A PCR Assay for the HTT Gene in Homo sapiens and a Possible Daphnia pulex Homolog with Gel Electrophoresis

By: A47461396, A47937464, A48336446, & A46397765

LB 145 Cell and Organismal Biology

Title page written by: A47461396

Revised by: A46397795

Finalized by: A47937464

Abstract

Written by: A47461396

Revised by: A46397795

Finalized by: A47937464

The HTT gene is known as the cause of Huntington’s Disease (HD), a disease characterized by movement, cognitive, and psychological disorders (Walker, 2007). An HD positive individual will have upwards of 36 CAG repeats on their HTT gene. Using a derived PCR method and two HTT gene primers (Andrew et al., 1994), 100bp ladder, on a wild-type HTT gene, CAG repeats will be evaluated and expected to be at a minimum of 130 bp and a maximum of 205 bp. We hypothesize that the application of Andrew’s two-primer method, which targets the CAG region of the HTT gene, will support successful amplification and isolation of the gene through proper annealing and binding of the two primers (Andrew et al., 1993). Also, the possible presence of a Daphnia pulex HTT gene will be investigated using our established PCR method, primers (Andrew et al., 1994), and primers from a PCR procedure applied to the homologous HTT gene of the Drosophila melanogaster (Jackson et al., 1998). We hypothesize that both primer sets will yield similar gel electrophoresis results because the Drosophila melanogaster DNA is considered a homolog for 75% of human diseases (Pandey et al., 2011). We predict that PCR analysis of the Daphnia pulex HTT gene will result in larger base pair lengths to the D. melanogaster results from Jackson (1998) because of the more lengthy Daphnia pulex potential HTT gene region with bands being around 5003 base pairs (Sturm et al., 2009). The assays were successful in amplifying the CAG region of the HTT gene using the Andrew method and our calculated band size was 161 base pairs. Evidence was found in support of the D. Pulex genome having a possible HTT gene homolog, having a band calculated at 2059 using primers derived from D. melanogaster HTT gene primers. These results are not only significant in PCR diagnostic testing for HD but also in their application for the development of therapeutic drugs to treat HD and research in how HTT homolog genes affect their species.

Discussion

Written by: A46397795

Revised by: A47461396

Finalized by: A48336446

Summary of Experiment

Huntington’s disease, a rare autosomal dominant progressive, neurodegenerative disorder, has devastating effects on both physical and mental health, resulting in disordered movements, cognitive decline, and emotional disturbance (Duyao et al, 1993). The disease is centered in the basal ganglia, particularly the areas controlling movement known as the caudate and the putamen, which then spreads to other parts of the brain (Mangiarini et. al, 1996). Huntington’s disease is the product of an excess amount of CAG trinucleotide repeats on the HTT gene of chromosome 4, ranging from 36-120 total repeats. Normal, wild-type individuals have between 10-35 CAG trinucleotide repeats on their HTT gene. The excess CAG repeats on the gene are relatively easy to detect in patients that are not on the borderline between the two ranges. The CAG repeat region controls the production of the huntingtin protein. Little is known about this protein typically found in neurons, more specifically dendrites and cell bodies, although researchers are still attempting to find the actual function of huntingtin, many researchers believe that it is correlated to brain development. (DiFiglia et al., 1995; Li and Li, 2004 ). PCR is the most effective way to test for Huntington’s disease because the procedure allows for researchers to identify the number of CAG trinucleotide repeats present in an individual’s HTT gene (Andrew et al., 1994). The question being addressed in this study is how effective a PCR test can be in diagnosing a patient with Huntington’s disease. We have hypothesized that by using known primers provided by standardized diagnostic methods (Andrew et al., 1993), we will successfully run a PCR procedure on human DNA and identify the target region on the HTT gene by running a PCR cocktail through gel electrophoresis in a variation of the procedure performed in the 1994 study by Andrew et al..

In addition to testing human DNA for the HTT gene, we will test a homologous organism for the HTT gene as well. Since fruit flies (drosophila melanogaster) and Daphnia (daphnia pulex) have an amino acid similarity of 59.1%, we will use primers designed to test for the HTT gene in fruit flies to test for the HTT gene in Daphnia as outlined in the methods section (Sturm. et al., 2009). Daphnia was chosen as the homolog because of its genetic similarity to the fruit fly, and fruit flies provide homologous genes for 75% of human diseases (Pandey et al., 2011). The procedure for the tests run on Daphnia DNA will be identical to that of our primary experiment on human DNA. Due to these similarities, we hypothesize that by using the procedure based on a previous study done in 1995 by Leeflang et al. as outlined in the methods section, we will be successful in a PCR assay and rendering bands of the Daphnia pulex DNA using the primers described above. The D. pulex DNA will be mixed into a PCR cocktail and run through gel electrophoresis in order to test for the presence of an HTT gene similar to that of a human.

Original Predictions

Using a polymerase chain reaction (PCR) we isolated the CAG region of the HTT gene which will be analyzed using gel electrophoresis (GE) on each PCR assay. The assay was carried out using two specifically designed primers used previously in the study by Andrew et al. Based on this information, we originally predicted that our results would support our hypothesis, and that we would render copies of our targeted 168 base pair (based on an average 21 repeat individual) DNA region because tested and published primers and protocols were used as outlined in the methods section with our predicted results depicted in Figure 4 of the Figures & Tables section below (Andrew SE et al. 1994). For the homolog tests, we originally predicted that our PCR procedures would function correctly resulting in the amplification of the target HTT gene sequences yielding a 5,003 bp product, because the two published Drosophila melanogaster primers were used on D. pulex DNA, these predictions are depicted in (Fig. 6).

Results and Ultimate Findings

All human HTT trials took place after a successful control amplification of the 1Rz gene in the Enterobacteria phage lambda. The HTT region of the human genome was copied via PCR using published primers and methods previously shown to target this region (Andrew et al, 1994). This PCR assay was then run through GE and 3 faint bands were amplified. Upon further inspection this unexpected amplification was most likely the result of nonspecific binding of the reverse primer downstream of the CCG repeat region which is downstream of the CAG repeat region. The lowest molecular weight band has a base pair length of 161 which is close to the expected length of 168. This length means that the individual has (23) CAG repeats indicating the absence of Huntington’s Disease, thus supporting our hypothesis (Walker, 2007).

For the homolog tests, due to their 59.1% similarity in amino acids, published Drosophila melanogaster primers were used to determine if there could possibly be a HTT homolog in Daphnia pulex DNA (Sturm et al., 2009; Jackson et al., 1998). A PCR and GE assay was performed on D. pulex DNA using D. melanogaster primers. This assay amplified a band length of 2,059 base pairs. Likely, this large difference between the expected and actual band length was a result of a distorted ladder. The gel was run at 135V, which may have been too high since the loading dye was not visible after and must have run off the gel. Based on information provided by the manufacturer, Invitrogen Life Technologies (Grand Island, New York), the 500 bp band of the ladder generally migrates together with the loading dye so it was assumed that the first visible ladder band from bottom was the 650 bp marker. Interpretation of the remaining ladder bands led to the calculated band length and the trend line equation. Unfortunately, the trend line was likely incorrect because the visible band in Fig. 7A is located below the 1,650 bp ladder marker. Another gel electrophoresis analysis of Daphnia PCR samples would be required for a more conclusive indication of a HTT homolog.

Future Directions

Finalized by: A48336446

Certain elements of the assays conducted could have yielded clearer or more readable results. The 1Rz gene gel amplification of the Enterobacteria phage lambda could have been better read by replicating the assay with more separated ladder. This defect was most likely caused by an incorrect agarose gel concentration as well as a higher than ideal gel electrophoresis (GE) running voltage. The experiment should be replicated and the agarose concentration of the gel increased from 1% to 2% as well as the voltage decreased from 150 volts to 120 volts for an extended period of time (until the loading buffer migrates ¾ of the gel length). These changes would decrease the rate of mobility of the ladder which would allow for the markers in the ladder to separate further as well as become more defined because as mobility decreases, the separation within that range increases (Helling et al., 1974).

The clarity of the human HTT GE assay could have benefitted not only from a better separated latter as described above but also from less high molecular weight smearing. The most likely reason for this type of smear was an excessive extension time. The assay would be best if replicated with a shortened from a 1 minute to a 30 second extension time as shorter PCR products require shorter extension time (Yu & Pauls, 1992).

The readability of the D. pulex GE assay was most likely hindered by having part of the ladder run off the gel due to an excessive electrophoresis running duration. The readability of this assay could have benefitted by being replicated with the electrophoresis running time shortened from 40 minutes to 20 minutes as well as a running voltage lowered from 135 volts to 110 volts.