A 3D-printed Sanger sequencing activity addresses student misconceptions

ABSTRACT

The process of Sanger sequencing can be a challenging and unintuitive concept for students to master. In order to improve student learning, we developed a hands-on Sanger sequencing activity using 3D-printed models to incorporate tactile learning. These 3D models and the accompanying activity demonstrate the differences between gene amplification polymerase chain reaction (PCR) and Sanger sequencing, including the purpose and function of dNTPs and ddNTPs, both in terms of building and terminating the chain and in how the DNA sequence is read. After completing the activity, students self-reported high levels of both learning and enjoyment from the activity. Students were also asked to discuss what misconceptions they had prior to this activity that were addressed and provide suggestions for improving this activity. A majority of the misconceptions are related to the function and differences between dNTPs and ddNTPs, with others related to the function of primers, the high-quality region of sequencing, and the purpose of DNA fragment sizes. Overall, student responses indicate that this activity was enjoyable, improved student learning, and addressed specific misconceptions regarding Sanger sequencing. The use of online dice rolling software or additional computational analysis was a common suggestion from students to improve this activity further in future semesters.

INTRODUCTION

Sanger sequencing is a fundamental molecular biology technique that is still used regularly due to its low cost, fast turnaround time, and high accuracy. Instructors in an advanced molecular biology course at our home institution identified Sanger sequencing as a major topic in which student learning could be improved. Sanger sequencing, like many concepts at the molecular level, is primarily taught using images as the prominent external representation (ER) (1, 2). The use of multiple ERs, such as images or models, has been demonstrated to improve student interactions and enhance instruction of complex scientific techniques (3, 4). We developed a hands-on Sanger sequencing activity composed of 3D-printed models to expand our current course materials to better address student misconceptions. While other hands-on activities have been previously developed to improve student understanding of Sanger sequencing using paper clips, candy, and paper (5–8), our activity has advantages due to the details in the 3D model design. The 3D-printed dNTPs can be easily connected via their side arms, are sturdy enough for repetitive use in multiple course sections or semesters, and each dNTP (A, T, C, and G) can be differentiated by touch due to the raised letter on the surface. Previous research has shown that 3D models can help with visualization by breaking down a complex process into discrete components in order to lower the cognitive load (9).

This activity was designed for a small enrollment (≤32 students) molecular biology course for non-major undergraduate and graduate students (400/500 level) (10, 11) but could be adapted for introductory molecular biology or genetics courses for majors. The use of an online interactive polling system (e.g., Poll Everywhere) would facilitate the adaptation of this activity in a large enrollment course by providing class-wide feedback instead of the instructor checking each group’s progress individually. Major course topics include DNA cloning and screening techniques, including Sanger sequencing. Course instructors had repeated anecdotal experiences of student confusion regarding the use of primers in different applications (specifically, gene amplification PCR, PCR screening, or Sanger sequencing) and what the Sanger sequencing chromatograph peaks represent in terms of their molecular reaction products. To improve student learning outcomes (Box 1), we designed a tactile activity for students to complete during the lab period. Before this activity, students are introduced to Sanger sequencing in lecture, including primer design, how the process works, and comparisons to polymerase chain reaction, which was introduced in an earlier lecture. The full activity, including 3D-model print files and student survey, can be found in the Appendix ( Files S1 to S10). There are no safety issues with the plastic models.

Box 1. Learning outcomes.

Exploration and concept invention learning outcomes (pre-work):

• Identify the directionality of the two strands of DNA.

• Identify the correct base pairing of DNA.

• Diagram the action of DNA polymerase.

• Describe the purpose of ddNTPs in Sanger sequencing and how they differ from dNTPs.

• Outline the steps in the process of Sanger sequencing.

• Outline differences between Sanger sequencing and PCR.

Application learning outcomes (model activity):

• Select an appropriate primer to sequence a designated section of DNA sequence.

• Describe how dNTPs and ddNTPs are added during a Sanger sequencing PCR reaction.

• Describe the products of a Sanger sequencing PCR reaction.

Course activities that rely on visual ERs can be inaccessible to blind and visually impaired students (12). To address accessibility concerns, this activity was designed to meet the Universal Design for Learning considerations: 1.2: Support multiple ways to perceive information, 4.2: Optimize access to accessible materials and assistive and accessible technologies and tools, and 7.3: Nurture joy and play (13). With this activity, we aimed to develop an accessible classroom tool that addresses student misconceptions related to Sanger sequencing.

PROCEDURE

Students are provided with a laminated template DNA sequence and choose from three possible sequencing primers by base pairing with the template sequence. Students then roll a 20-sided die; if a 2–19 is rolled, then the appropriate dNTP is added to the chain, and the students continue rolling. If a 1 or 20 is rolled, the students add the appropriate ddNTP and the chain is complete; this represents the typical ratio of 1:10 ddNTPs:dNTPs. 3D-printed ddNTPs lack a 3′ arm and contain a rhinestone signifying fluorescence. The raised letters printed on each dNTP or ddNTP, the arm(s) representing the phosphodiester bond, and the rhinestone representing ddNTP fluorescence can be distinguished by fingertips, making this an activity friendly to visually impaired students. Additionally, the lack of one arm on the 3D-printed ddNTPs reinforces the concept of chain termination, as additional nucleotides cannot be physically added to the chain. After completing this process six times, students order the chains by size to simulate how they would run through capillary gel electrophoresis (Fig. 1). Alongside this activity, students complete an accompanying worksheet, including reflection questions. Students complete the hands-on activity in pairs, but complete the worksheet and survey individually. Students were asked to self-report their enjoyment and learning (10-point Likert scales), as well as to complete open-ended questions on the misconceptions this activity helped with and any further comments or suggestions they had. Student survey data were collected for this activity in Spring 2024 from three course sections totaling 67 students. Access to student data was approved under IRB #27245. After the course had concluded, student data were de-identified and analyzed using GraphPad Prism (14) (Likert scale) and MAXQDA (15) (open-ended). The coding strategy for the free-response answers was developed and implemented according to Braun and Clarke’s six steps of thematic analysis (16). PEB and SHC served as coders. The coders each read the free-response answers and determined codes de novo independently prior to an initial meeting to agree on a coding scheme. The coders had similar initial categories; further discussion led to the clearly defined categories of “dNTPs vs ddNTPs,” “ddNTP probability,” “only ddNTPs are read,” “primer function,” “high quality region,” “fragment sizes,” “Sanger vs PCR,” and “none” for the question on student misconceptions and “liked/enjoyed,” “dice,” “computation,” “primer,” “groups,” “lecture,” “template,” “didn’t help,” and “N/A or didn’t answer” for the question on student suggestions. After the codebook was defined, each coder independently coded the free responses. The coders met one final time to compare their results and reach consensus on any statements for which differences in coding occurred.

Fig 1.

Example of terminated chains created from this activity.

CONCLUSION

Quantitative data from student surveys showed students enjoyed the activity and learned more about Sanger sequencing from participating (Fig. 2). Open-ended responses to the question “What is one specific misconception you had that is now clearer after participating in this activity?” revealed seven concept areas that this activity addressed (Table 1). The most common misconceptions were related to ddNTPs: how they are different from dNTPs, how the probability of ddNTP incorporation works, and how the fluorescence of ddNTPs is read. Additional misconceptions involved the function of primers, the high-quality region of Sanger sequencing reads, and the purpose of the DNA fragment lengths. Notably, only one student discussed a misconception related to comparing Sanger sequencing and PCR. In previous semesters, instructors had noted that students commonly conflate Sanger sequencing and PCR due to their similar use of primers and dNTPs. This activity and the accompanying worksheet were designed to address this misconception among others. The lack of student comments regarding misconceptions related to Sanger sequencing and PCR indicates that either discussions before this activity addressed this misconception or perhaps that it is a lingering misconception that students are not aware of. When asked for additional comments or suggestions, 14 students indicated they liked or enjoyed the activity, 2 students indicated the activity did not help their learning, and 10 students provided specific suggestions for improvements (Table 2). The most common suggestions involved the dice used to simulate ddNTP probabilities; students suggested the use of online dice rolling software to speed up the activity and lower the noise level in the room (Table 3). Alternatively, dice trays or felt pads could be provided to students to dampen the noise of dice rolling. Students also made suggestions involving the use of computational tools and modifications to the DNA template and primer sequences, which were interesting but beyond the scope of the learning goals of the activity for this course. These quantitative and qualitative reflections indicate this hands-on activity was successful as a learning tool and addressed student misconceptions about Sanger sequencing.

Fig 2.

Self-reported student enjoyment (n = 67, average = 7.87, standard deviation = 2.14) and learning (n = 66, average = 7.22, standard deviation = 2.59) of this Sanger sequencing activity on a 1–10 Likert Scale.

TABLE 1.

Survey data collected from students, in which they were asked to discuss a specific misconception this activity cleared up (n = 64)^a

Code	No. of comments	Example student quote
dNTPs vs ddNTPs	15	“This activity cleared up my misconception between ddNTPs and dNTPs. I was unclear the difference between the two and the impact and role each play in Sanger sequencing.”
ddNTP probability	14	“How the ddNTPs add to the sequence. It was really fun to see how long some of the sequences were are how short some of them were. It was just luck of the draw and was a cool way to see how Sanger sequencing works.”
Only ddNTPs are read	10	“I didn't realize the sanger sequencing machine only recognized the last nucleotide with the fluorescence, I thought it recognized the whole sequence up until the last ddNTP.”
Primer function	10	“The orientation where should add the primer.”
High-quality region	7	“I now understand the logic following the lack of quality in signal at the beginning of the sequencing reaction better after participating in this activity.”
Fragment sizes	7	“I originally did not understand how length was important to the sequencing and thought it had to do more with the repetition of sighting ddNTPs.”
Sanger vs PCR	1	“I was still a little shaky upon the a couple of the differences of PCR and Sanger Sequencing methods but thing defintlety cleared up some of those differences and similarities for me.”
None	12	“The process of Sanger sequencing was sufficiently explained in lecture and I am unaware of any misconceptions that were cleared up by this activity.”

^a Student comments could receive more than one code.

TABLE 2.

Survey data collected from students, in which they were asked to provide any additional comments or suggestions for improving the activity (n = 66)^a

Code	No. of comments	Example student quote
Liked/enjoyed	14	“It was a lot of fun and I wish there were more models like this to explain how these genetic processes work!”
Suggestion	10	See Table 3
Didn’t help	2	“This personally didn't help me since the lecture made sense but I can see how this can be beneficial, no suggestions :)”
N/A or didn’t answer	41	“N/A”

^a Student comments could receive more than one code.

TABLE 3.

Specific suggestions for improving the activity collected from student survey data (n = 10)^a

Code	No. of comments	Example student quote
Dice	4	“Die rolling sounds can be a little annoying when many groups are completing this activity.”
Computation	3	“Incorporate some computational aspect involving the statistical distribution of fragment lengths to show why certain sequence regions are higher vs lower quality.”
Primer	2	“We could have multiple primers that work for the sequence just to try out more!”
Groups	1	“Instead of 1 bag per 4 people...1 bag per group of two. Working between 4 people gets things done quicker but it can be more difficult to corrdinate answers and discussions. If 1 person is slower than the rest of the group, it can get confusing and that person can be left behind. This can make asking questions difficult and embarrassing.”
Lecture	1	“Maybe this would be a good activity to do in the class portion rather than the lab. It would be easier to understand along with the presented lecture material.”
Template	1	“Shorter parent sequence. The activity took a lot of space.”

^a Student comments could receive more than one code.

While we believe the use of dice and 3D-printed models enhanced student learning due to their tactile nature (3, 4, 9), these tools could be replaced with less expensive materials, including paper cut-outs and free online dice rolling software. This activity can be adapted to fit the specific needs of students and instructors to improve student learning outcomes related to Sanger sequencing.

Limitations

This student experience with the activity presented here relies on self-reported answers from students, including both quantitative data on their perceived learning gains and qualitative data on misconceptions that this activity addressed. After this activity, students are assessed on Sanger sequencing in this course; however, utilization of pre- and post-concept tests or other rigorous assessment tools to quantify student learning outcomes was outside the scope of this project and not part of our approved Institutional Review Board submission.

ETHICS APPROVAL

This project was completed in compliance with the North Carolina State University Human Subjects Institutional Review Board (IRB), which approved a waiver of informed consent (eIRB submission #27245).

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jmbe.00209-24.

S1. jmbe.00209-24-s0001.pdf.

Activity worksheet, answer key, and assembly instructions.

S2. jmbe.00209-24-s0002.stl.

A dNTP 3D.

S3. jmbe.00209-24-s0003.stl.

A ddNTP 3D.

S4. jmbe.00209-24-s0004.stl.

T dNTP 3D.

S5. jmbe.00209-24-s0005.stl.

T ddNTP 3D.

S6. jmbe.00209-24-s0006.stl.

G dNTP 3D.

S7. jmbe.00209-24-s0007.stl.

G ddNTP 3D.

S8. jmbe.00209-24-s0008.stl.

C dNTP 3D.

S9. jmbe.00209-24-s0009.stl.

C ddNTP 3D.

S10. jmbe.00209-24-s0010.stl.

Primer 1 3D.

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