Rachel Chung, Year 2 Research
Conducting genetic experiments for high school or science fair projects can be challenging because of high costs as well as lack of equipment and training. In practice, studying genetics in the lab can pose barriers to students. Therefore, the purpose of this study is to determine whether a genetic experiment kit developed by miniPCR can mitigate some of these limitations and serve as an appropriate high-school-level science project. In this study, a miniPCR Sleep Lab kit was used to test whether it can identify the PER3 gene. The PER 3 gene controls circadian rhythm, or whether a person is a “morning lark” or “night owl”. Consequently, it was determined that the miniPCR Sleep Lab did not serve as a robust model for a high-school level PCR kit.
Biological clocks exist in all living systems, including humans. Composed of proteins that interact with cells in different parts of the human body, the majority of the tissue and organ contains biological clocks that regulate various functions in the body system, especially the cycle of circadian rhythms. Transcription-translation negative feedback loops (Andreani et al. 2015) of multiple genes are involved in the functioning of the biological clocks. By binding to their promoters, two transcription factors BMAL1 and CLOCK(clock gene) form heterodimers and activate the transcription stage of Cryptochrome (Cry) and Period (Per) genes (Menet, Jerome S et al. 2014). When CRY and PER proteins accumulate in the cytoplasm gradually, proteins including these and other then form complexes which translocate to the nucleus and cause the BMAL1–CLOCK mediated expression of the Cry and Per genes to shut down (Reddy et al. 2021). The repetition of this transcription/translation negative feedback loop every 24 hours inside the cells consequently forms the biological clock in cells of the human body. The Period 3 circadian clock gene (Per3), the focus of these feedback loops, has variations among humans, thus indicating that the gene is polymorphic. Studies(Archer SN, et al. 2003) have found that a variable number tandem repeat (VNTR) in Per3 is the factor that determines how the biological clock sets in different people. A VNTR is a sequence that repeats multiple times within a gene. The variation of the sequence is either repetition of 4 times or 5 times in one allele (Hida, A., Kitamura, S., Kadotani, H. et al.).
In a different study (Archer, Simon N, et al.), 1590 participants including 707 males and 883 females were asked to complete the Horne–Östberg (HÖ) questionnaire (Morningness-Eveningness Questionnaire) for diurnal preference and provide a DNA sample. From the DNA sample, the frequencies of the PER3 4- and 5-repeat alleles were examined in separate age groups (18–29, 30–39, 40–49 and 50+years of age). Consequently, the 4-repeat allele was significantly more frequent in evening types, and the 5-repeat allele more frequent in morning types. Yet, only the results of the age group of 18-29 was found to be significant, thus the study suggests that diurnal preference in young people is more closely associated with this polymorphism than it is in other age groups.
When students conduct genetic experiments, they face budgetary constraints and may not have access to advanced technology and training. Due to its ease of use and low cost, miniPCR Sleep Lab™ – Morning lark or night owl kits are commonly used in student genetics projects. Previously, another student aimed to determine whether there is a correlation between the presence of the PER gene and trait anxiety. Initially, this project was aimed at expanding on these results, however, due to troubleshooting issues the goal of the project became to evaluate the miniPCR kit altogether. Thus, this project aimed to determine whether the miniPCR kit serves as a good model for high school science genetics projects/highschool PCR kit, in terms of result replicability and trouble-shooting.
Materials and Methods
In the first and the second trial, only one participant’s sample was used. In the third trial, five participants were selected using convenience sampling.
Following through the student guide of Sleep Lab™ – Morning lark or night owl (2019) from the miniPCR official website, first, 50 μl of X-Tract DNA Extraction Buffer was added to each 200μl tube using 20~200μl micropipette. After that, participants were asked to swab the inside of their cheeks with the end of the toothpicks gently for around 20 seconds and dip those toothpicks into the tube. Then, those tubes marked with their initials were put into the miniPCR machine in Heat Block mode for 10 minutes at 95 ̊C. The next step was to add the 3μl of heated student DNA extract sample, 5μl of 5X EZ PCR Master Mix, and 20μl of Sleep Lab Primers in one tube, then to run each student’s tubes in miniPCR mini8 thermal cycler using the miniPCR software. The PCR protocol parameters include the following, 30 cycles repeated: 30 seconds of initial denaturation, 20 seconds of denaturation, 20 seconds of annealing, and 20 seconds of extension.
As the thermal cycler is running for 60 minutes, 2.0% agarose gels were prepared using the GelGreen® Agarose Tabs™ from miniPCR bio™. For one gel, 20mL of water was mixed together, then put in the microwave for 60 seconds until the solution became transparent. The gel was then poured into the gel casting trays with combs and cooled for 10-15 minutes. After the
gel was solidified, it was placed in the blueGel™ electrophoresis system and covered with 40mL of 1X TBE Buffer. One lane was always loaded with 10μl 100bp DNA Ladder, and other lanes were loaded with 14 μl PCR product from student PCR samples, one lane per each sample. Once it was ready to run, the lid was put on and the power turned on for approximately 20 minutes. At last, the results were observed and recorded.
Figure 1. The first trial of Gel electrophoresis results for PER3 genotypes
Figure 2. The second trial of Gel electrophoresis results for PER3 genotypes with two DNA samples
Figure 3. The third trial of Gel electrophoresis results for PER3 genotypes of five participants
In the first trial, although it is merely possible to observe the allele sequence of the DNA sample placed on the left of the 100bp DNA ladder, the sequence has spread out further than the DNA ladder (Figure 1.). In the second trial, four samples are displaying their sequence, two of them showing ⅘-repeat and other two of them showing 4-repeat (Figure 2.). In the third trial, each sequence of the five samples looks the same, yet the DNA ladder is not stretched enough compared to the samples’ sequence (Figure 3.).
This study used miniPCR Sleep Lab™ to determine whether it’s possible to identify the Per3 gene in order to determine whether it serves as an adequate model for high school students to conduct science genetic projects with a low budget. In the first trial, the DNA extract example extended further down than the DNA ladder (Fig. 1), indicating that it was too light to have spread out too far, thus showing that there was not enough DNA extracted. In the second trial, two different methods were used to extract DNA samples: spitting, and swabbing from the inside of the cheek. Each method had two samples, and although it was extracted from a single participant, each set of samples of different methods displayed a different sequence. Two samples from the spitting method showed 4-repeat, and the other two samples retrieved from swabbing inside of the cheek showed ⅘-repeat (Fig. 2). In the third trial, all samples from five different participants displayed their sequence in a noticeable shade, however, the samples have extended further down than the DNA ladder. In other words, although 20 minutes had passed (followed the protocol according to the miniPCR student guide), the DNA ladder had not fully extended. Therefore, it is hard to determine which sequence the samples displayed.
The first and the third trial results (Fig.1 and Fig.3) were hard to determine which gene sequence the DNA samples displayed, and the second trial result (Fig.2) displayed a possible sequence for the gene, yet it is still inaccurate as samples from the same participant yielded different sequences. Some limitations that have affected this study include small sample size and a limited amount of reagents. About 6 participants in the study retrieved DNA samples, as the whole process from the DNA extraction, and running PCR to the gel electrophoresis was very time-consuming. In addition, due to a limited amount of the high-cost reagents, only a few trials were conducted in the study. As multiple attempts at troubleshooting were made, the availability of reagents made it difficult to conduct more trials.
No similar studies were done in this area, making this study more challenging. The results have demonstrated how difficult it can be to identify a Per3 gene with the miniPCR Sleep Lab™ kit, although the guide was carefully followed. At last, this study introduces suggestions for students who utilize miniPCR technology for their high school science projects. First, in the DNA extraction process, participants must swab the inside of their cheeks for a considerable amount of time to retrieve the DNA sample that will expand adequately in the gel electrophoresis process. Without enough DNA samples extracted, it is easy for the sample to extend longer than the DNA ladder in the gel electrophoresis step, in which the gene sequence cannot be read. Furthermore, the proper use of a micropipette serves a great significance in the entirety of the experiment. Genetic experiments deal with small volumes of reagents, thus when mixing and injecting the amount of reagents and samples using a micropipette, keeping the volume concise according to the miniPCR guide is fundamental in every process. In the final step, the gel electrophoresis process takes longer than 20 minutes for a DNA ladder to fully expand, and it should be comparable to the expected results diagram in the guide. These are the suggestions that were not mentioned in the miniPCR guide. Future research could be done to make this experiment less challenging for high school students. It could also expand on how to make the PCR and Gel electrophoresis technology more accessible for high school educational purposes, as only a limited number of students get to try these technologies prior to college.
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MiniPCR. miniPCR Sleep Lab™ – Morning lark or night owl. August 2019. MiniPCR. Student guide.
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Archer SN, et al. “A length polymorphism in the circadian clock gene Per3 is linked to delayed sleep phase syndrome and extreme diurnal preference.” Sleep. 2003 Jun 15;26(4):413-5.
Andreani, Tomas S et al. “Genetics of Circadian Rhythms.” Sleep medicine clinics vol. 10,4 (2015): 413-21. doi:10.1016/j.jsmc.2015.08.007
Hida, A., Kitamura, S., Kadotani, H. et al. Lack of association between PER3 variable number tandem repeat and circadian rhythm sleep–wake disorders. Hum Genome Var 5, 17 (2018). https://doi.org/10.1038/s41439-018-0017-7
Menet, Jerome S et al. “CLOCK:BMAL1 is a pioneer-like transcription factor.” Genes & development vol. 28,1 (2014): 8-13. doi:10.1101/gad.228536.113
Reddy, Sujana, et al. “Physiology, Circadian Rhythm.” StatPearls, StatPearls Publishing, 2021. PubMed, http://www.ncbi.nlm.nih.gov/books/NBK519507/.
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