Development of a High Quality, Rapid, Efficient and Economical DNA Extraction Protocol from Climate Resilient Pearl Millet Crop Without Liquid Nitrogen

Main Article Content

Supriya Ambawat
Subaran Singh
C. Tara Satyavathi
Rajbala Meena
R. C. Meena
Vikas Khandelwal

Abstract

Extraction of good quality genomic deoxyribonucleic acid (DNA) from plants is a major prerequisite for molecular biology experiments. An efficient genomic DNA protocol must be simple, fast and cost effective with high yield and purity. Presence of polyphenols, polysaccharides and secondary metabolites in some plants hamper with DNA extraction making it a very laborious, difficult and time consuming procedure. Here, we portrayed a modified protocol based on the cetyl trimethyl ammonium bromide (CTAB) method to isolate DNA from climate resilient pearl millet leaf tissues having higher amount of polysaccharides. It also excludes the use of expensive chemicals and equipments like proteinase K, liquid nitrogen and tissue lyser. This method includes extraction of DNA using a buffer (pH 8.0) containing 200 mM Tris-HCl, 20 mM ethylenediamine tetracetic acid (EDTA),1.4 M NaCl, 2% CTAB, 2% sodium dodecyl sulphate (SDS) and 1.0 % β-mercaptoethanol followed by purification of DNA with phenol, chloroform, isoamyl alcohol and finally precipitation of DNA by sodium acetate and isopropanol. Good quality genomic DNA with sharp and clear bands was obtained from 48 pearl millet genotypes using this protocol. The yield of DNA varied from 105.2 to 328.3 ng/μl. The purity of DNA sample ranged from 1.74 to 1.95 based on the absorbance at A260/A280 ratio indicating that it’s free from ribonucleic acid (RNA) and protein contamination. PCR analysis using simple sequence repeat (SSR) primers exhibited consistent and reliable amplification products ranging from 150 to 650 bp.This study reveals a fast, simple, efficient, specific, reproducible, reliable and cost effective method for extraction of DNA from small to large number of plant samples amenable to PCR amplification and could be stored for longer duration.

Keywords:
CTAB, genomic DNA extraction, Pennisetum glaucum, polyphenols, polysaccharides, secondary metabolites.

Article Details

How to Cite
Ambawat, S., Singh, S., Satyavathi, C. T., Meena, R., Meena, R. C., & Khandelwal, V. (2020). Development of a High Quality, Rapid, Efficient and Economical DNA Extraction Protocol from Climate Resilient Pearl Millet Crop Without Liquid Nitrogen. International Journal of Environment and Climate Change, 10(12), 85-94. https://doi.org/10.9734/ijecc/2020/v10i1230287
Section
Original Research Article

References

Ambawat S, Satyavathi CT, Meena R, Khandelwal V, Meena RC. Molecular characterization and genetic diversity analysis of released hybrids and varieties of pearl millet [Pennisetum glaucum (L.) R. Br.]. Curr J Appl Sci Tech. 2020;39(31):92-104.

Kale SS, Kadu TP, Chavan NR, Chavan NS. Rapid and efficient method of genomic DNA extraction from sweet sorghum [Sorghum bicolor (L.)] using leaf tissue. International J Chem Studies 2020;8(1):166-1169.

Sahu SK, Thangaraj M, Kathiresan K. DNA Extraction protocol for plants with high levels of secondary metabolites and polysaccharides without using liquid nitrogen and phenol. ISRN Molecular Biology. 2012;6(4):1-6.

Xin Z, Chen J. A high throughput DNA extraction method with high yield and quality. Plant Methods 2012;8(1):1-7.

Murray MG, Thompson WF. Rapid isolation of high molecular weight DNA. Nucleic Acids Res. 1980;8:4321-4325.

Dellaporta SL, Wood J, Hicks JB. A plant DNA minipreparation: version II. Plant Mol Biol Rep. 1983;1:19-21.

Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW. Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location and population dynamics. Proc Natl Acad Sci USA. 1984;81:8014-8018.

Doyle JJ, Doyle JL. Isolation of Plant DNA from fresh tissue. Focus. 1990;12:13-15.

Huang J, Ge X, Sun M. Modified CTAB protocol using a silica matrix for isolation of plant genomic DNA. Bio Techniques. 2000;28:432-434.

Allen GC, Vergara MAF, Krasnyanski S, Kumar S, Thompson WF. A modified protocol for rapid DNA isolation from plant tissue using cetyltrimethyl ammonium bromide. Nature Protocols. 2006;1:2320-2325.

Ahmed I, Islam M, Arshad W, Mannan A, Ahmad W, Mirza B. High-quality plant DNA extraction for PCR: An easy approach. Journal of Applied Genetics. 2009;50:105-107. Available: http://dx.doi.org/10.1007/BF03195661

Amani J, Kazemi R, Abbasi A, Salmanian A. A simple and rapid leaf genomic DNA extraction method for polymerase chain reaction analysis. Iranian Journal of Biotechnology. 2011;9:69-71.

Chen WY, Cui HR, Bao JS, Zhou XS, Shu QY. A simplified rice DNA extraction protocol for PCR analysis. Rice Sci. 2006;13 (1):67-70.

Thomas J, Dominic VJ. Isolation of high quality DNA from pulverized rice grain samples by tri-phase method. Int J Sci Res 2015;4(11):524-527.

McCarthy PL, Berger PH. Rapid identification of transformed wheat using a half-seed PCR assay prior to germination. Biotechniques. 2002;32:560-564.

Kang HW, Yong GC, Unghan Y, Moo YE. A rapid DNA extraction method for RFLP and PCR analysis from a single dry seed. Plant Mol Biol Rep. 1998;16:1-9.

Zidani S, Ferchichi A, Chaieb M. Genomic DNA extraction method from pearl millet (Pennisetum glaucum) leaves. African Journal of Biotechnology. 2005;4(8):862-866.

Sathelly KM, Govindannagari R, Pandey S, Kompelli SK, Visarada KBRS, Kaul T. A Simple and Efficient method for high quality DNA extraction from sweet sorghum [Sorghum bicolor (L.) Moench]. Annals of Plant Sciences. 2014;3(7):761-764.

Ambawat S, Kumar R, Singh S, Yadav R. An Easy, Quick and cost effective method of high quality DNA extraction from mungbean [Vigna radiata (L.) Wilczek] Without Liquid Nitrogen. Int J Curr Microbiol App Sci. 2017;6(9):2695-2703.

Ogunkanmi AL, Oboh B, Onifade B, Ogunjobi AA, Taiwo IA, Ogundipe OT. An improved method of extracting genomic DNA from preserved tissues of Capsicum annuum for PCR amplification. Eur Asian Journal of BioSciences. 2008;2(14):115-119.

Sharma P, Joshi N, Sharma A. Isolation of genomic DNA from medicinal plants without liquid nitrogen. Indian J Exp Bio 2010;48:610-614.

Ferdous J, Hanafi MM, Rafii MY, Muhammad K. A quick DNA extraction protocol: Without liquid nitrogen in ambient temperature. African Journal of Biotechnology. 2012;11(27):6956-6964.

Maniatis T, Fritsch ER, Sambrook J. Quantification of DNA and RNA. In: Molecular cloning: A laboratory manual cold spring harbor laboratory press: NewYork. 1982;448-469.

Chandra A, Tewari S. Isoaltion of genomic DNA from Stylo species without liquid nitrogen suitable for RAPD and STS analysis. Cytologia. 2007;72:287.

Sperisen CF, Gugerli Büchler U, Mátyas G. Comparison of two rapid DNA extraction protocols for gymnosperms for application in population genetic and phylogenetic studies. Genetics. 2000;7:133-136.

Khan IA, Awan FS, Ahmad A, Khan AA. A modified mini-prep method for economical and rapid extraction of genomic DNA in plants. Plant Mol Biol Rep. 2004;22:89a-89e.

Biswas K, Biswas, R. A modified method to isolate genomic DNA from plants without liquid nitrogen Current Sci. 2011;100:1622-1624.

Duan YB, Zhao FL, Chen HD, Li H, Ni DH, Wei PC et al. A simplified genomic DNA extraction protocol for pre-germination genotyping in rice. Genet Mol Res. 2015;14(2):6369-6375.

Cheng FS, Brown SK, Weeden NF. A DNA extraction protocol from various tissues in woody species. Hortscience. 1997;32(5):921-922.

Sari VK, Murti RH. An effective method for DNA extraction of mature leaf of sapodilla (Manilkara zapota (L.) van Royen). Agrivita. 2015;37(1):18-23. DOI:10.17503/Agrivita-2015-37-1-p018-023.

Paterson AH, Brubaker CL, Wendel JF. A rapid method for extraction of Cotton (Gossypium spp) genomic DNA suitable for RFLP or PCR analysis. Plant Mol Biol Rep. 1993;11:122-127.

Clark MS. In: Plant molecular biology-A laboratory manual; springer-verlog berlin Heidelberg. New York.1997;305-328.

Aljanabi SM, Forget L, Dookun A. An improved rapid protocol for the isolation of polysaccharide and polyphenol-free sugarcane DNA. Plant Mol Biol Rep. 1999;17:1-8.

Ausubel F, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA et al. Short protocols in molecular biology. Wiley,New York. 1997;2.9:2-11.

Puchooa DA. Simple, rapid and efficient method for the extraction of genomic DNA from lychee (Litchi chinensis Sonn.). African Journal of Biotechnology. 2004;3:253-255.

Rawat S, Joshi G, Annapurna D, Kumar AN, Karaba NN. Standardization of DNA extraction method from mature dried leaves and ISSR-PCR conditions for melia dubia Cav.-A fast growing multipurpose tree species. American J Plant Sci. 2016;7:437-445.

Sajib AA, Bhuiya MAI, Huque R. A Simple, efficient and rapid method for good quality DNA extraction from rice grains. Rice Sci. 2017;24(2):119-122

McKiernan HE, Danielson PB. Molecular Diagnostics. Academic Press, Cambridge. 2017;1(3):371-394.