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A Modified CTAB Protocol for Genomic DNA Extraction from Citrus Spp Ishfaq M, et al., J Biotech Res Biochem 2020, 3: 010 DOI: 10.24966/BRB-0019/100010 HSOA Journal of Biotech Research & Biochemistry Research Article problematic during PCR reactions. Different protocols were used for A Modified CTAB Protocol for genomic DNA extraction but CTAB is most commonly used with modifications [1]. After the discovery of DNA, a broad field was Genomic DNA Extraction from introduced to extract DNA from plants as well as different crops. This Citrus is due to find out the genetic differences among the same species of spp. (Orange) plants. After the extraction of DNA from orange plants, it has become easy to find out the differences of activated genes in one variety but not in other one. The development and application of sampling and Muhammad Ishfaq and Muhammad Imran Qadir* DNA extraction protocols include adaptations to plain procedures Department of Molecular Biology and Biotechnology, Bahauddin Zakariya to suit various inoculum sources. Soil is taken into account to be a University, Multan, Pakistan posh environment and dealing with DNA recovered from soil is commonly problematic due to the presence of PCR-inhibiting chemical components in soil. Therefore, PCR-based studies require extensive DNA extraction methods and careful purification of macro molecule Abstract extracts so as to get rid of humic acids and other contaminants [2]. The revolution in the best growth and high yield of plants and This possibility, therefore, compels the difference of techniques to crops is due to genetic modification. We explain a simple and sensitize detection. The employment of a two-step PCR with nested efficient method for the genomic DNA extraction from citrus woody primers increases both the sensitivity and also the specificity of the plants. Harvesting of fresh leaves from orange plants is done as it PCR significantly, which might overcome problems with relatively contains low level of poly phenyl. DNA extraction is relatively easy impure DNA extracts and low pathogen numbers. from citrus woody than non-citrus plants. CTAB is used in this method of DNA extraction. Ethanol and NaCl are used to remove It is essential to extract high quality DNA during work on it. plants polysaccharides. Same volume of isopropanol is added into Extraction of DNA from woody plants is not easy as they contain high supernatant to make DNA pellet. Use 70% ethanol to wash DNA polysaccharides level. But in case of citrus plants, DNA extraction pellet. Then pellet dissolve in TE buffer. 50-500micro g/g DNA pellet is relatively easy. A simple but important protocol is used to extract is used as a sample. Then PCR & RFLP analysis are carried out. This DNA then can be stored for long durations. Changes and DNA from Citrus spp. A proper sequence of steps is present in DNA modifications can also be done to get the best quality and high yield extraction through CTAB method. CTAB, extraction buffer, liquid from woody plants as well as all kinds of crops. nitrogen, chloroform, ethanol, isopropanol, 70% ethyl alcohol, NaCl, Keywords: CTAB; DNA pellet; Genomic DNA extraction; PCR; TE Buffer, PCR & RFLP are the steps and protocol used in this DNA RFLP, TE buffer extraction method [3]. For getting best quality DNA, all steps which are arranged in a proper sequence must be followed. Introduction Materials and Methods In Watson & F Crick had made a structural model of DNA. Plant material They suggested that DNA consists of nitrogen bases (A, T, G, C), Fresh leaves were harvested from citrus plants (orange) from Phosphate backbone and De-oxy ribose sugar. Historical background bio-park in BZU Multan. Then these leaves were kept in ice box to of pants and crops shows that cross breeding was mostly adapted make them safe from degradation. Fresh leaves were selected as they method to make changes in their variety. Genomic DNA extraction contain low level of polysaccharide (Figure 1). is the first step for studying the molecular and genetic aspects of any organism. DNA extraction from plant sources is difficult due to the large amount of polysaccharides and poly-phenols which become *Corresponding author: Qadir MI, Department of Molecular Biology and Biotechnology, Bahauddin Zakariya University, Multan, Pakistan, Tel: +92 0308 6752329; E-mail: [email protected] Citation: Ishfaq M, Qadir MI (2020) A Modified CTAB Protocol for Genomic DNA Extraction from Citrus spp. (Orange). Biotech Res Biochem 3: 010. Received: September 21, 2020; Accepted: September 26, 2020; Published: October 05, 2020 Copyright: © 2020 Ishfaq M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits un- Figure 1: Fresh leaves were selected as they contain low level of polysaccharide. restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Ishfaq M, Qadir MI (2020) A Modified CTAB Protocol for Genomic DNA Extraction from Citrus spp. (Orange). Biotech Res Biochem 3: 010. • Page 2 of 4 • Equipments and reagents • Add 5 micro L DNA solution in each sample to test on 0.8% agarose • Mortar & pestle • Store the DNA solution at -20C • Centrifuge • Micro-pipettes Gel documentation system & DNA display • Eppendorf tubes • Water bath A complete procedure is followed to make agarose gel which act • Liquid Nitrogen as carrier for DNA. Gel documentation is a system interlinked to computer used to display the proper sequencing of DNA. • Extraction buffer 100 mM Tris-HCl (pH 8) • mM NaCl Results and Discussion • 50 mM EDTA(pH 8) • 0.5% B-mercaptoethanol There are a lot of methods which are used to extract DNA but this • 1.5% (W/V) CTAB protocol of DNA extraction is cost effective and efficient. Removal of • Chloroform-isoamyl alcohol (24:1) polysaccharides from citrus plants leaves is easy by using this method. • TE buffer (pH 8) As the DNA pellets formed then quickly dissolve in TE buffer so this • 10mM Tris-HCl thing is a good way to the protection of DNA from degradation [4]. • 1 mM EDTA Low level of polysaccharides in citrus fruits plants (e.g. orange) is • 10 mg/mL RNase helpful for isolating high quality DNA via PCR and PFLP (Figure • 70% ethanol 2). When DNA obtained from Citrus plants leaves treated with 0.8% agarose gel, the sample present in the well appeared bright under UV • 5 M NaCl light [5] 70% ethanol play an important for the proper washing of • Agarose DNA pellets to remove the debris. Citrus spp. mostly preferred for DNA extraction protocol DNA extraction as these species are treated by using CTAB DNA extraction method (Figure 3). • Grind 5-8 g of fresh leaves of orange tree with pestle &mortar by using liquid nitrogen (or EDTA) • Transfer fine powder into 50 ml centrifuge tube and add 15ml extraction buffer • Gently invert and put all the tubes in water bath • Put off from water bath. Add 15 ml chloroform isoamyl- alcohol (24:1) and invert the tubes gently Figure 2: Low level of polysaccharides in citrus fruits plants (e.g. orange) is helpful • Centrifuge at 9000 rpm for 10 min for isolating high quality DNA via PCR and PFLP. • Transfer upper layer in another clean tube while debris is discard out • Add equal volume of isopropanol, gently invert then put the tubes in incubator at -20 for 30 min • Again centrifuge at 9000 rpm for 10 min • Supernatant is discarded while lower precipitates remain left • Wash the DNA pellets with 3 ml 70% ethanol (2- 3 times) • Air dry the pellets for 20-30 min • Add 3ml TE buffer & 10 mg/ml RNase • Incubate at 37C Figure 3: Quality test of 8 DNA samples of Citrus spp. On 0.8% agarose gel. Lane 1, • Transfer DNA solution in fresh tube and add 3 ml phenol- DNA/ Hind III DNA marker; 2-7, fresh mature leaves; 8, withered old leaves (stored chloroform isoamyl alcohol (25:24:1) in a handbag for 1 week); 9, frosted old leaves (harvested in January 2001), samples harvested from the field. • Gently invert and again centrifuge at 9000 for 15 min • Supernatant transfer into clean tube, add 1 ml of 5M NaCl and 4 ml ether in it Conclusion • Centrifuge at 9000 rpm for 15 min There are many hurdles to extract DNA from plants but in most • Supernatant is discarded, bottom layer is carefully transferred into cases accumulated amount of polysaccharides is responsible to resist clean tube against qualitative DNA extraction [6-10]. SDS (Sodium Dodecyl • Add chilled isopropanol and remove the DNA pellet with a hook Sulfate (SDS) is an important chemical which is used for cleaning • Wash the DNA pellets via 70% ethanol purpose. In case of DNA extraction, SDS is used for cell lysis. It is • Add 1ml TE buffer to dissolve the DNA pellet also used for denaturing of proteins in electrophoresis in SDS-Page • Dilute 15 micro L DNA solution for qualitative analysis (Figure 4). Volume 3 • Issue 2 • 100010 J Biotech Res Biochem ISSN: 2694-0019, Open Access Journal DOI: 10.24966/BRB-0019/100010 Citation: Ishfaq M, Qadir MI (2020) A Modified CTAB Protocol for Genomic DNA Extraction from Citrus spp. (Orange). Biotech Res Biochem 3: 010. • Page 3 of 4 • References 1. Qadir MI, Malik SA (2010) Comparison of alterations in red blood cell count and alterations in hemoglobin concentration in patients suffering from rectal carcinoma undergoing 5-fluorouracil and folic acid therapy. Pharmacologyonline 3: 240-243. 2. Qadir MI, Noor A (2018) Anemias. Rare & Uncommon Diseases. Cam- bridge Scholars Publishing. Newcastle, England. 3. Qadir MI, Javid A (2018) Awareness about Crohn’s Disease in biotechnol- ogy students. GloAdv Res J Med Medical Sci 7: 62-64.
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