1 A clicker-based case study that untangles student thinking about the processes in the central dogma – Supporting File 2. 1 Supporting File 2. Clicker questions used in the activity, distribution of student answers, and 2 explanations for the range of student answers. 3 4 Clicker questions are presented below with the correct answers after peer discussion 5 indicated in blue text. The weighted average (WA) of student responses from 8 different 6 classes is indicated in parentheses. Percentages that do not equate to 100 are due to 7 missing responses. Each question is followed by a brief explanation of the observed clicker 8 responses. 9 10 11 Q1: Is difference #5 a possible cause of DMD in Liam?* 12 A. Yes (85%) 13 B. No (14%) 14 15 Difference #5 represents a missense mutation: in Elijah (unaffected) the DNA encodes for 16 GAU (aspartate) and in Liam (affected) the DNA encodes for GAG (glutamate). This 17 question is an exploratory question, and most students answer A: “Yes” because, without 18 more information, this difference is a possible cause of the disease. In the next slide, 19 students will learn that aspartate and glutamate are similar in structure. 20 21 22 Q2: If difference #1 caused DMD, we would predict the mRNA levels in Liam to be 23 ______the mRNA levels in Elijah? 24 A. the same as (8%) 25 B. higher than (12%) 26 C. lower than (65%) 27 D. different in some way from (15%) 28 29 This question is asking students to examine the promoter region of the gene and to predict 30 how this type of mutation would affect the mRNA levels. The correct answer is D, but often 31 students arrive at C, thinking that a mutation will always reduce transcription. This 32 question can be used to discuss effects of mutations in promoter regions in general, and 33 that a mutation does not always have to down regulate a promoter. 34 35 36 Q3: You have now done the experiment and measured the mRNA levels in both Elijah 37 and Liam. If these are your results, would you conclude that difference #1 a likely 38 cause of DMD in Liam? * 39 A. Yes (9%) 40 B. No (90%) 41 42 The students observe that there is no change in the amount of mRNA as a result of the 43 promoter mutation and can therefore conclude this difference is likely not a cause of DMD 44 in Liam. The majority of students answer correctly (B). 45 1 * Percentages that do not equate to 100 are due to students omitting responses to a particular question or selecting an answer that is not part of a given question. 1 A clicker-based case study that untangles student thinking about the processes in the central dogma – Supporting File 2. 1 2 Q4: When DNA polymerase reaches the nucleotides encoding the premature stop 3 codon it will…* 4 A. stop when it reaches the first nucleotide encoding the premature stop codon. (31%) 5 B. stop when it reaches the last nucleotide encoding the premature stop codon. (42%) 6 C. not be affected by this base change and will continue to read through the nucleotide 7 difference. (26%) 8 9 This question is the first of several that address the effect of a premature stop codon on the 10 processes in the central dogma. The majority of students will incorrectly answer that the 11 DNA polymerase will stop prematurely (either A or B). The next slide promotes discussion 12 of the consequences of having DNA polymerase that stopped at every stop codon in the 13 genome. 14 15 16 Q5: When DNA polymerase reaches the nucleotides encoding the premature stop 17 codon it will…* 18 A. stop when it reaches the first nucleotide encoding the premature stop codon. (8%) 19 B. stop when it reaches the last nucleotide encoding or the premature stop codon. 20 (16%) 21 C. not be affected by this base change and will continue to read through the nucleotide 22 difference. (75%) 23 24 After the previous discussion, students vote again on the effect of a stop codon on DNA 25 polymerase. There is typically a shift towards the correct answer (C). 26 27 28 Q6: What do you predict will be the effect of the premature stop codon on mRNA 29 size? It will result in: 30 A. a shorter mRNA in Liam. (49%) 31 B. a longer mRNA in Liam. (5%) 32 C. the same size mRNA in both Liam and Elijah. (46%) 33 34 This question asks the students to predict the outcome of a premature stop codon on 35 mRNA size. Roughly half the students incorrectly choose (A), thinking that a premature 36 stop codon will stop transcription and result in a shorter mRNA. 37

2 1 A clicker-based case study that untangles student thinking about the processes in the central dogma – Supporting File 2. 1 Q7: The above DNA sequence is being transcribed by an RNA polymerase (red 2 square). The premature stop codon mutation in Liam’s DNA is indicated in blue. 3 What will the RNA polymerase do when it reaches the nucleotides encoding the 4 premature stop codon? It will: * 5 A. stop when it reaches the first nucleotide encoding the premature stop codon. (9%) 6 B. stop when it reaches the last nucleotide encoding the premature stop codon. (24%) 7 C. not be affected by this base change and will continue to read through nucleotide 8 difference. (66%) 9 10 This question asks about the effect of a premature stop codon on RNA polymerase. The 11 format used is intentionally similar to that used asking about DNA polymerase (Q4 and 5). 12 Although how DNA polymerases function has already been addressed, many students still 13 answer this question incorrectly and vote that RNA polymerase will stop prematurely 14 (either A or B). 15 16 17 Q8: What do you predict will be the effect of the premature stop codon on protein 18 size? It will: * 19 A. result in a smaller dystrophin protein in Liam. (83%) 20 B. result in a larger dystrophin protein in Liam. (5%) 21 C. result in the same size protein in both Liam and Elijah. (11%) 22 23 This question addresses the impact of a premature stop codon on translation. By this point 24 in the lesson most students have recognized a stop codon will result in a shorter protein 25 and the majority of the students arrive at the correct answer. 26 27 28 Q9: Which of the five nucleotide differences are mutations? * 29 A. All five are mutations. (58%) 30 B. The three in the exons (difference #2, #3, #5) (16%) 31 C. The two in exons that change the amino acid sequence (differences #3, #5) (20%) 32 D. The one in the exon that causes DMD (difference #5) (5%) 33 34 The final clicker question addresses the students’ conceptual difficulty that nucleotide 35 differences are only mutations if they are in a coding region or impact phenotype. While 36 many students correctly answer that all of these nucleotide changes are mutations, a subset 37 still think that mutations are restricted to coding regions (answer B) or must change the 38 amino acid sequence (answer C). This question allows for a discussion on what defines a 39 mutation.

3 * Percentages that do not equate to 100 are due to students omitting responses to a particular question or selecting an answer that is not part of a given question.