- Email: [email protected]
PCR amplification of the genomic DNA from the seeds of Ceylon ironwood, Jatropha, and Pongamia
biomass and bioenergy 33 (2009) 1724–1728
Available at www.sciencedirect.com
http://www.elsevier.com/locate/biombioe
Short communication
PCR amplification of the genomic DNA from the seeds of Ceylon ironwood, Jatropha, and Pongamia Vigya Kesari, Medhavi Sudarshan, Archana Das, Latha Rangan* Department of Biotechnology, Indian Institute of Technology Guwahati, North Guwahati 781 039, Assam, India
article info
abstract
Article history:
Plant source for fuel that replaces fossil fuels is a topical subject and has gained
Received 1 October 2008
prominence as ‘‘Biofuel crops’’. Successful DNA extraction from seed yielding appropriate
Received in revised form
DNA quality for PCR amplification will allow molecular genetic investigations in such
31 March 2009
crops. Standardized protocols for DNA isolation failed to yield high quality DNA from dried
Accepted 9 August 2009
seeds that are rich source of triglycerides. In this paper, we report a protocol for isolation of
Available online 27 August 2009
genomic DNA from three potential biofuel crops using SDS extraction step, followed by precipitation and purification to remove polysaccharides, proteins and polyphenols which
Keywords:
are abundant in storage tissues like seeds. The average yield of DNA among the biofuel
DNA isolation
crops varied from 15 to 25 mg kg1 tissue. Spectrophotometric and electrophoretic analysis
Jatropha curcas
indicated that the isolated DNA was highly pure and of high molecular weight amenable
Mesua ferrea
for PCR amplification and restriction endonucleases. This procedure may prove useful for
Pongamia pinnata
other oilseed crops of commercial importance.
RAPD PCR
ª 2009 Elsevier Ltd. All rights reserved.
Restriction enzymes Taq DNA polymerase
1.
Introduction
DNA marker technology has been rapidly developing and many techniques that seemed unfeasible before are now routinely used. In anticipation of new marker technology, we have been exploring basic approaches for extracting whole genomic DNA from dried seeds, as a part of a case study on a biodiesel plants (Pongamia pinnata, Mesua ferrea and Jatropha curcas). The ability to identify relevant gene(s) involved in fatty acid biosynthesis and the ability to reliably extract good quality DNA from seed samples is a fundamental step in the application of genetic techniques to the success of biofuel crops.
So far, the seeds which are rich source of triglycerides had very little attention from plant molecular biologists. The isolation of high quality DNA is a prerequisite for any molecular biological studies when using the seed source. The seeds of these plants contain exceptionally high amount of polysaccharides, polyphenols, and other secondary metabolites that can hamper DNA isolation, amplification, restriction digestion and subsequent molecular cloning [1–5]. Polysaccharides and phenolic compounds bind to nucleic acids during DNA isolation and interfere with subsequent reactions [6]. A number of methods are available and are being developed for the isolation of nucleic acid from seeds and
Abbreviations: EDTA, ethylenediaminetetraacetic acid; EtBr, Ethidium bromide; PCR, Polymerase chain reaction; RAPD, Random amplification of polymorphic DNA; SDS, Sodium dodecyl sulphate; TE, tris–EDTA buffer. * Corresponding author. Department of Biotechnology, Indian Institute of Technology Guwahati, Room No. 202, O Block, Guwahati 781 039, Assam, India. Tel.: þ91 361 2582214; fax: þ91 361 2582249. E-mail address: [email protected] (L. Rangan). 0961-9534/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.2009.08.005
biomass and bioenergy 33 (2009) 1724–1728
dehydrated tissues [7–9]. It has been observed that increasing the ionic strength of the extraction buffer by the addition of NaCl lead to increased efficiency in the removal of polysaccharide contaminants and improves the DNA yield [10–12]. Higher concentration of b-mercaptoethanol prevents oxidation of the secondary metabolites in the disrupted seed tissues. SDS an anionic detergent binds and denatures the proteins. Substituting ethanol for isopropanol has been shown to increase the yield and purity of DNA [13,14]. Because the seed contain high amounts of many different substances, it is unlikely that just one nucleic acid isolation method suitable for all seed can ever exit. We describe here an efficient protocol for isolating high molecular PCR amplifiable DNA from dried seeds of three well studied potential biofuel crops viz; P. pinnata (Karanj), M. ferrea (Nahar) and J. curcas (Ratanjyot). DNA was isolated from dried seeds following SDS method of McCouch [15] after its modification. While adapting the standard protocols of McCouch and CTAB method of Doyle and Doyle [16], it was observed that the final DNA preparation was brown having lot of mucilage and was recalcitrant to restriction digestion and/ or amplification. Hence we modified the original protocol of McCouch. The quality and yield of DNA is also appropriate for RFLP analysis. The isolation procedure developed here confirms that for each plant species and tissues being studied, isolation protocols needs to be tailored and optimized [17,18] suited to the needs of molecular characterization. The procedure described here for seeds is simple, rapid, effective and reproducible in different laboratories and can be scaled up as desired. The protocol described here can also be used for several other oilseed crops of commercial importance. To our knowledge this is the first reported protocol for isolation of DNA from seeds of three potential biofuel crops.
Isopropanol, 20 C; Absolute ethanol, 20 C; 70% (v/v) ethanol; TE: 10 mM Tris–HCl, pH 8; 1 mM EDTA, pH 8.0; 10 mg/mL RNase A (Sigma); Random decamer primers (Operon Technologies, Inc., USA); dNTPs (10 mM) (Finzymes); Taq DNA polymerase (5 U/mL) (Finzymes); Taq DNA polymerase buffer (Finzymes); EcoRI and BamHI restriction enzymes (Bangalore Genei, India); Restriction endonuclease buffer (Bangalore Genei, India); DNA extraction buffer: 100 mM Tris–HCl (pH 8.0), 25 mM EDTA (pH 8.0), 1.5 M NaCl, 1.25% SDS and add 5% b-mercaptoethanol (v/v) immediately before use.
2.3.
Materials and methods
2.1.
Plant material
For DNA extraction healthy ripened seeds from mature trees were used (w5 g fresh weight). Seeds of P. pinnata (approximately 2 in number) were collected during the month of April (Sila Forest Range, North Guwahati, 26 140 600 N, 91 410 2800 E, Assam). Similarly seeds of M. ferrea (approximately 3 in number) and J. curcas (approximately 5 in number) were collected during the month of October and July respectively from Tezpur, Assam (26 370 3200 N 92 470 8700 E) and were stored in plastic bags on ice for all future use. The samples were later removed from the plastic bags and placed in a well-spread manner over the shelves of a dehydrator-oven and dried at 50 C for 24 h; stored in sealed containers and kept in 20 C. The dried tissues are mechanically ground to a powder and used for DNA extraction.
2.2.
Reagents and solutions
1 M Tris Base (pH 8.0); 0.5 M EDTA (pH 8.0); 10% sodium dodecyl sulphate (SDS); 5 M NaCl; b-mecaptoethanol; 5 M potassium acetate; 3 M sodium acetate (pH 5.2); phenol:chloroform (1:1 v/v);
DNA quantification and restriction digestion
DNA concentration was measured by obtaining the A260/280 ratio with a spectrophotometer. DNA quality and concentration was also checked by running 2 mL of DNA from each sample on a 1% agarose gel containing 0.5 mg/mL of EtBr. Restriction digestion was done according to supplier’s instructions. Around 2 mg of DNA was restricted with BamHI and EcoRI and visualized on 1% agarose gel.
2.4.
PCR amplification
PCR amplification of the isolated DNA was done using random primers obtained from Operon Technologies, USA. PCR amplification was performed in 25 mL reaction volume (50 ng of DNA, 1 U Taq DNA polymerase, 0.2 mM dNTPs, 1.5 mM MgCl2, 5 pmol of decanucleotide primers) using a DNA Thermal cycler (Applied Biosystems, USA) according to the procedure of Williams et al. [19]. Amplified products were loaded in 1.5% agarose gel containing 0.5 mg/mL of EtBr and documented by a gel documentation system (Bio Rad, USA).
3. 2.
1725
Results and discussion
Seeds are storage organs and hence rich in proteins, lipids, polysaccharides, alkaloids and other secondary metabolites. These compounds can interfere with DNA isolation and successive amplification. We have therefore successfully isolated DNA from dried seeds of P. pinnata, M. ferrea and J. curcas by following SDS method of McCouch et al. [15] after its modification. One gram of powdered tissue was homogenized with 5 mL of freshly prepared DNA extraction buffer (preheated to 65 C) in a 15 mL polypropylene tube. Care was taken not to allow the powder to thaw at any point before homogenizing with extraction buffer. This was done by keeping the temperature below 0 C so as to inactivate the oxidizing enzymes during the homogenization. The contents were mixed well by shaking the tube vigorously and incubating at 65 C in a water bath for 20 min with occasional mixing. About 1.5 mL of 5 M potassium acetate (CH3COOK) was added, agitated and incubated on ice for 20 min. Tubes were then centrifuged at 2795 g for 20 min at 4 C. The aqueous clear phase was transferred to a fresh 15 mL centrifuge tube to which 2/3 volume of ice-cold 2-propanol was added and incubated at 20 C for 2 h to precipitate the DNA. Fibrous DNA settled as pellet can be separated from supernatant (containing impurities) by
1726
biomass and bioenergy 33 (2009) 1724–1728
centrifugation at 2795 g for 20 min at 4 C. The DNA pellet washed with 70% ethanol (EtOH), vacuum dried for 10 min, is finally resuspended and dissolved in 1 mL Tris–EDTA (TE) buffer. RNase A (10 mg/mL) was added to each sample and incubated at 37 C for 30 min to digest ribonuclease. Further extraction was done with equal volumes of phenol:chloroform to remove the unwanted impurities including proteins. The tubes were centrifuged at 11,180 g for 10 min and the aqueous layer was transferred to a fresh 2 mL eppendorf tube. To this 1/10 volume of sodium acetate (CH3COONa) and 2 volumes of absolute ethanol was added and incubated at 20 C for an hour. The fibrous DNA can be directly hooked out from the solution using an improvised hook (Pasteur pipette bent at the tip), washed with 70% EtOH, vacuum dried to remove traces of EtOH and resuspended in TE buffer (amount of TE buffer used decided by the size of the DNA pellet). Alternatively if the DNA precipitate is not hookable from the salt solution, can be centrifuged at 2795 g for 10 min, washed with 70% EtOH, airdried and then resuspended in TE buffer. Care was taken not to over dry the DNA pellet making it difficult to dissolve in TE buffer. DNA samples are stored in 4 C refrigerator for shortterm use and in 20 C freezer for long-term. The original SDS protocol of McCouch et al. employed for rice DNA extraction makes use of low salt concentration (0.5 M NaCl) and isopropanol for final DNA precipitation. Further the DNA preparation employing standard protocols was found to contain lot of mucilage and was also recalcitrant to restriction digestion and PCR amplification. The reason for this is that tree species often contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA [20,21] but are easily identified, because they make the DNA pellet sticky and gelatinous (polysaccharides) or often impart a brown colour (polyphenolics). Polysaccharides and polyphenols interfere with polymerases, ligases, and restriction enzymes [20], and their removal is essential for reliable downstream molecular manipulations. Thus the critical change in our modified DNA isolation procedure turned out to be the combined use of high concentration of NaCl and potassium acetate that significantly increased the efficiency of proteins, secondary metabolites and polysaccharide removal. Further the use of ethanol in contrast to isopropanol was found to be better in precipitating the final DNA yield in the solution. The yield of DNA was >3 folds over the method of McCouch et al. [15] while maintaining the purity, as assessed by spectrophotometer and gel electrophoresis. Also, to prevent residual ribonucleosides from acting as primers during the thermal reaction [5], we used an extended RNase treatment of 30 min at 37 C. This was sufficient to degrade RNA into small ribonucleosides that are not detectable by gel electrophoresis. DNA purity is a concern in extraction procedures. Restriction endonuclease digestion requires fairly clean and large quantities of DNA [22]. Random amplified polymorphic DNA (RAPD) and related technologies require less DNA, but purity is necessary to ensure repeatability and confidence [19]. Spectrophotometer measurements of DNA samples at 260 nm and 280 nm gave an absorbance ratio (A260/A280) of 1.54–1.97 indicating high purity (Fig. 1). The quality of DNA was also checked by agarose gel electrophoresis. We observed conspicuous
Fig. 1 – Purity index of genomic DNA from seeds of biofuel crops as shown by UV absorption spectra.
bands of high molecular weight RNA-free DNA about 30,000 bp with little shearing (Fig. 2). The DNA yields ranged from 20 to 25 mg kg1 of seed for P. pinnata, 20–23 mg kg1 for M. ferrea and 15–20 mg kg1 for J. curcas sufficient enough to carry 250–500 typical RAPD reactions. We successfully digested 2 mg of isolated DNA with 10 U of BamHI and 10 U of EcoRI. The digestion was complete as checked after 4 hr of incubation in water bath at 37 C (Fig. 2). The DNA isolated with this
Fig. 2 – Agarose gel electrophoregram of digested DNA from biofuel crops. DNA undigested (lanes 2, 5, 8), digested with BamHI (lanes 3, 6, 9) and digested with EcoRI (lanes 4, 7, 10). Lane 1: Lambda DNA HindIII marker; Lanes 2, 3, 4 - P. pinnata; Lanes 5, 6, 7 - M. ferrea; Lanes 8, 9, 10 - J. curcas.
biomass and bioenergy 33 (2009) 1724–1728
1727
Fig. 3 – RAPD profile of DNA isolated from seeds of biofuel crops with (a) primer OPA-01 (50 CAGGCCCTTC30 ); (b) OPA-03 (50 AGTCAGCCAC30 ); (c) OPA-05 (50 AGGGGTCTTG30 ). M: DNA Marker (100 bp ladder) Lane 1: P. pinnata; Lane 2: M. ferrea; Lane 3: J. curcas.
method was successfully amplified by RAPD PCR (Fig. 3). These results are superior to those reported by McCouch et al. [15] and Doyle and Doyle [16], as the DNA extracted from the study material using our modified protocol reported in the current study could amplify the DNA fragments of <500 bp size by PCR. Furthermore, it has been confirmed that the high quality DNA thus extracted could be useful to screen the levels of genetic diversity using more advanced and sophisticated PCR techniques like SSR and AFLP (our unpublished results). Although our experiments were carried out exclusively with three potential oil yielding tree species, we believe that this modified method of McCouch et al. [15] will be applicable to all types of oil yielding plants, though it is conceivable that some specimens will present their own specific purification challenges that might require modifications to our procedure. In our laboratory, this method has been used to extract DNA from leaves and seeds of plants from Fabaceae, Euphorbiaceae, Zingiberaceae and Guttiferae. In addition, this procedure is affordable and does not require sophisticated equipment, making it a superior choice relative to expensive commercial kits for DNA extraction. The PCR amplifiable high quality DNA extracted from the modified SDS protocol reported in current study have the potential to play a very important role in developing strategies for further improvement of biofuel crops through DNA polymorphism, genome mapping, identification of the QTLs and other plant breeding approaches such as marker assisted breeding. These approaches when complemented with ongoing conventional breeding programs will contribute to the application of sustainable management practices
especially in the wastelands. The DNA extraction procedure described here for seeds is simple, rapid, effective and reproducible in different laboratories and can be scaled up as desired.
Acknowledgements VK thanks Ministry of Human Resources Development (MHRD), Government of India for fellowship. Sincere thanks to Dr Konwar, Tezpur University for kind supply of germplasm material related M. ferrea and J. curcas. LR acknowledges funding by the Department of Science and Technology, Government of India SERC Fast Track Young Scientist Scheme.
references
[1] Webb DM, Knapp SJ. DNA extraction from a previously recalcitrant plant genus. Plant Molecular Biology Reporter 1990;8:180–5. [2] Demeke T, Adams RP. The effects of plant polysaccharides and buffer additives on PCR. BioTechniques 1992;12:332–4. [3] Jobes DV, Hurley DL, Thien LB. Plant DNA isolation: a method to efficiently remove polyphenolics, polysaccharides, and RNA. Taxon 1995;44:379–86. [4] Weishing K, Nybom H, Wolff K, Meyer W. DNA fingerprinting in plants and fungi. Boca Raton, Florida: CRC Press; 1995. p. 44–59.
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biomass and bioenergy 33 (2009) 1724–1728
[5] Porebski S, Bailey LG, Baum BR. Modification of CTAB DNA extraction protocol from plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter 1997;15:8–15. [6] Pirttila AM, Hirsicorpi M, Kamarainen T, Jakkola L, Hohtala A. DNA isolation method for medicinal and aromatic plants. Plant Molecular Biology Reporter 2001;19. 273a–273f. [7] Tomas HT, Tanksley SD. A rapid and inexpensive method for isolation of total DNA from dehydrated tissue. Plant Molecular Biology Reporter 1989;12:106–9. [8] Chunwongse J, Martin GB, Tanksley SD. Pre-germination genotypic screening using PCR amplification of half seeds. Theoretical and Applied Genetics 1993;86:694–8. [9] Kang HW, Cho YG, Yoon UH, Eun MY. A rapid DNA extraction method for RFLP and PCR analysis from a single dry seed. Plant Molecular Biology Reporter 1998;16:1–9. [10] Tibbits JFG, McManus LJ, Spokevicius AV, Bossinger G. A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees. Plant Molecular Biology Reporter 2006;24:81–91. [11] Smith JF, Sytsma KJ, Shoemaker JS, Smith RL. A qualitative comparison of total cellular DNA extraction protocols. Phytochemistry Bulletin 1991;23:2–9. [12] Fang G, Hammer S, Grumet R. A quick and inexpensive method for removing polysaccharides from plant genomic DNA. BioTechniques 1992;13:52–6. [13] Bult C, Kallersjo M, Suh Y. Amplification and sequencing of 16S/18S rDNA from gel purified total plant DNA. Plant Molecular Biology Reporter 1992;10:272–84.
[14] Syamkumar S, Jose M, Sasikumar B. Isolation and PCR amplification of genomic DNA from dried capsules of Cardamom (Elettaria cardamom M.). Plant Molecular Biology Reporter 2005;23. 417a–417e. [15] McCouch SR, Kochert G, Yu ZH, Wang ZY, Khush GS, Coffman WR, et al. Molecular mapping of rice chromosomes. Theoretical and Applied Genetics 1988;76:815–29. [16] Doyle JJ, Doyle JL. A rapid DNA isolation procedure from small quantities of fresh leaf tissue. Phytochemistry Bulletin 1987;19:11–5. [17] Bekesiova I, Nap JP, Mlynarova L. Isolation of high quality DNA and RNA from leaves of the carnivorous plant Drosera rotundifolia. Plant Molecular Biology Reporter 1999; 17:269–77. [18] Syamkumar S, Lowarence B, Sasikumar B. Isolation and amplification of DNA from rhizomes of turmeric and ginger. Plant Molecular Biology Reporter 2003;21. 171a–171e. [19] Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 1990;18: 6531–5. [20] Mannerlo¨f M, Tenning P. Screening of transgenic plants by multiplex PCR. Plant Molecular Biology Reporter 1997; 15:38–45. [21] Ostrowska E, Muralitharan M, Chandler S, Volker P, Hetherington S, Dunshea F. Technical review: optimizing conditions for DNA isolation from Pinus radiata. In Vitro Cellular and Developmental Biology - Plant 1998;34:108–11. [22] Doyle JJ, Dikson EE. Preservation of plant samples for DNA restriction endonuclease analysis. Taxon 1987;36:715–22.
Available at www.sciencedirect.com
http://www.elsevier.com/locate/biombioe
Short communication
PCR amplification of the genomic DNA from the seeds of Ceylon ironwood, Jatropha, and Pongamia Vigya Kesari, Medhavi Sudarshan, Archana Das, Latha Rangan* Department of Biotechnology, Indian Institute of Technology Guwahati, North Guwahati 781 039, Assam, India
article info
abstract
Article history:
Plant source for fuel that replaces fossil fuels is a topical subject and has gained
Received 1 October 2008
prominence as ‘‘Biofuel crops’’. Successful DNA extraction from seed yielding appropriate
Received in revised form
DNA quality for PCR amplification will allow molecular genetic investigations in such
31 March 2009
crops. Standardized protocols for DNA isolation failed to yield high quality DNA from dried
Accepted 9 August 2009
seeds that are rich source of triglycerides. In this paper, we report a protocol for isolation of
Available online 27 August 2009
genomic DNA from three potential biofuel crops using SDS extraction step, followed by precipitation and purification to remove polysaccharides, proteins and polyphenols which
Keywords:
are abundant in storage tissues like seeds. The average yield of DNA among the biofuel
DNA isolation
crops varied from 15 to 25 mg kg1 tissue. Spectrophotometric and electrophoretic analysis
Jatropha curcas
indicated that the isolated DNA was highly pure and of high molecular weight amenable
Mesua ferrea
for PCR amplification and restriction endonucleases. This procedure may prove useful for
Pongamia pinnata
other oilseed crops of commercial importance.
RAPD PCR
ª 2009 Elsevier Ltd. All rights reserved.
Restriction enzymes Taq DNA polymerase
1.
Introduction
DNA marker technology has been rapidly developing and many techniques that seemed unfeasible before are now routinely used. In anticipation of new marker technology, we have been exploring basic approaches for extracting whole genomic DNA from dried seeds, as a part of a case study on a biodiesel plants (Pongamia pinnata, Mesua ferrea and Jatropha curcas). The ability to identify relevant gene(s) involved in fatty acid biosynthesis and the ability to reliably extract good quality DNA from seed samples is a fundamental step in the application of genetic techniques to the success of biofuel crops.
So far, the seeds which are rich source of triglycerides had very little attention from plant molecular biologists. The isolation of high quality DNA is a prerequisite for any molecular biological studies when using the seed source. The seeds of these plants contain exceptionally high amount of polysaccharides, polyphenols, and other secondary metabolites that can hamper DNA isolation, amplification, restriction digestion and subsequent molecular cloning [1–5]. Polysaccharides and phenolic compounds bind to nucleic acids during DNA isolation and interfere with subsequent reactions [6]. A number of methods are available and are being developed for the isolation of nucleic acid from seeds and
Abbreviations: EDTA, ethylenediaminetetraacetic acid; EtBr, Ethidium bromide; PCR, Polymerase chain reaction; RAPD, Random amplification of polymorphic DNA; SDS, Sodium dodecyl sulphate; TE, tris–EDTA buffer. * Corresponding author. Department of Biotechnology, Indian Institute of Technology Guwahati, Room No. 202, O Block, Guwahati 781 039, Assam, India. Tel.: þ91 361 2582214; fax: þ91 361 2582249. E-mail address: [email protected] (L. Rangan). 0961-9534/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.2009.08.005
biomass and bioenergy 33 (2009) 1724–1728
dehydrated tissues [7–9]. It has been observed that increasing the ionic strength of the extraction buffer by the addition of NaCl lead to increased efficiency in the removal of polysaccharide contaminants and improves the DNA yield [10–12]. Higher concentration of b-mercaptoethanol prevents oxidation of the secondary metabolites in the disrupted seed tissues. SDS an anionic detergent binds and denatures the proteins. Substituting ethanol for isopropanol has been shown to increase the yield and purity of DNA [13,14]. Because the seed contain high amounts of many different substances, it is unlikely that just one nucleic acid isolation method suitable for all seed can ever exit. We describe here an efficient protocol for isolating high molecular PCR amplifiable DNA from dried seeds of three well studied potential biofuel crops viz; P. pinnata (Karanj), M. ferrea (Nahar) and J. curcas (Ratanjyot). DNA was isolated from dried seeds following SDS method of McCouch [15] after its modification. While adapting the standard protocols of McCouch and CTAB method of Doyle and Doyle [16], it was observed that the final DNA preparation was brown having lot of mucilage and was recalcitrant to restriction digestion and/ or amplification. Hence we modified the original protocol of McCouch. The quality and yield of DNA is also appropriate for RFLP analysis. The isolation procedure developed here confirms that for each plant species and tissues being studied, isolation protocols needs to be tailored and optimized [17,18] suited to the needs of molecular characterization. The procedure described here for seeds is simple, rapid, effective and reproducible in different laboratories and can be scaled up as desired. The protocol described here can also be used for several other oilseed crops of commercial importance. To our knowledge this is the first reported protocol for isolation of DNA from seeds of three potential biofuel crops.
Isopropanol, 20 C; Absolute ethanol, 20 C; 70% (v/v) ethanol; TE: 10 mM Tris–HCl, pH 8; 1 mM EDTA, pH 8.0; 10 mg/mL RNase A (Sigma); Random decamer primers (Operon Technologies, Inc., USA); dNTPs (10 mM) (Finzymes); Taq DNA polymerase (5 U/mL) (Finzymes); Taq DNA polymerase buffer (Finzymes); EcoRI and BamHI restriction enzymes (Bangalore Genei, India); Restriction endonuclease buffer (Bangalore Genei, India); DNA extraction buffer: 100 mM Tris–HCl (pH 8.0), 25 mM EDTA (pH 8.0), 1.5 M NaCl, 1.25% SDS and add 5% b-mercaptoethanol (v/v) immediately before use.
2.3.
Materials and methods
2.1.
Plant material
For DNA extraction healthy ripened seeds from mature trees were used (w5 g fresh weight). Seeds of P. pinnata (approximately 2 in number) were collected during the month of April (Sila Forest Range, North Guwahati, 26 140 600 N, 91 410 2800 E, Assam). Similarly seeds of M. ferrea (approximately 3 in number) and J. curcas (approximately 5 in number) were collected during the month of October and July respectively from Tezpur, Assam (26 370 3200 N 92 470 8700 E) and were stored in plastic bags on ice for all future use. The samples were later removed from the plastic bags and placed in a well-spread manner over the shelves of a dehydrator-oven and dried at 50 C for 24 h; stored in sealed containers and kept in 20 C. The dried tissues are mechanically ground to a powder and used for DNA extraction.
2.2.
Reagents and solutions
1 M Tris Base (pH 8.0); 0.5 M EDTA (pH 8.0); 10% sodium dodecyl sulphate (SDS); 5 M NaCl; b-mecaptoethanol; 5 M potassium acetate; 3 M sodium acetate (pH 5.2); phenol:chloroform (1:1 v/v);
DNA quantification and restriction digestion
DNA concentration was measured by obtaining the A260/280 ratio with a spectrophotometer. DNA quality and concentration was also checked by running 2 mL of DNA from each sample on a 1% agarose gel containing 0.5 mg/mL of EtBr. Restriction digestion was done according to supplier’s instructions. Around 2 mg of DNA was restricted with BamHI and EcoRI and visualized on 1% agarose gel.
2.4.
PCR amplification
PCR amplification of the isolated DNA was done using random primers obtained from Operon Technologies, USA. PCR amplification was performed in 25 mL reaction volume (50 ng of DNA, 1 U Taq DNA polymerase, 0.2 mM dNTPs, 1.5 mM MgCl2, 5 pmol of decanucleotide primers) using a DNA Thermal cycler (Applied Biosystems, USA) according to the procedure of Williams et al. [19]. Amplified products were loaded in 1.5% agarose gel containing 0.5 mg/mL of EtBr and documented by a gel documentation system (Bio Rad, USA).
3. 2.
1725
Results and discussion
Seeds are storage organs and hence rich in proteins, lipids, polysaccharides, alkaloids and other secondary metabolites. These compounds can interfere with DNA isolation and successive amplification. We have therefore successfully isolated DNA from dried seeds of P. pinnata, M. ferrea and J. curcas by following SDS method of McCouch et al. [15] after its modification. One gram of powdered tissue was homogenized with 5 mL of freshly prepared DNA extraction buffer (preheated to 65 C) in a 15 mL polypropylene tube. Care was taken not to allow the powder to thaw at any point before homogenizing with extraction buffer. This was done by keeping the temperature below 0 C so as to inactivate the oxidizing enzymes during the homogenization. The contents were mixed well by shaking the tube vigorously and incubating at 65 C in a water bath for 20 min with occasional mixing. About 1.5 mL of 5 M potassium acetate (CH3COOK) was added, agitated and incubated on ice for 20 min. Tubes were then centrifuged at 2795 g for 20 min at 4 C. The aqueous clear phase was transferred to a fresh 15 mL centrifuge tube to which 2/3 volume of ice-cold 2-propanol was added and incubated at 20 C for 2 h to precipitate the DNA. Fibrous DNA settled as pellet can be separated from supernatant (containing impurities) by
1726
biomass and bioenergy 33 (2009) 1724–1728
centrifugation at 2795 g for 20 min at 4 C. The DNA pellet washed with 70% ethanol (EtOH), vacuum dried for 10 min, is finally resuspended and dissolved in 1 mL Tris–EDTA (TE) buffer. RNase A (10 mg/mL) was added to each sample and incubated at 37 C for 30 min to digest ribonuclease. Further extraction was done with equal volumes of phenol:chloroform to remove the unwanted impurities including proteins. The tubes were centrifuged at 11,180 g for 10 min and the aqueous layer was transferred to a fresh 2 mL eppendorf tube. To this 1/10 volume of sodium acetate (CH3COONa) and 2 volumes of absolute ethanol was added and incubated at 20 C for an hour. The fibrous DNA can be directly hooked out from the solution using an improvised hook (Pasteur pipette bent at the tip), washed with 70% EtOH, vacuum dried to remove traces of EtOH and resuspended in TE buffer (amount of TE buffer used decided by the size of the DNA pellet). Alternatively if the DNA precipitate is not hookable from the salt solution, can be centrifuged at 2795 g for 10 min, washed with 70% EtOH, airdried and then resuspended in TE buffer. Care was taken not to over dry the DNA pellet making it difficult to dissolve in TE buffer. DNA samples are stored in 4 C refrigerator for shortterm use and in 20 C freezer for long-term. The original SDS protocol of McCouch et al. employed for rice DNA extraction makes use of low salt concentration (0.5 M NaCl) and isopropanol for final DNA precipitation. Further the DNA preparation employing standard protocols was found to contain lot of mucilage and was also recalcitrant to restriction digestion and PCR amplification. The reason for this is that tree species often contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA [20,21] but are easily identified, because they make the DNA pellet sticky and gelatinous (polysaccharides) or often impart a brown colour (polyphenolics). Polysaccharides and polyphenols interfere with polymerases, ligases, and restriction enzymes [20], and their removal is essential for reliable downstream molecular manipulations. Thus the critical change in our modified DNA isolation procedure turned out to be the combined use of high concentration of NaCl and potassium acetate that significantly increased the efficiency of proteins, secondary metabolites and polysaccharide removal. Further the use of ethanol in contrast to isopropanol was found to be better in precipitating the final DNA yield in the solution. The yield of DNA was >3 folds over the method of McCouch et al. [15] while maintaining the purity, as assessed by spectrophotometer and gel electrophoresis. Also, to prevent residual ribonucleosides from acting as primers during the thermal reaction [5], we used an extended RNase treatment of 30 min at 37 C. This was sufficient to degrade RNA into small ribonucleosides that are not detectable by gel electrophoresis. DNA purity is a concern in extraction procedures. Restriction endonuclease digestion requires fairly clean and large quantities of DNA [22]. Random amplified polymorphic DNA (RAPD) and related technologies require less DNA, but purity is necessary to ensure repeatability and confidence [19]. Spectrophotometer measurements of DNA samples at 260 nm and 280 nm gave an absorbance ratio (A260/A280) of 1.54–1.97 indicating high purity (Fig. 1). The quality of DNA was also checked by agarose gel electrophoresis. We observed conspicuous
Fig. 1 – Purity index of genomic DNA from seeds of biofuel crops as shown by UV absorption spectra.
bands of high molecular weight RNA-free DNA about 30,000 bp with little shearing (Fig. 2). The DNA yields ranged from 20 to 25 mg kg1 of seed for P. pinnata, 20–23 mg kg1 for M. ferrea and 15–20 mg kg1 for J. curcas sufficient enough to carry 250–500 typical RAPD reactions. We successfully digested 2 mg of isolated DNA with 10 U of BamHI and 10 U of EcoRI. The digestion was complete as checked after 4 hr of incubation in water bath at 37 C (Fig. 2). The DNA isolated with this
Fig. 2 – Agarose gel electrophoregram of digested DNA from biofuel crops. DNA undigested (lanes 2, 5, 8), digested with BamHI (lanes 3, 6, 9) and digested with EcoRI (lanes 4, 7, 10). Lane 1: Lambda DNA HindIII marker; Lanes 2, 3, 4 - P. pinnata; Lanes 5, 6, 7 - M. ferrea; Lanes 8, 9, 10 - J. curcas.
biomass and bioenergy 33 (2009) 1724–1728
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Fig. 3 – RAPD profile of DNA isolated from seeds of biofuel crops with (a) primer OPA-01 (50 CAGGCCCTTC30 ); (b) OPA-03 (50 AGTCAGCCAC30 ); (c) OPA-05 (50 AGGGGTCTTG30 ). M: DNA Marker (100 bp ladder) Lane 1: P. pinnata; Lane 2: M. ferrea; Lane 3: J. curcas.
method was successfully amplified by RAPD PCR (Fig. 3). These results are superior to those reported by McCouch et al. [15] and Doyle and Doyle [16], as the DNA extracted from the study material using our modified protocol reported in the current study could amplify the DNA fragments of <500 bp size by PCR. Furthermore, it has been confirmed that the high quality DNA thus extracted could be useful to screen the levels of genetic diversity using more advanced and sophisticated PCR techniques like SSR and AFLP (our unpublished results). Although our experiments were carried out exclusively with three potential oil yielding tree species, we believe that this modified method of McCouch et al. [15] will be applicable to all types of oil yielding plants, though it is conceivable that some specimens will present their own specific purification challenges that might require modifications to our procedure. In our laboratory, this method has been used to extract DNA from leaves and seeds of plants from Fabaceae, Euphorbiaceae, Zingiberaceae and Guttiferae. In addition, this procedure is affordable and does not require sophisticated equipment, making it a superior choice relative to expensive commercial kits for DNA extraction. The PCR amplifiable high quality DNA extracted from the modified SDS protocol reported in current study have the potential to play a very important role in developing strategies for further improvement of biofuel crops through DNA polymorphism, genome mapping, identification of the QTLs and other plant breeding approaches such as marker assisted breeding. These approaches when complemented with ongoing conventional breeding programs will contribute to the application of sustainable management practices
especially in the wastelands. The DNA extraction procedure described here for seeds is simple, rapid, effective and reproducible in different laboratories and can be scaled up as desired.
Acknowledgements VK thanks Ministry of Human Resources Development (MHRD), Government of India for fellowship. Sincere thanks to Dr Konwar, Tezpur University for kind supply of germplasm material related M. ferrea and J. curcas. LR acknowledges funding by the Department of Science and Technology, Government of India SERC Fast Track Young Scientist Scheme.
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