Isolation of HTT gene via PCR assay and gel electrophoresis

Linda Chen, Kealan Millies-Lucke, Hayden Stoub, Mellissa Ungkuldee

Trailer: https://www.youtube.com/watch?v=ZYKhEURRSaE&feature=youtu.be

Full Video: https://www.youtube.com/watch?v=KyDO0TecWGM

Abstract

Huntington’s Disease (HD) is a neurodegenerative disease caused by a mutation in the HTT gene. This mutation affects the production of the protein huntingtin (HT) which results in psychological, behavioral, and psychomotor symptoms. The purpose of this study was to isolate and clone the HTT gene using polymerase chain reaction (PCR) methods adapted from prior studies (MacDonald et al., 1993). The amplified genes were analyzed via gel electrophoresis. We hypothesized that by utilizing a set of published primers (MacDonald 1993) along with theory from published literature (Jama, 2013), we could isolate a 35-155 bp segment of an individual’s HTT gene because of the denaturing, annealing, and extension of primers in the targeted gene region. Results were found to contain non-specific binding, which is consistent with previously published papers and believed to be caused by the CAG nucleotide sequence of the primer binding to multiple sections of the target DNA (Jama, 2013)  Huntington’s Disease has many detrimental effects, including motor degeneration. Typical HD speech pathology results from loss of motor control of the muscles involved in speech (Murray, 2001). This study sought to examine the psycho-social impacts of Huntington’s Disease speech pathology by using a clear, athletic mouthguard to emulate this lingual impairment. We hypothesized that this customized mouthguard would increase the difficulty of understanding subjects in everyday speech because of its design (Olsen et al, 1997). Speech efficacy was analyzed with a speech test using The Harvard Sentences (IEEE, 1969) adapted from Nicholas (1993). A statistically significant difference was found between the time it took to communicate an idea with a mouthguard worn compared to without. We also hypothesized that the aforementioned decreased coherence would created increased stress in subjects, because of the disconnect between normal cognitive speech function and impaired speech mechanics (Walker, 2007). Stress was measured by monitoring blood pressure, pulse, and a perceived stress scale. No statistically significant difference was found between average blood pressure and pulse changes during retail interaction when speaking with a mouthguard compared to speaking naturally. However, a statistically significant difference was found in stress levels comparing weeks with a speech impediment with weeks without.

Discussion

Experiment Summary

The mutation chiefly responsible for Huntington’s Disease is within the polyglutamine (poly-q) repeat section, coded for by a CAG repeat. Due to the mutation’s nature, HD may manifest with partial (35-40 poly-q repeats) or full (41+ repeats) penetrance (Walker, 2007). The purpose of our experiment was to isolate the portion of the HTT gene within the human genome that includes the CAG repeat sequence. We hypothesized that by using a published PCR methodology (MacDonald, 1993) along with PCR methods utilizing non-specific binding derived from published literature to fit our specific needs (Jama et al, 2013), we could isolate a segment of an individual’s HTT gene. Non-specific binding was sought after, as this would allow for the targeting of the various possible CAG repeats that an individual might have.

In addition to primer design, we brought a humanistic perspective to our laboratory experiments. Motor control loss of the muscles involved in speech occurs frequently in HD patients (Murray, 2001). The main purpose of the study was to observe and quantitatively measure stress caused by the disconnect between the cognitive and and motor centers responsible for speech. We hypothesized that the daily use of a customized mouthguard would increase the difficulty of understanding subjects in everyday speech because of its design (Nozaki et al, 2013). We also hypothesized that decreased coherence would create increased stress in subjects, because of the disconnect between normal cognitive speech function and impaired speech mechanics (Folkow, 1982).

Ultimate Findings

PCR was first performed on lambdaphage genome in order to isolate and clone the Rz gene as our experiment’s positive control. The predicted band length was 380 bp, and gel electrophoresis successfully indicated bands of length 384 bp. This demonstrated the validity of techniques and materials used in the PCR, creating a positive control for the PCR process. Primers used by Jama et al. produced PCR samples ranging from 39-159 bp in length. As predicted, this elongated PCR sample showed less variation in electrophoresis band display through successful trials due to the further minimized percentage of wild-type CAG repeat variation in respect to the entire PCR sample (Jama et al, 2013). We expected to obtain bands 35-155 bp long, and obtained bands of 26 bp. This fell into our margin of error, supporting our hypothesis of non-specific binding.

In our psycho-social experiment, a mouthguard used in this study was chosen to best emulate the linguistic deficit observed in HD cases. By inhibiting mobility and normal speech mechanics, this mouthguard simulated HD speech impairment while still remaining hidden within the user’s mouth. Keeping the physical appearance of the subject unchanged was crucial, as this ensured that the subject was seen as having a genuine speech impediment, rather than as a model. Prior research has indicated that speech impairments typical of neurodegenerative diseases such as HD result in decreased syntactical complexity in everyday speech (Murray, 2001). We originally predicted that there would be a statistically significant difference in speech efficacy when wearing a mouthguard rather than not (Nozaki et al, 2013). We found a statistically significant difference in the length of time it took to successfully say a repeatable sentence with mouthguard usage in comparison to natural speaking. However, we did not find a statistically significant difference in the number of ties a patient had to repeat themselves while wearing a mouthguard than without. This may have been impacted by the fact that the listeners became more comfortable with adjusting their listening to the impediment. Additionally, listeners may have also used context clues to successfully repeat the sentence back to the reader, which may create a difference in what they actually heard in comparison to what they believed the sentence to be.

In the Psycho-Social Impacts of Huntington’s Disease Speech Pathology Experiment, we chose to measure stress through blood pressure and pulse modulation because cardiovascular effects are both common and quantifiable. We predicted that significantly higher blood pressure and higher pulse rate would be indicative of the amount of stress brought on by a situation where clear communication is critical to have, yet unavailable (Eliasson et al, 1983). Our data showed no statistically significant difference between heart and pulse rate when comparing the experimental weeks with the control weeks. A possible source of error may include the blood pressure monitoring wrist cuff, which may not have had accurate and consistent measurements.

Finally, a Perceived Stress Scale (Cohen, 1983) was used at the end of each week to determine whether or not speech inhibition had an impact on overall stress levels. We predicted that the average stress scale score would be higher for the experimental weeks than the control week, due to the stressful nature of speech inhibition (Nicholas et al, 1993). Statistical tests showed a significant difference between the scores of the control and experimental weeks, supporting our hypothesis (Figure 7). Other factors out of our control may have contributed to increased stress levels, including academics that possibly increased in vigor each week as the university semester neared an end.

Future Directions

Sequencing of the proposed non-specific binding products will offer confirmation that there was in fact multiple binding sites to the CAG repeat. A confluence of autosomal dominance and late onset symptoms creates difficult family planning situations. This is further exacerbated by the fact that current diagnostics are only useful after the presentation of advanced symptoms such as CT scans to detect for loss of brain mass. Further studies are being done to determine the feasibility of using PCR with non-specific and chimeric binding as a more cost-efficient diagnostic tool for earlier detection.

While we found that wearing a mouthguard had a negative impact on speech capability, we were unable to successfully determine manifested stress through the use of physiological factors. The use of a more precise and consistent research-grade blood pressure cuff may give better results. Furthermore, to find even more conclusive data, using a different listener during each of the experimental weeks for the reading of the Harvard Sentences may have given better results. It is possible that listeners became adaptive towards the speech impediment, which may have impacted the number of times the listener asked the reader to repeat themselves. Listeners may have also used context clues in repeating the sentence. To prevent this issue, a future test may utilize non-obvious syntax and randomized words.

Finally, obtaining perceived stress scores would ideally only be impacted by the use of a mouthguard. A future experiment might try to compare mouthguard-wearers with non-mouthguard wearers during the same week. This would establish a baseline during the same week of a semester, potentially lessening the impact that academic rigor could have on stress.

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Figure 4: Amplified sections of the HTT gene using PCR and gel electrophoresis with gDNA negative control. Both a 100bp ladder and 10 Kb+ ladder were used for reference, the 100bp ladder was chosen for the semi-log plot for its clarity. Lane 2 contained product from previously successful control PCR reaction solution for amplification of the Rz lambdaphage gene. Lane 3 contained PCR reaction solutions created as a control. Lanes 4-5 contained PCR reaction solutions of  isolated CAG repeat section fragments, presenting a “smear” pattern leading up to very strong bands. Primers used included Fprimer (5’-ATGAAGGCCTTCGAGT- CCCTCAAGTCCTTC-3’) and Rprimer (5’-AAACTCACGGTCGGTGCAGCGGCTCCT- CAG-3’). 50µl reaction solutions consisting of 1.65µl human DNA, 5µl 10X PCR buffer, 2µl Fprimer (100µM), 2µl Rprimer (100µM), and 200µM dNTP were run through PCR at 95°C for 45 s, 58.25°C (lane 4) or 60.0°C (lane 5) for 45 s, and 72°C for 45 s. An additional 3 min. at 95°C for denaturing before cycles and 3 min. at 72°C for final extension were added to ensure optimal product amplification. Lane 6 consisted of the aforementioned PCR reaction solution without genomic DNA template run at annealing temperature 60°C as a control.