Acta agriculturae Slovenica, 119/4, 1–8, Ljubljana 2023 doi:10.14720/aas.2023.119.4.16094 Original research article / izvirni znanstveni članek A rapid and efficient DNA extraction method from high oily content seeds: Ricinus communis L.- apt for PCR based assay Elham R. S. SOLIMAN 1, 2 Received September 09, 2023; accepted November 27, 2023. Delo je prispelo 9. septembra 2023, sprejeto 27. novembra 2023 1 Cytogenetics and Molecular Genetics Unit, Botany and Microbiology Department, Faculty of Science, Helwan University, Cairo, Egypt 2 Corresponding author, e-mail: Elham_soliman@science.helwan.edu.eg A rapid and efficient DNA extraction method from high oily content seeds: Ricinus communis L.- apt for PCR based assay Abstract: Ricinus communis seeds harbor high oil and polyphenolics contents that hinder DNA extraction. Here, a rapid and efficient protocol for isolating total DNA from R. communis seeds was developed. The current method implies the use of repeated cycles of freeze/heat shock for the seed tis- sue to lyse the cells. DNA isolated with this protocol was suc- cessfully used as a template for PCR amplification of the inter- nal transcribed spacer (ITS) region of the rRNA encoding gene that widely used for molecular identification of different plant species. As far to our knowledge, this study is the first one that report the efficient use of freeze/heat shock repeated cycles for isolation of a high-quality DNA from plant cells. The current protocol would support the subsequent analysis for seed lot pu- rity analysis. Key words: castor bean seed, cell lysis, heat shock, ITS, PCR, sequencing Hitra in učinkovita metoda DNK ekstrakcije iz semen klo- ščevca (Ricinus communis L.), bogatih na oljih, primerna za PCR analizo Izvleček: Semena kloščevca so bogata na oljih in poli- fenolih kar ovira ekstrakcijo DNK. V tej raziskavi je bil razvit hiter in učinkovit protokol za izolacijo celokupne DNK iz se- men kloščevca. Metoda uporablja ponavljajoče se cikle zmr- zovanja in segrevanja, kar povzroči lizijo celic v tkivu semena. DNK izolirana po tem protokolu je bila uspešno uporabljena kot osnova za PCR namnoževanje ITS regij v rRNK, ki kodirajo gene, na širokouporabljene pri molekularni identifikaciji raz- ličnih rastlinskih vrst. Kolikor nam je znano, je to prva raziska- va, ki poroča o učinkoviti rabi ponavljajočih se cikličnih šokov zmrzovanja in segrevanja za izolacijo visoko kakovostne DNK iz rastlinskih celic. Ta protocol bo pripomogel k hitri analizi čistosti semen. Ključne besede: kloščevec, lizija celic, vročinski šok, ITS, PCR, sekvenciranje Acta agriculturae Slovenica, 119/4 – 2023 2 E. R. S. SOLIMAN 1 INTRODUCTION Castor bean (Ricinus communis L.) seed is one of the most common oil seed plants that is widely used eco- nomically in biodiesel production, cosmetics industry, lubricants, biomedical applications and as a rich animal fodder (Patel et al., 2016; Sturtevant et al., 2019; Sánchez et al., 2019; Awais et al., 2020). The seeds are externally protected by hard, brittle, mottled brown and shinning testa. The outermost layer of the seeds coat is the waxy cuticle, which represents the first barrier to water im- bibition. The mature desiccated seed coat is rich in the oxidative products of polyphenolic compounds includ- ing phenolic acids, tannins, and flavonoids which are the source of the brown color of the mature seed coat, and play a significant role in plant disease resistance. Also, peroxidases and other antioxidant scavenging enzymes are commonly found in seeds coats (Moïse et al., 2005). Employing genomics and molecular technologies promise to accelerate our knowledge of seeds and thus open new potentials for uses and conservation. The low concentration of DNA present in seed tissues makes it difficult to isolate intact DNA. Additionally, the DNA in seeds can be tightly bound to various proteins, polyphe- nols, and sugars. These biochemical interactions further complicate the process of extracting high quality DNA from seeds (Roy Davies, 1977; Sliwinska, 2006; Lee et al., 2020). The isolation of a good quality DNA is a pre- requisite for any molecular biology work because pro- teins, polyphenols and polysaccharides impurities that co-precipitate with the DNA may hinder any enzymatic action, such Taq DNA polymerase in Polymerase Chain Reaction (PCR) and subsequent sequencing (Lakay et al., 2007; Wnuk et al., 2020) and endonucleases in genotyp- ing and blotting techniques (Brown, 2001; Harju et al., 2004; Shiraishi and Iwai, 2020). PCR based technique is successfully used to detect R. communis candidate genomic loci that are associated with important agronomic traits (Fan et al., 2019). PCR is essential for specific gene detection that prevailed in molecular identification and characterization (Manju- nath and Sannappa, 2014). Enhancing the current seed traits to add value or to overcome an existing problem may be achieved by the generation of genetically modi- fied plants. This wouldn’t be achieved without isolating a good quality DNA that would serve as a template for a PCR reaction. The production of transgenic R. communis plants expressing the Bacillus thuringiensis cry1Aa gene help in lepidopteran insect pest management that were responsible for 30–50 % of yield losses (Muddanuru et al., 2019). CTAB (Cetyl trimethylammonium bromide) DNA based extraction protocol and tailored modification ver- sions of it, is widely used for isolating DNA from different plant tissues. However efficient, the CTAB is harsh and toxic in nature for human health and should be followed by phenol purification to ensure sufficient DNA purity. The phenol is volatile and can burn the skin (Doyle and Doyle, 1990; Shukla et al., 2018; Aboul-Maaty and Oraby, 2019). The used CTAB based protocol is time consuming as it requires long incubation time reached up to 60 min at 65  0 C (Novaes et al., 2009). Alternatively, the commer- cial DNA extraction kits is an efficient substitute, despite the associated cost burden. So, a rapid efficient isolation method is of demand to facilitate any genomics and mo- lecular biology work. Therefore, the current study was designed for devel- oping a rapid and efficient seed DNA isolation method that implies repeated cycles of freeze/heat shock to lyse the seed cells. Subsequent removal of the coagulated pro- teins and lipids by chloroform extraction was made and finally, high quality DNA was precipitated by ethanol. 2 MATERIALS AND METHODS 2.1 PLANT MATERIALS Ten different samples of R. communis seeds were collected from their wild habitat in Egypt. Their DNA was extracted according to the newly developed protocol and used as a template for PCR amplification to ampli- fy the ITS region of the corresponding rRNA encoding gene (Cheng et al., 2016). 2.2 MATERIALS AND EQUIPMENTS - 1.5 ml Eppendorf tubes - Mortar and pestle - Tips - Cooling centrifuge (Hettich MIKRO 22) - Crushed ice - UV-transilluminator (Vilber Lourmat-Germany) - Digital balance (RADWAG Wagi Elektroniczne, AS 220/C/2) - Micropipettes - Vortex (VELP SCIENTIFICA) - Water bath (MLW W21) - Gel electrophoresis unit - Thermal cycler (Biometra, Germany) 2.3 REAGENTS - Lysis buffer: 2 % Triton X-100 (ADVENT), 100 Acta agriculturae Slovenica, 119/4 – 2023 3 A rapid and efficient DNA extraction method from high oily content seeds: Ricinus communis L.- apt for PCR based assay mM NaCl (POWER CHEMAL), 1 % SDS (Sodium Dodecyl Sulfate), 10 mM Tris-HCl (pH 8.0) (OX FORD) and 1 mM EDTA (pH 8.0) (Hoffman and Winston, 1987, Harju et al., 2004) - Ice cold 99 % ethanol (POWER CHEMICAL) or isopropyl alcohol - 70 % ethanol - 1 × TAE buffer - Ethidium bromide (ALPHA CHEMIKA) - Chloroform (SIGMA-ALDRICH) - 3 M sodium acetate pH 5.2 - TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0, autoclaved) - Agarose (molecular grade, Cleaver Scientific Ltdm CAS 9012-36-6) 2.4 THE DETAILED PROTOCOL 1. In 1.5 ml Eppendorf tubes, 0.1 g crushed de- coated R. communis seeds were combined with 400 μl of the DNA lysis buffer. And then vortexed vigorously in a bench top vortex for up to one minute, followed by incu- bation on ice. a. TIP: Avoid using a large amount of seed sample because the polysaccharides, polyphenols, and their de- rivatives in the seed would increase concurrently. These substances could interfere negatively with proper DNA isolation, reducing the isolated amount. b. TIP: Rather than using a mortar and pestle, the sample could be crushed inside a round end 2 ml Eppen- dorf tube using a glass rod. c. TIP: Grinding in liquid nitrogen would facilitiate the grinding process and increase DNA integrity. 2. After two minutes on crushed ice, the tubes were immersed abruptly in a 95 °C water bath for 5 minutes with interval vortexing. 3. After repeating the freeze/heat shock procedure exactly as described above, the tubes were vigorously vortexed for a continuous 30 seconds. TIP: The solution takes on the appearance of a milky emulsion. 4. 400 μl of chloroform was added and vigorously vortexed for one minute, followed by centrifugation at 4 °C, 5 min, 10,000 rpm (RCF: 17,507). TIP: Chloroform and isoamyl-alcohol can be mixed in a ratio of (24:1 v/v). 5. The aqueous layer was transferred to a new tube and the chloroform purification was repeated twice. TIP: The chloroform purification should be repeat- ed whenever necessary till the aqueous fraction is clear. 6. To allow DNA precipitation, the clear aqueous layer was transferred to a new tube containing 1 ml of ice-cold 99 % absolute ethanol and 40 µl of 3 M sodium acetate pH 5.2. After 15 minutes on ice, the tubes were centrifuged for 10 minutes at 4 °C and 10,000 rpm. TIP: Instead of absolute ethanol, isopropyl alcohol may be used. 7. Following the removal of supernatants, DNA pel- lets were washed with 0.5 ml of 70 % ethanol and quickly centrifuged at 4 °C for 2 minutes at 10,000 rpm. The pellet was then air dried for 5 minutes. TIP: To expedite the drying of the DNA pellet, in- vert the Eppendorf on a piece of clean. tissue. 8. Resuspend the DNA in 30 μl TE buffer [10 mM Tris, 1 mM EDTA (pH 8.0)]. The samples were stored at -20 °C until they were used. TIP: It is recommended to dissolve the DNA pellet in a small volume of TE buffer to ensure that the DNA is adequately concentrated. If the sample was dissolved in a larger volume, it resulted in a low concentration of DNA. Reprecipitate your DNA and resuspend it in a reduced volume of TE buffer. 2.5 QUANTIFICATION AND VISUALIZATION OF DNA The concentration of isolated DNA was determined using a NanoDrop® ND-1000 UV-Vis Spectrophotometer (Thermo Scientific-USA). The optical density (OD) at A260 and A280 was used to determine the purity. Elec- trophoresis of samples was performed on a 1 % agarose gel in 1× TAE (Tris-Acetate- Ethylenediaminetetraacetic acid) buffer containing 0.5 µg ml -1 ethidium bromide. The DNA was visualized and photographed with the aid of a UV-transilluminator embedded in Gel Documenta- tion system (Virballurmate-Germany). 2.6 PCR AMPLIFICATION AND SEQUENCING The PCR was carried out according to the manufac- turer’s instructions in a 50 μl reaction volume using (BI- OLINE cat # BIO-21108) Mytaq Red DNA polymerase master mix. ITS1 (5’ TCCGTAGGTGAACCTGCGG 3’) and ITS4 (5’ TCCTCCGCTTATTGATATGC 3’) primers were used. The reaction mixture contains 1 × mytaq Red DNA polymerase master mix, 2.0 μl of each primer at a concentration of 10 pm l -1 , 1.0 μl of DNA template, and 0.25 μl of MyTaq TM DNA polymerase (5U μl -1 ). The fol- lowing PCR cycles were run in a Thermal Cycler (Biom- etra, Germany): Initial denaturation was carried out for 3 minutes at 95 °C, followed by 35 cycles of 20 seconds at 95 °C (denaturation), 20 seconds at 55 °C (annealing), 30 seconds at 72 °C (extension), and then a final extension Acta agriculturae Slovenica, 119/4 – 2023 4 E. R. S. SOLIMAN for 10 minutes at 72 °C. On a 1 % agarose gel, the PCR products were separated. The amplified fragments were excised and gel pu- rified according to the manufacturer’s protocol using the PCR-M clean up system (VIOGENE cat# PF1001). Following that, their sequences were determined at GATC Company using an ABI 3730xl DNA sequencer and an ITS4 primer. The obtained nucleotide sequenc- es were validated against the NCBI database using the MEGA blast tool (https://blast.ncbi.nlm.nih.gov/Blast. cgi). Geneious 11.1.5 software was used to align all se- quences. The following sequences were deposited in the NCBI GenBank: MN880879, MN880880, MN880881, MN880882, MN880885, MN880886, MN880887, MN880888, MN880889, and MN880890. 3 RESULTS The following is a description of a rapid DNA isola- tion protocol called the “ES DNA isolation method” (Fig. 1). The method is based on the use of repeated freeze- and-heat shock cycles to disrupt the cell wall and release genomic DNA from crushed de-coated R. communis seeds in lysis buffer. Following that, two rounds of chlo- roform purification were performed to remove co-bound proteins and lipids to the DNA following cell lysis. After transferring the aqueous phase to a clean tube, the DNA is easily precipitated using absolute (Abs.) ethanol and 3 M sodium acetate (pH 5.2). Following a 70 % ethanol wash, the air-dried pellet was dissolved in TE buffer to ensure the extracted DNA was preserved for the long term. The isolated DNA’s purity and lack of degradation are demonstrated by gel electrophoresis (Fig. 2). Figure 2: Gel electrophoresis of DNA isolated from the de- coated R. communis seeds used in the current protocol; “ES DNA isolation method” . Lanes 1 to 10 refers to the ten samples used. λ DNA refers to serial dilution of undigested λ DNA for quality control and quantification of isolated DNA. The flourecent bands towards the bottom of the gel likely represent degraded RNA, which commonly co-purifies during DNA extraction protocols Table 1: Quantitative estimates of DNA purity and concentra- tion revealed by Nanodrop Spectrophotometer Sample ID Concentration ng µl -1 A260/A280 readings (DNA Purity) Rc1 95.1 2 Rc 2 81.4 2.57 Rc 3 49.5 2.3 Rc 4 55.6 2.28 Rc 5 79.7 1.99 Rc 6 52.8 2.11 Rc 7 56.9 2.3 Rc 8 60.1 2.5 Rc 9 66.3 1.99 Rc 10 55.7 2.44 Figure 1: Flow chart representation of the current R. com- munis seed DNA isolation protocol termed “ES DNA isolation method” Acta agriculturae Slovenica, 119/4 – 2023 5 A rapid and efficient DNA extraction method from high oily content seeds: Ricinus communis L.- apt for PCR based assay The A260/280 ratio, which ranges between 1.99 and 2.57 for all R. communis seed DNA samples examined, indicates the quality of the isolated DNA (Table 1). The isolated DNA concentration ranges between 49.5 and 95.1 ng µl -1 . The yield and purity of the isolated DNA were sufficient for performing polymerase chain reaction amplification (Fig. 3). All samples were amplified to the expected product size of 700 bp. All PCR products were sequenced and the nucleo- tide sequences verified using the National Center for Bio- technology Information’s (NCBI) website. Alignment of the ITS sequences was performed to demonstrate their homology (Fig.4). The matched sequences revealed that all samples shared a significant degree of homology. The results demonstrates that the sequenceable PCR products obtained are of a high grade. Figure 3: PCR-amplification of the ITS-rRNA encoding gene of the different R. communis seeds using the DNA extracted by the current method “ES DNA isolation method” as a template. Lanes 1 to 10 represent the ten R. communis samples. 100 bp ladder refers to the DNA ladder. NTC refers to non-DNA template of the PCR, no amplification means no PCR contamination Figure 4: Partial sequence alignments of the ITS-rRNA encoding gene sequences obtained from the 10 R. communis seeds using Geneious 11.1.5 software. The data confirms the quality of the obtained PCR products that could be sequenced Acta agriculturae Slovenica, 119/4 – 2023 6 E. R. S. SOLIMAN 4 DISCUSSION While a PCR-based assay requires adequate quality genomic DNA, a rapid isolation methodology is required to permit the processing of large numbers of samples. Be- cause the majority of polyphenolic chemicals in seeds are localized in their seed coats, de-coating the seeds reduces their interference with DNA isolation (Moïse et al., 2005). Repeated freeze-heat shock cycles are employed to shat- ter the cell wall and release genomic DNA from crushed de-coated R. communis seeds in lysis buffer, obviating the necessity for enzymatic or mechanical degradation (Har- ju et al., 2004). The lysis buffer contains mild detergents such as Triton X-100 and SDS and is frequently used to lyse cells, extract proteins, extract oils, and permeabilize live cell membranes by dissolving protein-lipid and lipid- lipid interactions without denaturing proteins (Johnson, 2013). However, freezing and heat shock may affect cell wall permeability and denature proteins, thereby expe- diting their removal, without the need for hazardous pol- yvinyl pyrrolidone (PVP), phenol, or β-mercaptoethanol (John, 1992; Shukla et al., 2018). Seed polysaccharides, polyphenols, and their deriv- atives may impair the integrity of isolated DNA from R. communis seed (Porebski et al., 1997). When cells are ly- sed, these compounds covalently link to DNA, impairing DNA integrity and impeding PCR amplification (Wnuk et al., 2020). Therefore, the 60 °C CTAB incubation may be particularly important to dissociate polysaccharides from DNA, allows efficient and thorough lysis of cells and breakdown of proteolytic enzymes that could otherwise degrade DNA. This contributes to high DNA yields with CTAB and improve purity of the extracted DNA. The current lysis protocol using repeated freeze-heat shock cycles may achieve comparable degree of enzymatic deg- radation and release of DNA from cellular components without prolonged optimum heated incubation (Carey et al., 2023). Chloroform can be used to remove these com- pounds during DNA extraction. Additional to removing denatured proteins, it aids in the removal of other col- ouring chemicals such as pigments and dyes. Chloroform facilitates the separation of lipids, proteins, and cellular detritus into the organic phase, while the recoverable DNA is dissolved in the aqueous phase and then easily precipitated with absolute (Abs.) ethanol. Because the low seed DNA content necessitates efficient DNA pre- cipitation, 3 M sodium acetate (pH 5.2) was added to the absolute ethanol during the precipitation process (Júnior et al., 2016; Heikrujam et al., 2020). Gel electrophoresis was used to determine the feasi- bility of the current DNA isolation approach. No degra- dation was seen on the gel. Additionally, this was also ob- served with the nanodrop measurements. The A260/280 ratio of isolated DNA varied between 1.99 to 2.57 for all R. communis seed DNA samples examined, indicating that the DNA was of acceptable quality (Nzilibili et al., 2018). The isolated DNA concentration ranged between 49.5 and 95.1 ng µl -1 , which was sufficient for down- stream applications such as PCR. Although the A260/280 ratio for high-quality DNA should be between 1.8 and 2, values greater than 2 have been observed previously for DNA samples isolated from various plant tissues us- ing a modified CTAB-based approach (Aboul-Maaty and Oraby, 2019). This could be attributed to ionic strength and altered pH of the solutions used in the extraction process (Wilfinger et al., 1997; Boesenberg-Smith et al., 2012). Despite the fact that the DNA isolated using the current approach had a slightly higher A260/280 ratio, the DNA isolated was effectively employed for the down- stream application, PCR. The extracted DNA was used as a template for PCR amplification of the ITS region of the rRNA encoding gene, which is commonly used for molecular identification and assessment of the molecular diversity of eukaryotic cells (Cheng et al., 2016; Yang et al., 2018; Ghareb et al., 2020; Soliman and A., 2021). PCR amplification of the extracted DNA samples yielded the expected product size of approximately 700 bp, as shown in Figure 3. The amplified PCR products were successful- ly sequenced, and the obtained sequences were deposited in the NCBI database (Fig. 4) (Soliman and A., 2021). These results demonstrate that the isolated DNA was of suitable quality for PCR amplification and downstream sequencing applications. 5 CONCLUSION This rapid DNA extraction protocol demonstrates the ability to isolate DNA from seed tissues even with- out relying on specific chemical reagents typically used for extraction, such as CTAB. By utilizing simple, readily accessible lysis buffer components and efficient physi- cal disruption of cells through freeze-heat cycles. This method provides an alternative approach to extract PCR- suitable DNA without requiring specialized chemicals. While CTAB is considered a staple reagent for high- quality DNA purification, this work shows that effective extraction is still achievable using very basic buffers and lysing techniques. The simplicity and accessibility of the reagents needed could make this rapid protocol easily adoptable, especially in resource-limited settings where procuring chemicals like CTAB may be difficult or cost- prohibitive. The proposed approach would facilitate the molecular investigation of seed lot quality and the char- acterization of germplasm. Acta agriculturae Slovenica, 119/4 – 2023 7 A rapid and efficient DNA extraction method from high oily content seeds: Ricinus communis L.- apt for PCR based assay 5.1 DECLARATIONS 5.1.1 List of abbreviations PVP, polyvinyl pyrrolidone. ITS-rRNA, Internal transcribed spacer of ribosomal RNA encoding gene. 5.1.2 Ethics approval and consent to participate “Not applicable” 5.1.3 Consent for publication “Not applicable” 5.1.4 Availability of data and material All data generated or analyzed during this study are included in this article. 5.1.5 Competing interests “The author declare that she has no competing in- terests” 5.1.6 Funding This study received no funding grant 5.1.7 Authors’ contributions ERSS: generates the idea, performs all experimental work, analyze the data, submit the sequences to the data- base, wrote the manuscript, revised it, and corresponding the publication. 5.1.8 Acknowledgements “I am very gratefully thanking Prof. Soliman M. S. A, Cyto-genetics and Molecular Genetics section, Bot- any and Microbiology department, Faculty of Science, Helwan University, (Egypt) for providing R. communis seeds. ” 6 REFERENCES Aboul-Maaty, N.A.F. and Oraby, H.A.S. (2019). 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