Cloning is a molecular biology technique that makes many identical copies of a gene. A gene is inserted into a circular piece of DNA called a plasmid in a typical cloning experiment. The plasmid is then introduced into bacteria, and the bacteria are selected for their ability to express the gene and produce protein.
1. Lack of Experience
In high school, students typically have only a few lab courses, and many are not exposed to laboratory research. This is the time to introduce them to the concept of scientific inquiry and real-world problems. Cloning a gene gives students the opportunity to be involved in a project that ultimately produces an identifiable end product, something they can write up in their lab book and display in class.
The GAPDH cloning exercise also introduces students to multiple wet lab procedures, including DNA isolation, PCR, gel electrophoresis, ligation, and heat shock transformation. It also requires students to be active participants in their own group’s decisions about upcoming steps, such as how much plasmid to transfer. In addition to these hands-on activities, the exercise provides opportunities for critical thinking through examining BLAST results and making judgment calls about sequence ambiguity and sequence quality.
This cloning and sequencing exercise is well suited for a wide range of undergraduate and early graduate-level molecular biology courses and advanced-level biotechnology classes at the high school or community college level. It can be modified to include a variety of sequence analysis options, including a BLAST search against the genomes of other plant species for comparison of the cloned gene with its relative positions in other organisms.
Finally, a great benefit of using plants is that instructors do not need to get institutional animal care and use committee approval to extract DNA from them. Plants also provide a convenient source of genetic material, as they are readily available from local gardens, plant nurseries, florists, and grocery stores. This allows students to conduct their cloning and sequencing outside of scheduled lab periods, which may be especially helpful for those students with scheduling conflicts.
After completing the cloning and sequencing exercise, students report that they better understand molecular techniques and have increased confidence in their ability to perform these tasks in future experiments. They have also become more aware of the importance of bioinformatics to the cloning process. The instructor has also observed that students are more accurate and efficient in pipetting, PCR preparations, and gel loading.
2. Lack of Equipment
Cloning genes is a complex subject that can overwhelm students. The subject matter requires students to use a wide range of skills, including pipetting, gel electrophoresis, PCR, and plasmid isolation. These experiments can be time-consuming and require students to work in groups. This type of learning is difficult for students who are not motivated to learn these skills in a traditional lecture format.
Another challenge is that the cloning process can be expensive. Students will need access to a DNA sequencing laboratory and an appropriate PCR instrument. Some schools have a DNA sequencer that students can use, but many do not. The costs of these instruments can be prohibitive for high school instructors who do not have funding to purchase new equipment.
Cloning can also be complicated because of the difficulty in creating a genetically identical strain of an organism that can reproduce. Additionally, animal cells and embryos are very fragile and must be handled carefully to prevent contamination or mutations. In addition, some animals are extremely rare or endangered, making it challenging to find the right animal to clone.
While it has been possible to clone animals, scientists have had trouble bringing a cloned animal back to life. Moreover, cloning animals can be very costly and takes a great deal of time and effort.
Despite these challenges, molecular cloning is an essential topic in biotechnology. In order to fully understand the topic, it is important for students to see how the process works and how each step fits together. Molecular cloning is a vast and complex field that can be overwhelming for students, especially those who do not have experience with molecular biology or genetics. For these reasons, 3D virtual laboratory simulations such as Labster can be helpful in addressing the challenge of teaching molecular cloning to high school students.
3. Poor Lab Management
Students can also have trouble with the wet-lab steps of DNA cloning, especially those that involve handling the nucleic acid intercalating stain ethidium bromide. Several alternatives to ethidium bromide, such as SYBR(r) safe DNA gel stain, are available. Students also have to prepare competent cells, perform PCR, analyze gel electrophoresis, purify their Gateway(r) PCR products, ligate, heat shock transform, and isolate their entry clone plasmid DNA. They must then run one or more diagnostic restriction enzyme digestions to verify their plasmid and set up a sequencing reaction.
Moreover, many students have difficulty distinguishing between vectors and plasmids. Plasmids are extra-chromosomal components found in bacterial cells that can transport foreign DNA molecules into host cells. Conversely, vectors are specialized molecules that deliver DNA molecules into host cells for expression.
Cloning can be a daunting concept for high school students because it can involve a long list of procedures and a lot of details that can be hard to remember. To help them, students can use Labster’s virtual laboratory simulation to practice molecular cloning. This 3D immersive experience provides a visual alternative to wet lab experiments that might be difficult for students to understand through text and images. The lab simulation allows students to practice important molecular concepts such as PCR and cloning without the need for an expensive microscope. Labster’s virtual laboratory also helps students develop critical thinking skills by requiring them to answer questions that challenge their understanding of key molecular concepts, such as how to calculate PCR and ligation volume, interpret gel electrophoresis results, and examine chromatograms from DNA sequencers.
4. Lack of Time
In molecular biology, cloning usually means copying something. While many students think that cloning involves bringing Dolly the sheep back to life, it is much more common for scientists to clone genes and other small pieces of DNA.
Cloning at the molecular level is complicated. The subject matter is highly advanced and often includes terms that are unfamiliar to students, such as plasmids and vectors, sequences, and PCR. Students who are unfamiliar with these concepts could find that the subject matter becomes overwhelming and that their interest in the subject quickly dissipates.
A 6- to 8-wk project-based exercise on cloning and sequencing a gene of interest, such as the plant Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is an excellent way to engage students in critical thinking while learning about the process. The exercise provides numerous opportunities to practice skills in the wet lab, including DNA isolation, PCR, gel electrophoresis, and the preparation of competent cells. It also requires students to perform multiple bioinformatics analyses, such as evaluating sequence quality, sequence editing, Basic Local Alignment Search Tool searches, assembling a contiguous sequence, and identifying introns and exons.
The GAPDH cloning exercise is a highly engaging and meaningful activity that combines wet lab and bioinformatics activities. It also integrates student-centered learning and assessment techniques. For example, students were given a pretest on the first day of the exercise that included 12 multiple-choice questions on topics related to gene cloning and sequencing (Supplemental Material 2, Questions 1-12). At the end of the exercise, students took a posttest that included these same questions and self-reported their confidence levels on each question (1 was lowest, 5 was highest). These pre-, mid-, and final tests showed that students learned a great deal about gene cloning and sequence analysis.