. 333(1):1-13. Review.

3.     Chum, P. Y., Haimes, J. D., André, C. P., Kuusisto, P. K., Kelley, M. L. 2012 Genotyping of Plant and Animal Samples without Prior DNA Purification. J. Vis. Exp. (67), e3844, DOI: 10.3791/3844.

4.     Ganguly, Joydev 2010 Genomic DNA of Developing and Senescing Paddy (Oryza sativa L.) Plants: Evaluation with Innovative Agarose Gel Cast and Inexpensive Extraction Protocol; B. Tech. (Biotechnology) Dissertation, (Biotechnology Department, Bengal college of Engineering and Technology, Durgapur), West Bengal University of Technology, Kolkata.

5.     Lakhotia S. C. 2008 Are biotechnology degree courses relevant? Curr. Sci, 94 (10), 1244-1245.

6.     Mala, Kanchan 2010 Genomic DNA of Aloe vera L.: Pot Grown vis-à-vis Tissue Cultured Plants; M. Tech. (Biotechnology) Dissertation, (Biotechnology Department, Bengal college of Engineering and Technology, Durgapur), West Bengal University of Technology, Kolkata.

7.     Nasar S. K. T. 2009 Degree courses in biotechnology; Curr. Sci, 96 (3), 323.

8.     Nasar S.K.T. and Nasar S. Farzaan D. 2009 Genomic DNA Extraction is About the Playing; The Science Creative Quarterly. Available at http://www.scq.ubc.ca/genomic-dna-extraction-is-about-the-playing/; Accessed on 10 November 2012.

9.     Nasar S.K.T. and Nasar S. Farzaan D. 2010 Genomic DNA Extraction is Play http://futureagricultureindia.blogspot.com/2010/04/genomic-dna-extraction-is-play.html; Accessed on 30 April 2010.

10.  Ogden, R.C., and Adams, D.A., (1987) Electrophoresis in agarose and acrylamide gels. Meth. Enzymol. 152, 61-87.

11.  Shirazu Yas, Lee Donna and Abd-Elmessih Esther 2009 The MacGyver Project: genomic DNA extraction and gel electrophoresis experiments using everyday materials. The Science Creative Quarterly. Available at http://www.scq.ubc.ca/the-macgyver-project-genomic-dna-extraction-and-gel-electrophoresis-experiments-using-everyday-materials/; Accessed on 10 November 2012.

12.  Tewari, Prasenjit 2011 Genomic DNA of Rice Cultivar: Ultrasimple Extraction and RFLP; M. Tech. (Biotechnology) Dissertation, (Biotechnology Department, Bengal college of Engineering and Technology, Durgapur), West Bengal University of Technology, Kolkata.

=============================================================

^{1, 2 & 3}Former B. Tech. (Biotechnology) students; ⁴Lab In-charge; ¹sourav.aka.peter@gmail.com, ²suman.biotech1411@gmail.com, ³srvhell@gmail.com, ⁴palash_muk@rediffmail.com,

*skt.nasar@gmail.com; Corresponding author, Former Honorary Professor (Biotechnology)

======================