We have developed a new DNA extraction experiment for high schools. It uses the concept “Life possesses not only unity, but also diversity” to teach Japanese and American students that various organisms have DNA in their bodies as a common chemical background. In this experiment, students extract DNA from representative organisms of the five kingdoms Monera, Protista, Fungi, Plantae, and Animalia (although the system of classification into five kingdoms has been replaced by three domains in current taxonomy, Japanese students are familiar with this classification system from their earlier education). After practice experiments with high school students and biology teachers were performed, the educational effects were evaluated by questionnaires. The results suggested that the DNA extraction experiments were effective to a certain extent and that, although several points should be improved, this experiment is adequate for practice in high schools.

Introduction

In the Japanese high school science curriculum, the importance of learning about DNA and its functions is central (Takemura & Kurabayashi, 2014). Decline in the scholastic ability of Japanese high school students and university undergraduates during the past few decades has been of great concern to the scientific community and to science educators (Rode, 1995; Katayama et al., 2004; Kobayashi, 2008). It is reported that molecular biological processes such as DNA replication and gene transcription are difficult for Japanese students to learn, as they are for students in other countries (Rode, 1995; Krebs, 1999; Balgopal, 2010; Takemura & Kurabayashi, 2014).

In 2012 a new national curriculum framework, the Course of Study (CS) for secondary schools, was introduced in Japan. Like the U.S. Next Generation Science Standards, the CS emphasizes the importance of teaching students that “Life possesses not only unity, but also diversity” (Ministry of Education, Culture, Sports, Science and Technology of Japan, 2009). The CS guidelines also consider the concept that “Existing organisms share a common origin” important for students' understanding of the two perspectives of unity and diversity. The present study emphasizes the genetic substance, DNA, which every living thing shares as part of its makeup. DNA is one of the famous substances that represent the unity of life; therefore, teaching materials or student experiments about DNA should be among the most appropriate “assistants” for biology teachers, not only under the new Japanese CS, but also in the United States. DNA extraction experiments for secondary school students – in which white supernatants, including DNA, are extracted from human cheek cells and from biological materials such as liver and broccoli – are included in the Japanese lower and upper high school biology curriculum, as they are in other countries (Hearn & Arblaster, 2010; Nishino, 2010; Alozie et al., 2012; Banba et al., 2013).

Purpose of the Study

We developed a new DNA extraction experiment to enable students to learn that various organisms have DNA in their bodies as a common chemical background. In this experiment, students extract DNA from representative organisms of the five kingdoms Monera, Protista, Fungi, Plantae, and Animalia (although the system of classification into five kingdoms has been replaced by three domains in current taxonomy, Japanese students are familiar with this classification system from their earlier education). As starting organisms, we used food materials familiar to Japanese students that could be purchased at low cost. The experiment can easily be adapted for American students by substituting food materials that are familiar to Americans.

Materials & Methods

Selection of Organisms for DNA Extraction

Biological materials representing Animalia that are used frequently for DNA extraction experiments include testes of salmon (Oncorhynchus keta) and liver of chicken (Gallus gallus domesticus); we chose chicken liver because it is readily obtainable from Japanese supermarkets at any time of the year. Frequently used biological materials representing Plantae include broccoli (Brassica oleracea var: italica) and banana (Musa acuminata); we used banana because its texture facilitates the experimental techniques. Biological materials commonly used to represent Fungi include shimeji mushroom (Lyophyllum shimeji) and shiitake mushroom (Lentinula edodes); we used shiitake because the area of the mycelial mat that holds the basidium in this mushroom is regarded as containing more DNA. Biological materials representing Protista include sea tangle (Saccharina japonica) and brown seaweed (Undaria pinnatifida); we used brown seaweed because its texture is softer and students can easily break it up. Biological materials representing Prokaryota include lactic acid bacterium (Lactobacillus sp.) and the bacterium called “natto-kin,” which is used to ferment soybeans to produce the common Japanese food “natto” (Bacillus subtilis var. natto); we chose the latter. All five food materials we selected were purchased at low cost from Japanese supermarkets.

Procedures of DNA Extraction for Organisms in Each Classification

To crush cells and extract DNA, a different method was used for each of the five types of organisms, as described below.

Natto-kin. The natto was mixed gently using a glass rod to avoid crushing the soybeans. The sticky material adhering to the soybeans, which included natto-kin (B. subtilis var. natto), also adhered to the glass rod. After a certain amount of sticky material had accumulated on the rod, it was removed using a spatula or bamboo skewer and put into a glass beaker. The same procedures were repeated until ~2 g of the sticky material was obtained, and then 20 mL of 10% NaCl solution was added to the beaker (Figure 1).

Figure 1.

Preparation of Bacillus subtilis var. natto (from commercially available natto) for DNA extraction. (A) The sticky material adhering to the soybeans was gathered on a glass rod. (B) Sticky material adhering on the glass rod was collected using a spatula and put into a glass beaker. (C) About 2 g of sticky material.

Figure 1.

Preparation of Bacillus subtilis var. natto (from commercially available natto) for DNA extraction. (A) The sticky material adhering to the soybeans was gathered on a glass rod. (B) Sticky material adhering on the glass rod was collected using a spatula and put into a glass beaker. (C) About 2 g of sticky material.

Brown seaweed. About 1 g of dried brown seaweed was (1) immersed in water for 5 minutes, (2) pressed against a paper towel to remove excess water, (3) shredded finely using scissors, (4) put into a mortar with 10 mL of 10% NaCl solution and ground, and (5) put into a glass beaker to which 10 mL of 10% NaCl solution was added (Figure 2).

Figure 2.

Preparation of Undaria pinnatifida for DNA extraction. (A) Commercially available dried brown seaweed was immersed in water. (B) Wet seaweed after immersion. (C) Water was removed by pressing the wet seaweed against a paper towel. (D) The seaweed was shredded using scissors. (E) The shredded seaweed was put into a mortar and ground. (F) The ground seaweed was put into a glass beaker.

Figure 2.

Preparation of Undaria pinnatifida for DNA extraction. (A) Commercially available dried brown seaweed was immersed in water. (B) Wet seaweed after immersion. (C) Water was removed by pressing the wet seaweed against a paper towel. (D) The seaweed was shredded using scissors. (E) The shredded seaweed was put into a mortar and ground. (F) The ground seaweed was put into a glass beaker.

Shiitake. The open “umbrella” portion of a raw shiitake mushroom was halved, shredded finely using scissors, and put into a mortar. The shredded mushroom and 10 mL of 10% NaCl solution were put into a mortar and ground lightly. The ground mushroom was then put into a glass beaker to which 10 mL of 10% NaCl solution was added (Figure 3).

Figure 3.

Preparation of Lentinula edodes for DNA extraction. (A) A shiitake mushroom broken in half. (B) The mushroom was shredded using scissors. (C) The shredded mushroom was put into a mortar and ground.

Figure 3.

Preparation of Lentinula edodes for DNA extraction. (A) A shiitake mushroom broken in half. (B) The mushroom was shredded using scissors. (C) The shredded mushroom was put into a mortar and ground.

Banana. A banana was peeled and ~10 g of banana fruit was put into a glass beaker, then crushed using a spatula, and 20 mL of 10% NaCl solution was added (Figure 4).

Figure 4.

Preparation of Musa acuminata for DNA extraction. (A) About 10 g of banana fruit. (B) The banana was crushed using a spatula. (C) Crushed banana.

Figure 4.

Preparation of Musa acuminata for DNA extraction. (A) About 10 g of banana fruit. (B) The banana was crushed using a spatula. (C) Crushed banana.

Chicken liver. About 10 g of chicken liver of convenient size was put into a bag and frozen. The bag was then placed in a glass beaker, which was immersed for 1 minute in hot water. The slightly thawed liver was cut to appropriate size using a kitchen knife and then put into a mortar and ground. The ground liver was put into a glass beaker to which 20 mL of 10% NaCl solution was added (Figure 5).

Figure 5.

Preparation of liver from Gallus gallus domestica for DNA extraction. (A) Frozen liver was put into a glass beaker, which was immersed in a vessel containing hot water for 1 minute to thaw the liver. (B) Thawed liver was cut to appropriate size using a kitchen knife. (C) Cut liver was placed into a mortar and crushed.

Figure 5.

Preparation of liver from Gallus gallus domestica for DNA extraction. (A) Frozen liver was put into a glass beaker, which was immersed in a vessel containing hot water for 1 minute to thaw the liver. (B) Thawed liver was cut to appropriate size using a kitchen knife. (C) Cut liver was placed into a mortar and crushed.

Common Procedures

Twenty milliliters of 1% sodium dodecyl sulfate (SDS) solution was added to each of the above Preparations (each already containing one of the biological materials and 20 mL of 10% NaCl solution). The contents were then transferred to a glass beaker, stirred by swinging gently, and left to stand for 10 minutes. The solution was then subjected to incubation for 5 minutes with ~50°C hot water and filtered using a tea strainer. About twice the volume of 99.5% ethanol was well cooled, and added to the filtrate after filtration (Figure 6).

Figure 6.

Common procedures. (A) Addition of 20 mL of 1% SDS aqueous solution. (B) Incubation with 50°C hot water (top view). (C) Incubation with 50°C hot water (side view). (D) Filtration using tea strainer. (E) Addition of 99.5% ethanol.

Figure 6.

Common procedures. (A) Addition of 20 mL of 1% SDS aqueous solution. (B) Incubation with 50°C hot water (top view). (C) Incubation with 50°C hot water (side view). (D) Filtration using tea strainer. (E) Addition of 99.5% ethanol.

Students observed that a white supernatant precipitated after a while, and the teacher instructed them to place a glass beaker containing a white supernatant on a diagram of the five-kingdom phylogenetic tree (Figure 7).

Figure 7.

Diagram of the five-kingdom phylogenetic tree, with spaces for photos of the selected representative organisms.

Figure 7.

Diagram of the five-kingdom phylogenetic tree, with spaces for photos of the selected representative organisms.

First Practice Experiment for High School Students

In May 2012 a practice experiment was performed in a Basic Biology class with 30 first-year students at a high school (“A”) in Tochigi Prefecture, Japan. The five-kingdom phylogenetic-tree diagram and the procedures for extracting DNA from each organism were distributed to each student. The experimental techniques were explained carefully, including the roles of SDS, ethanol, and the NaCl aqueous solution. The students were then divided into groups of five, and each member of a group performed DNA extraction on a different organism. Transparent disposable cups were used instead of glass beakers. After the DNA extraction operation was completed, a transparent disposable cup containing the extracted DNA was placed on a diagram of the five-kingdom phylogenetic tree. The students were asked to discuss whether the supernatants from the five species were the same.

The educational effects of this practice experiment were evaluated by a questionnaire distributed both before and after the practice session (Table 1). For questions 1–4, four selections were available: “I think so strongly” (score 4 for statistical analysis), “I think so somewhat” (score 3), “I do not particularly think so” (score 2), and “I do not think so at all” (score 1). Question 5 was a fill-in-the-blank (correct answer, score 1; incorrect answer, score 0). For question 6 (added to the questionnaire only after the practice session), four options were given: “I realized it very much,” “I realized it somewhat,” “I did not realize it particularly,” and “I did not realize it at all.”

Table 1.
Questionnaire for high school students.
Question 1 Do you think all living organisms have DNA? 
Question 2 Do you think all living organisms originated from the same ancestor? 
Question 3 Do you think DNA is a chemical substance? 
Question 4 Do you think living organisms have become diverse while maintaining unity? 
Question 5 Living organisms are widely classified into the following five groups: 
Question 6 Did you realize that the extracted material was DNA? 
Question 1 Do you think all living organisms have DNA? 
Question 2 Do you think all living organisms originated from the same ancestor? 
Question 3 Do you think DNA is a chemical substance? 
Question 4 Do you think living organisms have become diverse while maintaining unity? 
Question 5 Living organisms are widely classified into the following five groups: 
Question 6 Did you realize that the extracted material was DNA? 

Practice Experiment for Biology Teachers

In July 2012 a practice experiment for high school biology teachers, similar to the one for high school students described above, was conducted with 17 people who participated in a workshop held at a university (“B”) in Tokyo. The development and background of the practice experiment was explained before starting. In this case, the practice experiment was designed so that each biology teacher was able to perform the DNA extraction experiments for each of the five classes of organisms. To confirm that this practice experiment can be executed in high school classes and to ascertain its good points and potential improvements, a questionnaire was given only after the practice experiment (Table 2). For question 1, four options were given: “I wish to perform,” “I wish to perform when possible,” “I do not wish to perform particularly,” and “I do not wish to perform.” A descriptive reply was requested for questions 2–4.

Table 2.
Questionnaire for biology teachers.
Question 1 Do you wish to teach the DNA extraction experiments that we performed today in a Basic Biology class at the school where you are teaching? 
Question 2 Please present the reasons why you replied in that way. 
Question 3 With regard to the DNA extraction experiments that we performed today, please write positive points and points of potential improvement. 
Question 4 Please write any of your impressions of this activity. 
Question 1 Do you wish to teach the DNA extraction experiments that we performed today in a Basic Biology class at the school where you are teaching? 
Question 2 Please present the reasons why you replied in that way. 
Question 3 With regard to the DNA extraction experiments that we performed today, please write positive points and points of potential improvement. 
Question 4 Please write any of your impressions of this activity. 

Improved Practice Experiment for High School Students

From May to June 2013 an improved practice experiment was performed (1) in Basic Biology and Advanced Biology classes with 98 second-year students at a high school (“C”) in Tokyo and (2) in an Advanced Biology (Biology II) class with 36 third-year high school students at another high school (“D”) in Tokyo. The practice experiment itself was performed using the same processes described above, except for the following: broccoli was used instead of banana because it was reported that white supernatants extracted from bananas include much more foreign matter than those extracted from broccoli (Banba et al., 2013); shimeji mushroom was used instead of shiitake; 10 g of natto was directly added to the disposable cup, and 20 mL of 10% NaCl solution added, followed by incubation for 5 minutes at room temperature; and after removal of the sedimented soybean fractions, 20 mL of 1% SDS solution was added to the supernatant, including many B. subtilis var. natto.

The educational effects of this improved practice experiment were evaluated using the same questionnaire distributed before and after the practice session (Table 3). For questions 1 and 2, four selections were available: “I think so strongly” (score 4 for statistical analysis), “I think so somewhat” (score 3), “I do not particularly think so” (score 2), and “I do not think so at all” (score 1). Question 3 requested a descriptive reply (correct answer, score 1; incorrect answer, score 0). Questions 1, 2, and 3 in this improved practice experiment (Table 3) were identical to questions 3, 4, and 5 in the previous experiment (Table 1).

Table 3.
Questionnaire for high school students in improved practice experiment.
Question 1 Do you think DNA is a chemical substance? 
Question 2 Do you think living organisms have become diverse while maintaining unity? 
Question 3 Living organisms are widely classified into the following five groups: 
Question 1 Do you think DNA is a chemical substance? 
Question 2 Do you think living organisms have become diverse while maintaining unity? 
Question 3 Living organisms are widely classified into the following five groups: 

Statistical Analysis

The pre- and post-questionnaire scores were compared by paired t-test using Microsoft Excel 2007.

Results

First Practice Experiment for High School Students

When banana and chicken liver were used, all groups were able to extract DNA. For natto-kin, brown seaweed, and shiitake mushroom, few groups succeeded in DNA extraction. However, a teacher encouraged the students of such groups to observe other groups' successful DNA extractions from the five organisms; therefore, all students seemed to recognize that all living things, at least the representative five organisms, possessed DNA.

For questionnaires performed before and after the practice experiment, the replies to questions 1–4 were analyzed. The results showed that the number of students who replied “I think so strongly” increased significantly (Figure 8). The results of the analysis of replies to question 5 revealed that the number of students who answered correctly for all five groups increased after the practice experiment (Figure 9).

Figure 8.

Questionnaire results for questions 1–4 (high school students); 100% indicates 30 replies. Pre: Results of the questionnaire before the practice experiment. Post: Results of the questionnaire after the practice experiment. Numbers indicate the percentages replying “I think so strongly.” Significant differences were as follows: question 1, paired t-test, df = 29, t = −2.41, P < 0.05; question 2, paired t-test, df = 29, t = −3.07, P < 0.01; question 3, paired t-test, df = 29, t = −3.25, P < 0.01; and question 4, paired t-test, df = 29, t = −2.50, P < 0.05.

Figure 8.

Questionnaire results for questions 1–4 (high school students); 100% indicates 30 replies. Pre: Results of the questionnaire before the practice experiment. Post: Results of the questionnaire after the practice experiment. Numbers indicate the percentages replying “I think so strongly.” Significant differences were as follows: question 1, paired t-test, df = 29, t = −2.41, P < 0.05; question 2, paired t-test, df = 29, t = −3.07, P < 0.01; question 3, paired t-test, df = 29, t = −3.25, P < 0.01; and question 4, paired t-test, df = 29, t = −2.50, P < 0.05.

Figure 9.

Questionnaire results for question 5 (high school students); 100% indicates 30 replies. Pre: Results of the questionnaire before the practice experiment. Post: Results of the questionnaire after the practice experiment. Numbers indicate the percentages of correct answers. Significant difference: paired t-test, df = 29, t = −2.41, P < 0.05.

Figure 9.

Questionnaire results for question 5 (high school students); 100% indicates 30 replies. Pre: Results of the questionnaire before the practice experiment. Post: Results of the questionnaire after the practice experiment. Numbers indicate the percentages of correct answers. Significant difference: paired t-test, df = 29, t = −2.41, P < 0.05.

For question 6, “Did you realize that the extracted material was DNA?,” 13% of the students replied that they “realized it very much,” 50% “realized it somewhat,” and 37% “did not realize that, particularly” (Figure 10). Because the percentage of students who replied that they realized it was DNA (somewhat, very much) was 63%, clearly many students realized that the extracted material was DNA.

Figure 10.

Questionnaire results for question 6 (high school students); 100% indicates 30 replies. Numbers indicate the percentages for each answer.

Figure 10.

Questionnaire results for question 6 (high school students); 100% indicates 30 replies. Numbers indicate the percentages for each answer.

Practice Experiment for Biology Teachers

Many biology teachers were able to extract DNA from all five of these types of organisms. For question 1, 59% of the biology teachers replied that they “wanted to do it” and 41% replied that they “wanted to do it if possible” (Figure 11).

Figure 11.

Questionnaire results for question 1 (biology teachers); 100% indicates 17 replies. Numbers indicate the percentages for each answer.

Figure 11.

Questionnaire results for question 1 (biology teachers); 100% indicates 17 replies. Numbers indicate the percentages for each answer.

The results of descriptive questions 2 and 3 of the questionnaire are presented in Table 4. Question 2 related to the reason why respondents stated that they “wanted to do it” or “wanted to do it if possible.” Most respondents believed that students' understanding of the unity of life would be enhanced because DNA extraction is possible from the organisms of all five kingdoms.

Table 4.
Questionnaire for biology teachers: excerpts from replies to question 2.
These contents seem to be well suited for teaching about the commonness of living organisms because they are simple and inexpensive. 
It is interesting that the DNA of all five kingdoms of living organisms can be examined simultaneously. 
It can be performed simply and the unity of the five categories can be understood. 
If well prepared in advance, middle-ranking students of my school can understand and perform the contents. 
I wish to conduct these exercises if materials and instruments are made available and if the number of people is adjusted well. 
It is effective for learning that DNA is found in all living organisms. 
We learned methods and materials that can be implemented in a high school setting. We wish to promote cooperation between businesses and schools. 
It is possible to see and confirm using our hands that DNA is included. 
These contents seem to be well suited for teaching about the commonness of living organisms because they are simple and inexpensive. 
It is interesting that the DNA of all five kingdoms of living organisms can be examined simultaneously. 
It can be performed simply and the unity of the five categories can be understood. 
If well prepared in advance, middle-ranking students of my school can understand and perform the contents. 
I wish to conduct these exercises if materials and instruments are made available and if the number of people is adjusted well. 
It is effective for learning that DNA is found in all living organisms. 
We learned methods and materials that can be implemented in a high school setting. We wish to promote cooperation between businesses and schools. 
It is possible to see and confirm using our hands that DNA is included. 

The results of question 3, regarding the good points of the present experiment, showed that respondents mainly agreed that the possibility of DNA extraction from many familiar organisms, in particular from all five kingdoms, is an excellent learning tool (Table 5). Regarding improvements to the experiment, many respondents cited the method of confirming DNA extraction.

Table 5.
Questionnaire for biology teachers: excerpts from replies to question 3.
Good Points 
Materials that are familiar to the students and which represent the five kingdoms are used. 
We can realize that the same material is obtainable from different living organisms. 
Materials were carefully selected, yield is high, and DNA extraction from all five kingdoms was realized. 
All living organisms, some from each of the five kingdoms, are used for experiments. 
Experiments using SDS, a transparent disposable cup, and scissors might be accepted willingly by students at the school. 
We were able to understand that extraction of DNA from all living organisms is possible. 
Experiments are related to the five kingdoms. They are risk-free and they are simple. 
Familiar cooking ingredients can be used. 
Points Demanding Improvement 
We wish to use an activator with economically good performance for living organisms with poor yield, unless SDS is used. 
Please find a method by which students can notice that this is DNA. 
Functions of each chemical should be explained in the class. Students would not be impressed with white milk-like substances. We suggest that helical geometry by X-ray diffraction (photograph in the illustration) or competition for elucidation of the DNA structure be introduced. 
The issue here is whether the experiment can be promoted skillfully or not. Handling one or two types is no problem. The key is whether living organisms of five types can be handled successfully or not. 
Steps to confirm that this substance is DNA should be sought. Why this experiment is necessary should be shown in the guidance sheet to convince the students to promote the experiment smoothly. If the experiment is performed without knowing the reason, the students would only slightly question “why?” 
If collection from natto and shiitake is as easy as banana and liver, understanding would be enhanced. 
It is doubtful whether it is really DNA or not. 
Students would understand more easily and teachers would be able to teach them more easily if criteria were available showing what materials should be crushed and to what degree. 
Good Points 
Materials that are familiar to the students and which represent the five kingdoms are used. 
We can realize that the same material is obtainable from different living organisms. 
Materials were carefully selected, yield is high, and DNA extraction from all five kingdoms was realized. 
All living organisms, some from each of the five kingdoms, are used for experiments. 
Experiments using SDS, a transparent disposable cup, and scissors might be accepted willingly by students at the school. 
We were able to understand that extraction of DNA from all living organisms is possible. 
Experiments are related to the five kingdoms. They are risk-free and they are simple. 
Familiar cooking ingredients can be used. 
Points Demanding Improvement 
We wish to use an activator with economically good performance for living organisms with poor yield, unless SDS is used. 
Please find a method by which students can notice that this is DNA. 
Functions of each chemical should be explained in the class. Students would not be impressed with white milk-like substances. We suggest that helical geometry by X-ray diffraction (photograph in the illustration) or competition for elucidation of the DNA structure be introduced. 
The issue here is whether the experiment can be promoted skillfully or not. Handling one or two types is no problem. The key is whether living organisms of five types can be handled successfully or not. 
Steps to confirm that this substance is DNA should be sought. Why this experiment is necessary should be shown in the guidance sheet to convince the students to promote the experiment smoothly. If the experiment is performed without knowing the reason, the students would only slightly question “why?” 
If collection from natto and shiitake is as easy as banana and liver, understanding would be enhanced. 
It is doubtful whether it is really DNA or not. 
Students would understand more easily and teachers would be able to teach them more easily if criteria were available showing what materials should be crushed and to what degree. 

Improved Practice Experiment for High School Students

Few groups had succeeded in DNA extraction from all five organisms during the previous practice experiment. However, a change in the organisms used and improvement of the protocol for DNA extraction from natto resulted in many groups succeeding in DNA extraction from organisms of all five kingdoms.

Replies to the pre- and post-questionnaires (Table 3) were analyzed. The results obtained after the practice experiment showed that the number of students who replied “I think so strongly” to the question “Do you think DNA is a chemical substance?” increased, with significant differences in both “C” and “D” high schools (Figure 12, question 1). The numbers who replied “I think so strongly” to the question “Do you think organisms have become diverse while maintaining unity” increased in both high schools; however, in high school “C” there was no significant difference (P = 0.29; Figure 12, question 2). Those who answered correctly for all five groups increased, with significant differences (Figure 12, question 3) in both “C” and “D” high schools.

Figure 12.

Questionnaire results for questions 1–3; 100% indicates 98 (left; “C” high school) and 36 (right; “D” high school) replies. Pre: Results of the questionnaire before the practice experiment. Post: Results of the questionnaire after the practice experiment. Numbers indicate the percentages replying “I think so strongly” (questions 1 and 2) or “Correct” (question 3). Significant differences were as follows. “C” high school: question 1, paired t-test, df = 190, t = −2.84, P < 0.01; question 2, paired t-test, df = 185, t = −1.07, P = 0.29; question 3, paired t-test, df = 178, t = −10.7, P < 0.001. “D” high school: question 1, paired t-test, df = 72, t = −2.73, P < 0.01; question 2, paired t-test, df = 72, t = −4.20, P < 0.001; question 3, paired t-test, df = 57, t = −11.8, P < 0.001.

Figure 12.

Questionnaire results for questions 1–3; 100% indicates 98 (left; “C” high school) and 36 (right; “D” high school) replies. Pre: Results of the questionnaire before the practice experiment. Post: Results of the questionnaire after the practice experiment. Numbers indicate the percentages replying “I think so strongly” (questions 1 and 2) or “Correct” (question 3). Significant differences were as follows. “C” high school: question 1, paired t-test, df = 190, t = −2.84, P < 0.01; question 2, paired t-test, df = 185, t = −1.07, P = 0.29; question 3, paired t-test, df = 178, t = −10.7, P < 0.001. “D” high school: question 1, paired t-test, df = 72, t = −2.73, P < 0.01; question 2, paired t-test, df = 72, t = −4.20, P < 0.001; question 3, paired t-test, df = 57, t = −11.8, P < 0.001.

These results suggest that by using the DNA extraction exercise developed in this study, high school students learned to recognize that all living things possess DNA, that DNA is a chemical substance, and that organisms in the five kingdoms are diverse but are unified by DNA.

Discussion

The practice experiment by high school students and the results of the questionnaire suggest that the DNA extraction experiments developed in this study are effective, to a certain extent, for teaching the fact that “Life is diverse, but it also has unity.” The practical experiment and the results of questionnaires targeting biology teachers suggest that, although several points should be improved (Table 5), this experiment is adequate for practice in high schools.

None of the DNA extraction experiments developed to date have specifically examined representative organisms of five kingdoms. In the experiment developed for this study, DNA extraction is performed from five representative organisms, and a disposable cup (or beaker) containing the extracted DNA is placed on the five-kingdom phylogenetic tree (Figure 13), thus enhancing students' understanding of biodiversity as represented by the five-kingdom classification system and of the unity represented by the substance called “DNA.” One of our goals was for students to infer that DNA is a chemical substance representing the unity of life, and our results suggest that many students indeed realized this (Figures 8 and 12). A critical element is the use of organisms representing the five kingdoms, but the selection of these is adaptable. For example, natto is a traditional Japanese food that may be difficult to obtain in the United States, but American teachers could extract DNA from other popular bacteria, such as Lactobacillus species.

Figure 13.

Beakers containing extracted DNA, placed on the diagram of the five-kingdom phylogenetic tree in the last procedure to connect unity and diversity.

Figure 13.

Beakers containing extracted DNA, placed on the diagram of the five-kingdom phylogenetic tree in the last procedure to connect unity and diversity.

Several problems remain unresolved. In the first practical experiment for high school students, not all groups were successful in extracting DNA from each of the representative organisms from the five kingdoms. Although large quantities of DNA were extractable from bananas and chicken liver, many groups failed to extract DNA from brown seaweed, natto-kin, or shiitake mushroom. The questionnaire responses from biology teachers revealed their opinion that collection of DNA was difficult with some materials and that they preferred DNA collection to be simplified to the degree that it was for banana or liver samples (Table 5). A method that allows effective extraction of DNA from such biological materials should be developed. Our improved protocol addressed this problem by substituting broccoli and shimeji mushroom and by introducing several modifications for DNA extraction from natto-kin. However, DNA extraction from brown seaweed has yet to be improved, and several groups could not extract DNA from this organism.

More time is needed for preparing the biological materials of each of the five types, along with the necessary instruments and reagents, before the students can successfully perform the experiment. The need for such Preparations to be done simply and within a limited time is an important issue to be resolved in the future, along with improvement of DNA extraction efficiency and reinvestigation of biological materials.

In the current biology curriculum in Japanese high schools, as well as in American schools, biology teachers must address the three-domain system of classification rather than the five-kingdom system. In the future, we must mention the three-domain system and use representative organisms according to this classification or other appropriate ones, such as representatives of Amoebozoa, Opisthokonta, Excavata, and Archaeplastida.

DNA confirmation experiments to show students that the white supernatants being extracted are DNA (Nishino, 2010; Banba et al., 2013) should be used in the DNA extraction experiment presented here. However, this aspect of the experiment was not examined because of the limited lesson time in high school and the cumbersome operations for DNA extraction from organisms of the five types. In the present study, 63% of students at high school “A” replied that they realized that the extracted materials were DNA (Figure 10), but this may have been a rote response to instruction: “The teacher tells us the extracted material is DNA; therefore, it is DNA.” To ensure that students really understand that “Organisms are diverse, but they have unity,” confirmation experiments showing that the extracted material actually is DNA are extremely important. Indeed, the responses to questionnaires by biology teachers revealed similar opinions: “Please find a method by which students can notice that this is DNA”; “Steps to confirm that this substance is DNA should be sought”; and “It is doubtful whether it is really DNA or not” (Table 5). The diphenylamine method (Banba et al., 2013) in particular is considered simple and feasible, and we intend to implement it in DNA extraction experiments developed in future studies.

The handouts for biology teacher are available at the Takemura Laboratory website (http://www.takemura-lab.com). This work was supported by a Grant-in-Aid for Scientific Research (B) (nos. 22330253 and 25285251 to M.T.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Drs. Naoko Endo (Graduate School of Mathematics and Science Education, Tokyo University of Science), Chieko Uchiyama (Tokyo Metropolitan Ueno High School), and Miyuki Muramatsu (Seibi-Gakuen High School) for useful comments and discussions; students of “A,” “C,” and “D” high schools; and biology teachers for practice experiments and useful comments (shown in Tables 4 and 5). We also thank Mrs. Tomoko Kazama, Rei Kato, Mario Kurabayashi, Sayaka Kobori, Hiroki Kikuchi, Kiyoshi Sumita, and Yuta Yamamoto of our laboratory for their useful comments. We affirm that no conflict of interest exists in connection with this work.

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