Replication of Homo sapiens ZDHHC8 Linked to Schizophrenia By Exon Precise PCR Amplification

By: Anthony Allemon, Andrew Guthrie, Shannon Li, and Samantha Thacker

LB145 Cell and Molecular Biology

Tuesday/Thursday 7pm

Jessica Goldsworthy, Chelsey Klein, and Anthony Watkins

4/21/2015

Abstract

The mutation of the ZDHHC8 gene has been shown to correlate with the mental illness schizophrenia (Mukai et al., 2004). The mutation occurs between exon four and exon five on chromosome 22, which is caused by a microdeletion in the single nucleotide polymorphism (SNP) rs175174 (Faul et al. 2005; Chen et al., 2004). The purpose of our study is to properly isolate exon 1 to exon 8 of the Homo sapiens ZDHHC8 gene and replicate it using polymerase chain reactions (PCR). DHHC-type genes have been shown to increase radiosensitivity in mesothelioma cells, if the method of replication of ZDHHC8 is successful it could be useful for the production of ZDHHC8 to be used in conjunction with mesothelioma patient’s radiation treatments (Sudo et al 2012). The hypothesis of our research was that by using calculated temperatures in PCR, the primers Fl-ZDHHC8 and Rl-ZDHHC8 will efficiently form hydrogen bonds to the complementary sequences of the template DNA and taq polymerase will add the proper nucleotides to the 3’ end of the primers. Our products were analyzed using gel electrophoresis and were predicted to be 9312 base pairs (bp) long. The PCR was unsuccessful in copying exon 1 to exon 8 of the ZDHHC8 gene, however the PCR of the positive control was successful.

Figure

Figure 1: Amplification attempt of Human IB3 cells ZDHHC8 gene segment by PCR with various annealing temperatures. (Left) The forward primer, F1-ZDHHC8 (F1), is 3’-CAGCCCCGGGACGCGCCTCAAAC-5’ which pairs with the wild-type Homo sapien sequence 5’-GTCGGGGCCCTGCGCGGAGTTTG-3’. The F1 primer was designed to attach at base pair number 1 and will be 23 base pairs long. The reverse primer, R1-ZDHHC8 (R1), is 3’-TGGCCCCAAGTCCAGTGGCTTCTC-5’ which pairs with the wild-type Homo sapien sequence 5’-ACCGGGGTTCAGGTCACCGAAGAG-3’. The R1 primer was designed to bind to base pair 9288- 9312 and the R1 primer will be 24 base pairs long. The gene is located on chromosome 22. Through PCR, it is predicted that there will be about 1 trillion copies of the aspired ZDHHC8 gene, which is found by the equation 2ⁿ, where ‘n’ is the number of cycles ran in the thermocycler.The PCR cocktail contains 40.5 µl of nuclease-free water, 5 µl of 10x PCR buffer containing MgCl₂, 0.5 µl of Taq Polymerase, 1µl of dNTPs, 1 µl of 1F Primer, 1 µl of 1R Primer, and 1 µl of Homo sapien genomic DNA. The Denaturing temperature for PCR cycles will be at 95℃, the annealing temperature will be set on a gradient from 67.4-60.6℃ and then the final step, elongation, will be set to 72℃. The initial denaturing stage will be three minutes then 30 seconds for the rest of the cycles. The annealing and elongation will be set to 45 seconds. Once there are four cycles completed, the mixture is about 50% percent ZDHHC8 gene due to the fact that as more cycles go on there are more fragments of only the ZDHHC8 genes being copied. In wells 1-5 a temperature gradient was implemented from 67.4-60.6℃. Well 6 has the same temperature as well 1 because when loading well two there was uncertainty if the solution made it into the well. In result we ran the 67.4℃ cocktail again in well 6 to reduce potential errors. 0.4 g of agarose, 40 mL 1X of LB buffer, and 2µl of GloGreen were used to make the 1% agarose gel. In wells 1-6 there were no ZDHHC8 bands present, the gene was unable to be targeted in this allotted scenario. (Right) Semi-log plot measuring migration distance (cm) vs molecular size (bp) of the Human IB3 cells ZDHHC8 gene using 1 Kb Plus Ladder. The x-axis shows the migration distance in centimeters that the bands of the ladder had migrated from the well. The y-axis shows the number of base pairs present in the ladder at each distance. The equation of the semi-log plot is y = 8137x^-2.918 in order to determine the DNA band size, The bands found on the left are at 3.7 cm, therefore it should be having the band size to be 178.8 bp. The R² value of 0.95453 shows that the graph is near perfect fit represented by 1.

Discussion

Experiment Summary

Schizophrenia, a serious illness that affects the brain’s ability to decipher reality, affects around 1% of the world’s population. It is said to be associated with the mutated form of the ZDHHC8 gene on chromosome 22 ( Andreasen et al, 1991). The mutated form of the ZDHHC8 gene can be caused by any mutation that suppresses the wild-type gene such as a single substitution, microdeletion, or insertion (Chen et al, 2004).  The ZDHHC8 gene is specifically affected in the region between exon 4 and exon 5 where the single nucleotide polymorphism rs175174 resides (Mukai et al, 2004). Even though PCR has been used in other studies such as Evidence that the gene encoding ZDHHC8 contributes to the risk of schizophrenia (Mukai et al, 2004) to look specifically at the region from exon 4 to 5, we are addressing the question of whether PCR of the regions between exon 1 to exon 8 of the ZDHHC8 wild-type gene could be performed to analyze the gene through gel electrophoresis. We used E.coli and Lambda DNA as our controls because their base pair length are known, therefore, we can compare the ZDHHC8 gel electrophoresis band to the controls as well as to the ladder. We hypothesized that by using calculated temperatures in PCR, the primers Fl-ZDHHC8 and Rl-ZDHHC8 will efficiently form hydrogen bonds to the template ZDHHC8 DNA, elongate with taq polymerase that adds complementary nucleotides, and provide our desired DNA strand from exon 1 to exon 8 that views rs175174.  

Original Predictions

When we ran the ZDHHC8 through the gel, we expect that there will be around 9000 base pairs shown next to the ladder because the ZDHHC8 wild-type gene from exon 1 to exon 8 is 9312 base pairs long (Mukai et al, 2004). For our controls, we anticipated that E.coli would have 512 base pairs and lambda to have roughly 500 pair pairs (Rawool et al, 2015). In comparison, the ZDHHC8 target gene would be larger than both of the controls.                                                                                      

Ultimate Results and Findings

In order to have proper results, we ran multiple trials because we have to discover the proper temperature for the primers to anneal (Blake et al, 1992). We predicted that at a favorable temperature, the primers will anneal and we will expect that the taq polymerase will be able to copy roughly a million copies of the target portion of the ZDHHC8 gene from exon 1 to exon 8 because the primers will be able to induce the correct sequence of copying, hence supporting our hypothesis (Mukai et al, 2004).  Our experiment were based on the Case–control study and transmission disequilibrium test provide consistent evidence for association between schizophrenia and genetic variation in the 22q11 gene ZDHHC8 in which the researchers were able to create primers that successfully replicated their target region of the ZDHHC8 gene multiple times (Chen et al, 2004).  However in our experiment, we were unsuccessful in amplifying the ZDHHC8 gene with the time given to us and we had inconclusive findings as a result. We were able to successfully amplify the E.coli and Lambda control, but with no results on our human band for the ZDHHC8 gene our findings did not support our hypothesis as we did not find the correct annealing temperatures for the primers to anneal to the target DNA.

Weaknesses and Technical Difficulties

A weakness in our experimental design is that we failed to anticipate the appropriate time to obtain the control bands for both the E.coli and Lambda DNA. We were unable to run the number of trials for the ZDHHC8 gene as we had intended due to the time limited to us. A complication with obtaining bands could be caused by the overall G-C content of each primer. When calculating the total melting point of the primers, the G-Cs play the biggest role in the equation. Our forward primer annealing temperature will be 61.97℃ while our reverse primer will anneal at 58.5℃. There is a 3.47℃ difference between the two which could account for the weakness. If the annealing is temperature is too high, then the primers will not anneal. If the temperature is too low the primers may not anneal to the correct position, or at all (Markoulatos et, al 2002). Another possible weakness in our experimental design is that when PCR cocktail is in the annealing stage in the thermocycler, the gradient set up might not be the annealing temperature for the primers to anneal. According to the study High and Low Annealing Temperatures Increase Both Specificity and Yield in Touchdown and Stepdown PCR it is more effective to have a gradient on temperature for the annealing stage to get stronger results (Hecker et al, 1996). We had ran our experiment with multiple gradients, however, it took longer than expected to come to obtain the bands that we anticipated for. Another complication that we came across was keeping all the components needed for the PCR cocktail to be cooled and knowing the time of when the components are put in. In the article Observation of individual DNA molecules undergoing gel electrophoresis, it discusses that taq polymerase is extremely sensitive to heat, therefore it would need to have to put in right before it goes into the thermocycler (Smith et al, 2012). Taq polymerase will not be able to amplify the target gene when it sits at room temperature too long, resulting of having no bands when analyzing them through gel electrophoresis. Another complication that came across in our experiment was not adding enough MgCl₂. Taq needs Mg ions to help attach itself to the DNA strand in order for the Primers to anneal (Smith et al, 2012) . However, when there are over excess about of  Mg²⁺, there would be undesired PCR products shown when analyzing through gel electrophoresis. When there are low amounts of Mg²⁺, the PCR product would not be shown since the taq polymerase are unable to bind onto the DNA strand resulting its inability to amplify the target gene (Smith et al, 2012).

Future Directions

Consequently, an experiment that could be performed in the future if we were to continue our research is by using the exact same forward and reverse primers but afterwards splicing the bases near exon 3 and exon 6. This would make us focus on the smaller segment of the ZDHHC8 gene than from our current focus of exon 1 to exon 8. Additionally, it would be different from the study done in the research article Evidence that the gene encoding ZDHHC8 contributes to the risk of schizophrenia (Mukai et al, 2004) because we would not specifically be looking only between exon 4 and 5.

The gene ZDHHC8 has further applications in science beyond studying schizophrenia. According to the study ZDHHC8 knockdown enhances radiosensitivity and suppresses tumor growth in a mesothelioma mouth model it was found that when ZDHHC8 is paired with X-irridiation the combination of the two suppressed tumor growth (Sudo et al, 2012). It is not just the ZDHHC8 gene that has been thought to suppress tumors, the entire ZDHHC family is being linked to the suppression of tumors (Greaves et al, 2014)(Yan et al, 2013). This is significant to our research because if we can successfully isolate the ZDHHC8 gene, then tests can be run to see if this gene is specifically associated with tumor suppression. ZDHHC2 has been found to be associated with the knock down of the process (Planey et al, 2009). Another future experiment is to do a homolog. For example, the experiment can consist of amplifying the ZDHHC8 gene in zebrafish and daphnia to see either the zebrafish or the daphnia would have the same ZDHHC8 gene.  By doing these experiments, it can open the door for further research to be done with ethical means. If the gene is found in one of these organisms it could be an efficient way to run further tests on ZDHHC8 in the tumor suppression and schizophrenia aspects.