Abstract

Using commonly available materials, this tool allows students to extract DNA, exploring DNA chemistry and the principles of experimental design and execution. We take a “Choose Your Own Adventure” approach encouraging students to explore the protocol and vary individual steps. Students learn the science behind each step of extraction, how that science can allow us to identify and understand certain aspects of the structure of DNA, and how modifying experimental steps can change the observed results. The lesson is intended for an undergraduate setting, but we include adaptations to allow delivery of this lesson to a variety of ages from preschool through adult science events. The manuscript is in English, but we have included supporting materials in Anishinaabemowin, French, Spanish, Urdu, Arabic, Japanese, Mandarin, Hindi, Twi, and English, so that more learners can access these materials in their first language. We have included a supplemental figure showing the simplified structure of DNA using a color scheme that is effective with those with typical sight and colorblindness. We have also linked a video demonstration of the extraction that is available in both French and English and with closed captioning. Inclusion of materials in multiple languages and formats makes the material more user-friendly, allowing its direct inclusion in non-English speaking classrooms, and allows learners to understand that science is not limited to the “universal” scientific language and can be conducted in any language of choice.

Primary Image: This image highlights the basic steps of the extraction process, showing the experimental setup, the DNA precipitation, the product and variation observed amongst different group members.

Citation

Levesque DC, Wallis AL, Daypuk J, Petahtegoose J, Slobodian M, Sutherland-Hutchings AK, Black I, Vélez JM, Abood A, Wahbeh MH, Cejas RB, Cisneros AF, McNeil L, Konno K, McGregor L, Farooqi B, Bautista C, Rajpurohit S, Garg D, Zhu J, Yang G, Arthur S, Merritt TJS. 2023. An Interactive Protocol for In-Classroom DNA Extraction. CourseSource 10. https://doi.org/10.24918/cs.2023.37

Introduction

The central, and almost universal, role of deoxyribonucleic acid, DNA, as the informational molecule of life makes its extraction a great engagement tool in the classroom (and the community). Most students will know what DNA is, but many will have never seen the molecule for themselves. The tool described here is a standardized method for teachers to use to extract DNA and explore the properties of the molecule by modifying the protocol. We include suggestions for alterations in the protocol to allow students to use a “choose your own adventure” model to explore DNA properties and experimental design.

History of DNA Extractions

Our understanding of DNA essentially begins in 1869 with Swiss physician, Friedrich Miescher’s study of the chemical composition of cells (1). Miescher discovered a new molecule unlike anything previously recorded that differed from proteins and lipids. He gave this mysterious molecule the name nuclein since it was located within the nucleus of the cell. Interestingly this linguistic naming root is conserved in the current appellation of deoxyribonucleic acid. Miescher’s first successful crude extraction of DNA was performed with leucocytes (white blood cells) which he collected from bandages (2). At the time, the scientific community did not fully appreciate this amazing discovery. Many protocols have stemmed from this original one, evolving through time to incorporate modern innovations including centrifuges and quantification methods. Simple extraction methods, such as the one outlined in this experiment, include simply salting out the DNA and other methods of DNA precipitation (3).

DNA Structure and Function

The double helix of the DNA molecule is an icon of science and perhaps the most widely used and recognized symbol of modern biology and genetics. Two anti-parallel sugar-phosphate chains linked by phosphodiester covalent bonds make up the backbone of the helix. Each negatively charged phosphate group is attached to a deoxyribose sugar ring that also has a single nitrogenous base covalently linked to it. DNA contains a four-letter alphabet of these nitrogen bases: adenine (A), thymine (T), guanine (G), cytosine (C), and the information in DNA is encoded in a specific sequence of these bases. The two strands of the DNA molecule are held together by hydrogen bonds between nitrogen bases. This bonding, called “pairing,” between bases is specific: Gs pair only with Cs, and Ts pair only with As. This specificity allows either strand to serve as a template for DNA replication. The information contained in DNA is the basis of biological similarities and differences, coding for proteins, and regulating proper cell function including cell division and replication.

Biological Samples and Genome Size

In this experiment, we are isolating the genome, essentially all the DNA in an organism. Many students will understand the concept of a gene and that most genes are made of DNA. This experiment isolates total DNA in the sample and a review of DNA and genome content may be helpful. Remember that most of the DNA in a genome is not actually gene coding sequence. In part because of all the non-coding DNA, genome size varies tremendously across eukaryotes. Genome size also varies because of differences in the sets of chromosomes. Many eukaryotes are diploid, having pairs of homologous chromosomes. Other eukaryotes, polyploids, have multiple chromosome-sets which can lead to very large genome sizes (some diploid also have very large genomes). Domesticated plants are often polyploid as a result of hybridization and the large number of chromosomes seems to be related to faster growth and larger fruit size (4). Wild strawberries, for example, are diploid, but some commercial varieties, often used in DNA extractions like the one described here, are octoploid (5). This impressive ploidy may play a role in the commercial characteristics of fruits, such as size, fertility, flowering periods, taste, and certainly has a great impact on the total genome size (6, 7). The large genome size, and resulting large amount of DNA in the fruit, means that commercial strawberries are a particularly effective biological sample for the DNA extractions described here. This soft fleshy fruit is easy to pulverize and carries the natural enzymes pectinase and cellulase that help break down cell walls. Similarly, commercial bananas, another common choice for these DNA extractions, is triploid and has a relatively large genome (8).

Experimental Design

Experimental design is a crucial element in scientific investigation. Generating your own ideas and using them to frame questions that can be answered is the foundation of any experiment and a skill that can be taught and practiced. Allowing students to design and execute an experiment with variables of their choosing is a great method to use as this stimulates creativity and develops useful skills for the workplace like communication and organization. In this lesson, the “Choose Your Own Adventure” format permits students to exercise these abilities by asking questions around the effects on extraction yield, and even the purity, of modifications to the reagents used or the steps followed. To aid students in organizing their experiment, we created a flowchart (Supporting File S1) detailing the experimental steps and areas to mark down the modifications made to the original protocol. The flowchart and an experimental protocol have been translated into ten different languages to allow more students to practice science in the language they are most comfortable speaking.

Recommended Procedure

Pre-Class Preparation

Prior to the experiment day, students should have discussed and selected the variables of interests, enabling them to design their experiment. Multiple modifications can be made to the original protocol. Instructors can give students free reign or oversee to ensure things like controls and replication. The experiment can also be set up so that students work in groups or individually. The instructor should encourage students to formulate a hypothesis on the expected result from the altered protocol. We created a table of some common modifications and scientific explanations for the possible outcome to aid the instructor in assessing student’s comprehension of the topics discussed (Supporting File S4). In addition, we have created video demonstrations (in French and English, with captions) of the experiment that detail the procedure, it may be useful for students to watch the video prior to get an understanding of DNA chemistry and the steps to be performed.

Experiment Day

The experiment is easily performed step by step. If the group goes through each step together, the instructor can give a running commentary to students on the role of each reagent in the extraction and how this role reflects the structure and function of DNA.

1. Set up

The instructor can choose to prepare stations with allocated materials prior or allow students to gather the materials needed. In our experience, the latter helps students remember the experimental steps.

2. Sample homogenization

Students place approximately 30 g of sample in a plastic bag, expelling the air, and crush their sample until there are no discernable chunks of sample left. This mechanical disruption breaks up the solid structure of the cell walls of the sample and is the first step in releasing the DNA from the nucleus. This is one of the lengthiest steps and serves as a good time to engage students in experimental theory and DNA biology. Once the sample is thoroughly mashed, students add an equal mass of tap water about 30 mL, and a pinch of salt (1 g), and gently mix.

3. Sample filtration

Next, the students slowly pour the mixture on top of the filter and allow the homogenization liquid, containing the cells and DNA, to filter through the paper and into the collection cup (Figure 1A). Through experience, cheap, thin filters work best, allowing faster, but still effective, filtration—we are just removing large solids in this step. Depending on the age group with which this experiment is performed, students may speed the filtration by squeezing the liquid through the filter. To do so, cautiously remove the rubber band and gather the filter edges to create a pouch, then gently squeeze the filter into the cup to speed up this process. While this added step does speed the process and improve homogenate recovery, there is a chance the filter bursts. If a filter does burst and solids fall into the collection cup, the instructor can provide the student with an additional filter to repeat this step. This step is often omitted because of the risk of bursting a filter, but we have found that the risk adds a little excitement to the experiment.

4. Disruption of cell and nuclear membrane

Students then add a few drops of dish washing liquid detergent (0.1 mL) to their solution and gently mix. Detergent contains surfactants that possess a hydrophobic tail which has similar chemical properties as the phospholipid bilayer membrane in a cell. This similar structure allows surfactants to easily integrate themselves into the membrane, inducing mechanical strain, and disrupting the membrane. This disruption of lipids helps solubilize the cellular and nuclear membranes in an aqueous solution, releasing the chromatin from the nucleus (9). Because DNA is now released from the nucleus, the long strands are vulnerable to shearing from vigorous mixing. Sheared DNA will not precipitate, or at least aggregate, as efficiently. The negatively charged strands of DNA would be expected to repel each other and prevent precipitation, but the positive salt ions should coat and neutralize the negative charge of the phosphate backbone of the DNA allowing the DNA strands to coalesce. In our experience, the best method to avoid overmixing and creating bubbles is to move the cup in a circular motion on the countertop. Stop mixing as soon as students no longer see the dish detergent in the bottom of the cup.

5. Breakdown proteins

If proteinase is added, this is the time to swirl in this reagent. A few drops will suffice, around 0.1 mL. This enzyme will break down proteins by splitting the peptide bonds that link amino acid residues. Theoretically, a purer DNA sample should be attained as the histones from the chromatin and other cellular proteins should be digested.

6. DNA precipitation

At this stage, students should tilt the cup and carefully pour in a layer of alcohol approximately equal to the water volume (30 mL), lab grade ethanol is suitable, however commercial over-proofed alcohol and even isopropanol (available as rubbing alcohol) will work for this protocol. Pouring along the side of the cup forms two distinct liquid layers (Figure 1B). DNA is soluble in water, however the addition of ethanol to this mixture causes the DNA to precipitate from solution forming the long threads at the interphase of the two liquids. The precipitation is driven by the difference in the dielectric properties of water and alcohol, with the alcohol having a lower dielectric constant than water. This difference diminishes the relative strength of repulsion between the sugar phosphate groups that make up the helical DNA strands allowing the strands to aggregate and precipitate. The precipitation is facilitated by the Na²⁺ ions from the salt masking the negative charge of the DNA phosphate groups (10). Chilling the alcohol enhances aggregation and can improve extraction results. Gently place the cup on a countertop and allow the DNA to precipitate at the interface (Figure 1C). This step starts quickly, but precipitation can continue for a few minutes. When the DNA is visible as long mucus-like strands, allow students to make observations about the precipitation at this stage. The DNA can also be spun around the end of a wooden stick and removed from the cup (Figure 1D). The rough surface of a wooden stir stick works better than plastic stirrers. Students can now note their observations on the quantity of DNA extracted, the efficiency to pick-up the sample, whether there was consistency in all experimental steps, as well as ways to modify their experiment to obtain different relevant results.

Choose Your Own Adventure Format

Workings of the Choose Your Own Adventure Format

The DNA extraction described above is a great introductory tool to engage the public in science and it can be expanded to explore DNA biology and experimental design more completely. A “Choose Your Own Adventure” format encourages students to modify the protocol to their liking, formulate a hypothesis based on these changes, and propose logical reasonings for their observations. This format allows students to challenge the experimental protocol, to design an experiment tailored to their interests, and to determine if every step is crucial to obtain a DNA sample or improve DNA recovery. Later, the results can all be pooled across the entire class and analyzed in a group discussion. Team discussions allow students to develop their communication and cooperative skills to interact and encourage multiple perspectives and ideas to foster a stressless and open learning environment. The format of these discussions also helps engage students in the topic and become an active participant in their learning (11).

Possible Experiment Ideas

Modifications to this protocol are only limited by the imagination. Below are some ideas to inspire and potentially further investigate.

Altering the order of the steps may have an impact on the outcome of the protocol. For example, adding the detergent to the solution too early lowers total yield and the ability to “pick up” the DNA with a stir stick. The effect is possibly because the detergent is forced to interact with the complex homogenate rather than being focused on nuclear membrane, leading to a lower yield, and shearing of the DNA if it is released before filtration (12).

A change in the biological sample used, such as pineapple, may be of higher purity than the standard strawberry extraction due to the high content of natural protease found in pineapple. These enzymes can lead to the breakdown of the histones and other cellular proteins. Students can test this hypothesis by extracting DNA from both fruits, with replicates, and measuring the purity of the sample using the spectrophotometric analysis described in the adaptation section below (13).

Omitting a step can lead to interesting visual observations. Omitting the salt lowers yield, likely through loss of the shielding of the negative charge of the DNA phosphate groups by the sodium ions making the DNA strands repel each other (14).

Changing how a step is preformed can alter the results of the experiment, for example, the method used to add the ethanol can lead to changes in the amount of DNA precipitated. If the pour is gentle, there is less effective mixing between the two solutions, hence some DNA will remain shielded by water and won’t precipitate as readily. Meanwhile, if the pour is turbulent, mixing is more effective and will allow the ethanol to interact with more DNA and will in turn favor interactions between sodium ions and precipitate (15). This change can be observed in Figure 1.

Another avenue that can be explored in this experiment is to compare extraction yields from approximately equal volumes of wild and commercial fruit and note difference in yields between both species. Typically, domesticates species have altered genome size, as this can increase or reveal desired traits. Higher fruit yield, fruit size, changes in flowering period, have all been linked to genomic changes, including differences in ploidy, in some domesticated crops including strawberries, bananas, blueberries and watermelons (16–23). Including this kind of comparison in the lesson can help students understand complex concepts that involve uses of DNA extraction (genetic engineering), therefore associating this knowledge to real-world issues. We have created a table (Supporting File S5) with the most common domesticated/wild crops used in this experiment to help students choose a biological sample and formulate their hypothesis.

Inclusive Teaching Tools

Visual Demonstration

As a supplement to this paper, we have created a detailed video demonstration of the protocol to help students and instructors alike in visualizing and following along with the protocol. The links to the video, in either French or English, are here. Both versions have subtitles.

Translated Student Resources

A flowchart and summary protocol to help students organize and plan the experiment and follow each step are included in Supporting Files S1 and S2. We have translated the flowchart in ten different languages, and the protocol in eight, because we think that it is important to engage students in the language they are most comfortable. The flow chart is included in Anishinaabemowin, French, Spanish, Japanese, Urdu, Arabic, Mandarin, Hindi, Twi, and English. The protocol is included in French, Spanish, Japanese, Arabic, Mandarin, Hindi, Twi, and English. The protocol was not translated into Anishinaabemowin or Urdu, because some technical language made the translation problematic. This inclusion directly opens this material to roughly 2.7 billion people whose first language is not English. This supporting file will allow a wider range of audiences to conduct this experiment directly, without the need for independent translations, making it more accessible as a teaching resource for instructors and students alike.

Colour Sensitive Figure

We have also included a figure of the basic structure of DNA (Supporting File S3) to help students formulate their hypotheses with the “Choose Your Own Adventure” format. This figure was intentionally made with a color scheme suitable for typical sight and to accommodate most colorblindness. The choice of colour scheme allows a wider audience to benefit from this visual aid.

Teaching Discussion

Experimental Observations

In our test group of undergraduate students, the open group learning environment of this lesson promoted discussion and the participants actively exchanged qualitative observations. Instructors can build on such spontaneous conversations to challenge students to explain their observations and results in light of what the students have learned about DNA and the cell. In our experience, the success or failure of the extraction seemed to be more a function of the individual doing the extraction than changes in the protocol. This observation was termed The Slobodian Effect after Mitchell Slobodian, a graduate student who assisted in the lesson with a noted inability to successfully extract DNA (Figure 1C). It was unclear whether the effect was an issue of sample size or technique, but it led to an interesting discussion of both. These kinds of spontaneous discussions are exactly what the engaging nature of this experiment fosters, and the students finished the lab with a much better understanding of experimental design, and DNA biology, because of them.

Suggestions for Improvement and Adaptations

The experiment works well with non-specialist audiences and is not limited to a classroom setting. The experiment is engaging and straightforward to execute and gives students the opportunity to see DNA with the naked eye, taking the molecule out of the textbook and into their real-world experiences. The simplicity and robustness of the lessons makes a great tool to engage a wide variety of audiences, where the background knowledge can be scaled back accordingly.

To scale this experiment for a younger audience, the instructor can focus on promoting participant interest in science and experimentation. In the introduction, the instructor can highlight the almost universal use of DNA as the genetic material across species. In the methods, the instructor can focus on the standard protocol and prepare all materials, for example the sample filters, to accommodate shorter attention spans and less physical dexterity. Instructors can opt to use an approach we like to call “learner language,” a strategy to simplify some concepts and engage students. For example, saying “I learned that…” or “I found … really interesting” to begin introducing a new topic. Using this kind of language makes the instructor seem more enthusiastic about the science and will in turn help foster a positive learning environment where the audience feel more open and comfortable with the experiment. We have used this approach with participants as young as preschool age.

To scale this experiment to an older audience, the instructor can focus on reinforcing concepts around the properties of DNA and explore modifications of the standard protocol. In the introduction, the instructor can elaborate on the physicochemical properties of DNA and the purpose of each reagent in the protocol. To aid instructors in describing DNA, we have created a diagram of the simplified structure of DNA. The diagram, in both French and English, and color coded to be accessible to those with typical vision as well as individuals who are colorblind (Supporting File S3).

Instructors can also scale up the experiment for more advanced students by expanding on the basic protocol to explore the extracted DNA samples. Two potential expansions use the absorbance of ultraviolet light by DNA to explore the extraction and to compare genomes.

Instructors can have students explore the quality and potentially compare the variation of the standard DNA extraction protocol, by quantifying the relative concentrations of DNA and protein in their samples. Chromatin is primarily made up of protein and DNA and the ratio of their concentrations tells us something about the quality of the DNA extraction. We can quantify each using a spectrophotometer. The aromatic rings of the nitrogenous bases absorb energy at 260nm. The aromatic amino acids of proteins, tryptophan, phenylalanine, and tyrosine, absorb energy at 280nm. The ratio of absorbances can be used to explore the quality of the extracted samples. Values between 1.6 and 1.8 are considered of good quality for DNA, lower ratios indicate that more proteins have been retained in the DNA extractions (21).

As an example, we conducted our standard protocol and two variations and compared the DNA recovered with a sample recovered using a commercially available kit (Qiagen kit; DNeasy Blood and Tissue Kit, 69504) (Supporting File S6) We collected the DNA strands that precipitated out of solution, resuspended the DNA recovered from each protocol in water and quantified the absorbance of each sample at both 260 nm and at 280 nm and calculated their ratio. The commercial kit gave the highest purity extraction while the standard protocol and its variants gave similar values, with the complete protocol performing only marginally better in terms of purity. The complete protocol did, however, give the highest yield of the three variations, as roughly indicated by the absolute 260nm and 280nm values. Overall, the experiment shows that the standard procedure is effective at extracting large amounts of DNA and that variations in the protocol do influence DNA recovery.

Instructors can also expand the experiment to include comparison of different genomes. Genomes differ by many features, including size and content. One large-scale method to quantify these differences is by DNA melting temperature which can be measured through an expansion of the above spectrophotometric assay. DNA is typically a double stranded molecule, but it is possible to obtain single stranded DNA by cleaving the hydrogen bonds pairing the nitrogen bases. Melting temperature reflects genome content (24, 25) and students can use the measurement to explore differences between samples, for example between sample types. Separation can be accomplished by gently heating the DNA samples and measuring the absorbance of UV light at 260nm at a series of temperatures following established protocols (e.g., the protocol published in the Springer Protocol Handbook series). Instructors can use these measurements as a gateway to conversations about diversity in genetics and the mechanisms that led to these phenotypes. Many factors affect the melting temperature of a sample, for example the length of the molecule and the composition of the DNA recovered. These are suggestions of topics to discuss with the group and their relevance to why they may influence the melting temperature.

This DNA extraction protocol can also be adapted to engage older audiences in science, genetics, and DNA. In the introduction, the instructor can focus on explaining core concepts, like the universality of DNA, in concise, simple, and engaging language. In this way, adult and preschool audiences are not dissimilar. In the methods, the instructor can highlight that DNA can be extracted with materials that everyone might have on hand, over proofed vodka or rum, dish detergent, salt and contact lens solution (which contains protease). We have had success with this approach in “Nerd Nite” style events, extracting DNA from reagents literally found in a bar.

Online Adaptation

This experiment can be easily adapted to either in-person, online, or a hybrid delivery. The pandemic crisis has clearly shown the need for, and some advantages in, long-term online teaching options. This experiment is designed such that it can be easily conducted remotely with video conferencing platforms by students at home using common household items that can be purchased at any convenience or grocery store. With our test group, each student was tasked to take a picture of their extraction and share with the class. This allowed students to assess variations in experimental results across the classroom and aided discussing their findings amongst themselves.

Supporting Materials

• S1. Interactive DNA Extraction – Flowchart. A schematic of the extraction experiment, included in Anishinaabemowin, French, Spanish, Japanese, Urdu, Arabic, Mandarin, Hindi, Twi, and English.

• S2. Interactive DNA Extraction – Extraction Protocol. A written description of the steps in the DNA extraction, included in French, Spanish, Japanese, Arabic, Mandarin, Hindi, Twi, and English.

• S3. Interactive DNA Extraction – DNA Structure Diagram. A figure of DNA structure, included in French and English, and accessible to participants that are affected by typical colorblindness.

• S4. Interactive DNA Extraction – Expected Results Summary. A table outlining common modifications to the protocol and the scientific reasoning to the expected result.

• S5. Interactive DNA Extraction – Genome Size and Ploidy. A table with a generalization of genome size and ploidy potential biological samples that can be used in the protocol.

• S6. Interactive DNA Extraction – Absorbance Result Example. A table of absorbance values and the calculated ratio to assess the purity of the extraction from modified protocols.

Acknowledgments

We thank the students in Laurentian University winter 2021 term course, CHMI3217 Biochemistry of Nucleic Acids, for their participation in the experiment.

The authors of this paper also acknowledge that the majority of this paper was written at Laurentian University, which is located within the territory of the Robinson-Huron Treaty of 1850, on the traditional lands of the Atikameksheng Anishnawbek, and in close proximity to Wahnapitae First Nation. The student flowchart included in Supporting File S1 is translated into Anishinaabemowin.

References

1. Dahm R. 2005. Friedrich Miescher and the discovery of DNA. Dev Biol 278:274–288. doi:10.1016/j.ydbio.2004.11.028.
2. Dairawan M, Shetty PJ. 2020. The evolution of DNA extraction methods. Am J Biomed Sci Res 8:39–45. doi:10.34297/AJBSR.2020.08.001234.
3. Tan SC, Yiap BC. 2009. DNA, RNA, and protein extraction: The past and the present. BioMed Res Int 2009:e574398. doi:10.1155/2009/574398.
4. Blommaert J. 2020. Genome size evolution: Towards new model systems for old questions. Proc R Soc B Biol Sci 287:20201441. doi:10.1098/rspb.2020.1441.
5. Folta KM, Barbey CR. 2019. The strawberry genome: A complicated past and promising future. Hortic Res 6:97. doi:10.1038/s41438-019-0181-z.
6. Yildiz M. 2013. Plant responses at different ploidy levels, p 363–385. In Silva-Opps M (ed), Current progress in biological research. IntechOpen, Rijeka, Croatia. doi:10.5772/55785.
7. Vander Kloet SP. 1988. The genus Vaccinium in North America. Agriculture Canada, Ottawa, Canada.
8. Bredeson JV, Lyons JB, Prochnik SE, Wu GA, Ha CM, Edsinger-Gonzales E, Grimwood J, Schmutz J, Rabbi IY, Egesi C, Nauluvula P, Lebot V, Ndunguru J, Mkamilo G, Bart RS, Setter TL, Gleadow RM, Kulakow P, Ferguson ME, Rounsley S, Rokhsar DS. 2016. Sequencing wild and cultivated cassava and related species reveals extensive interspecific hybridization and genetic diversity. Nat Biotechnol 34:562–570. doi:10.1038/nbt.3535.
9. Lichtenberg D, Ahyayauch H, Goñi FM. 2013. The mechanism of detergent solubilization of lipid bilayers. Biophys J 105:289–299. doi:10.1016/j.bpj.2013.06.007.
10. He S, Cao B, Yi Y, Huang S, Chen X, Luo S, Mou X, 5Guo T, Wang Y, Wang Y, Yang G. 2022. DNA precipitation revisited: A quantitative analysis. Nano Sel 3:617–626. doi:10.1002/nano.202100152.
11. Ofstad W, Brunner LJ. 2013. Team-based learning in pharmacy education. Am J Pharm Educ 77:70. doi:10.5688/ajpe77470.
12. Dias RS, Magno LM, Valente AJM, Das D, Das PK, Maiti S, Miguel MG, Lindman B. 2008. Interaction between DNA and cationic surfactants: effect of DNA conformation and surfactant headgroup. J Phys Chem B 112:14446–14452. doi:10.1021/jp8027935.
13. Rizzi G, Dufva M, Hansen MF. 2017. Two-dimensional salt and temperature DNA denaturation analysis using a magnetoresistive sensor. Lab Chip 17:2256–2263. doi:10.1039/C7LC00485K.
14. Sweeney PJ, Walker JM. 1993. Proteinase K (EC 3.4. 21.14). p. 305–311. In Burrell MM (ed), Enzymes of molecular biology. Humana Press, Totowa, NJ. doi:10.1385/0-89603-234-5:305.
15. Kadoch B, Bos WJT, Schneider K. 2020. Efficiency of laminar and turbulent mixing in wall-bounded flows. Phys Rev E 101:043104. doi:10.1103/PhysRevE.101.043104.
16. Costich DE, Ortiz R, Meagher TR, Bruederle LP, Vorsa N. 1993. Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry. Theor Appl Genet 86:1001–1006. doi:10.1007/BF00211053.
17. Guo S, Zhang J, Sun H, Salse J, Lucas WJ, Zhang H, Zheng Y, Mao L, Ren Y, Wang Z, Min J, Guo X, Murat F, Ham B-K, Zhang Z, Gao S, Huang M, Xu Y, Zhong S, Bombarely A, Mueller LA, Zhao H, He H, Zhang Y, Zhang Z, Huang S, Tan T, Pang E, Lin K, Hu Q, Kuang H, Ni P, Wang B, Liu J, Kou Q, Hou W, Zou X, Jiang J, Gong G, Klee K, Schoof H, Huang Y, Hu X, Dong S, Liang D, Wang J, Wu K, Xia Y, Zhao X, Zheng Z, Xing M, Liang X, Huang B, Lv T, Wang J, Yin Y, Yi H, Li R, Wu M, Levi A, Zhang X, Giovannoni JJ, Wang J, Li Y, Fei Z, Xu Y. 2013. The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions. Nat Genet 45:51–58. doi:10.1038/ng.2470.
18. Sakhanokho HF, Rinehart TA, Stringer SJ, Islam-Faridi MN, Pounders CT. 2018. Variation in nuclear DNA content and chromosome numbers in blueberry. Sci Hortic 233:108–113. doi:10.1016/j.scienta.2018.01.031.
19. Zhang N, Bao Y, Xie Z, Huang X, Sun Y, Feng G, Zeng H, Ren J, Li Y, Xiong J, Chen W, Yan C, Tang M. 2019. Efficient characterization of tetraploid watermelon. Plants 8:419. doi:10.3390/plants8100419.
20. Wang Y, Nie F, Shahid MQ, Baloch FS. 2020. Molecular footprints of selection effects and whole genome duplication (WGD) events in three blueberry species: detected by transcriptome dataset. BMC Plant Biol 20:250. doi:10.1186/s12870-020-02461-w.
21. Tennessen JA, Govindarajulu R, Ashman T-L, Liston A. 2014. Evolutionary origins and dynamics of octoploid strawberry subgenomes revealed by dense targeted capture linkage maps. Genome Biol Evol 6:3295–3313. doi:10.1093/gbe/evu261.
22. Shulaev V, Sargent DJ, Crowhurst RN, Mockler TC, Folkerts O, Delcher AL, Jaiswal P, Mockaitis K, Liston A, Mane SP, Burns P, Davis TM, Slovin JP, Bassil N, Hellens RP, Evans C, Harkins T, Kodira C, Desany B, Crasta OR, Jensen RV, Allan AC, Michael TP, Setubal JC, Celton J-M, Rees DJG, Williams KP, Holt SH, Rojas JJR, Chatterjee M, Liu B, Silva H, Meisel L, Adato A, Filichkin SA, Troggio M, Viola R, Ashman T-L, Wang H, Dharmawardhana P, Elser J, Raja R, Priest HD, Bryant DW, Fox SE, Givan SA, Wilhelm LJ, Naithani S, Christoffels A, Salama DY, Carter J, Girona EL, Zdepski A, Wang W, Kerstetter RA, Schwab W, Korban SS, Davik J, Monfort A, Denoyes-Rothan B, Arus P, Mittler R, Flinn B, Aharoni A, Bennetzen JL, Salzberg SL, Dickerman AW, Velasco R, Borodovsky M, Veilleux RE, Folta KM. 2011. The genome of woodland strawberry (Fragaria vesca). Nat Genet 43:109–116. doi:10.1038/ng.740.
23. Edger PP, Poorten TJ, VanBuren R, Hardigan MA, Colle M, McKain MR, Smith RD, Teresi SJ, Nelson ADL, Wai CM, Alger EI, Bird KA, Yocca AE, Pumplin N, Ou S, Ben-Zvi G, Brodt A, Baruch K, Swale T, Shiue L, Acharya CB, Cole GS, Mower JP, Childs KL, Jiang N, Lyons E, Freeling M, Puzey JR, Knapp SJ. 2019. Origin and evolution of the octoploid strawberry genome. Nat Genet 51:541–547. doi:10.1038/s41588-019-0356-4.
24. Lucena-Aguilar G, Sánchez-López AM, Barberán-Aceituno C, Carrillo-Ávila JA, López-Guerrero JA, Aguilar-Quesada R. 2016. DNA source selection for downstream applications based on DNA quality indicators analysis. Biopreserv Biobank 14:264–270. doi:10.1089/bio.2015.0064.
25. Winder L, Phillips C, Richards N, Ochoa-Corona F, Hardwick S, Vink CJ, Goldson S. 2011. Evaluation of DNA melting analysis as a tool for species identification. Methods Ecol Evol 2:312–320. doi:10.1111/j.2041-210X.2010.00079.x.