Per3 Gene and Diurnal Preference - Lab Report Example

Per3 Gene and Diurnal Preference Introduction: Most of the living organisms possess a circadian rhythm, which is a series of interactions between the organism and multiple biological activities. Period homologue 3(PER3) is one of the inducer protein present in the circadian system. The studies have found that a biallelic VNTR (variable number tandem repeat) polymorphism is present in the human PER3 gene at the chromosome 1p36.23, and it contains two alleles with 4 or 5 tandem repeats of 54 bp (Nadkarni et al., 2005).This VNTR was evaluated as the important genetic factor for the chronotypes as it is associated with the diurnal preference (AM and PM people) (von Schantz, 2008). Many hypotheses were set to check whether these factors are intrinsic factors of the population and have effect on the associations (Archer et al., 2003). Some studies have also found that there is no association between PER3 gene and diurnal preference (Perea et al., 2014). In the given study, the effect of the PER3 on the diurnal preference of the people is analyzed. (Ebisawa et al., 2001).The association between the morning and evening preference were analyzed using the chi square test. The buccal cavity DNA is used for the analysis of PER3 gene. The PER3 gene is separated from the genome using PCR amplification technique (Viola et al., 2008). The final PCR product is analyzed using the agarose gel electrophoresis. Methods Preparation of human DNA 50 individuals were selected for the study and they were asked to rinse their mouth with 12ml of 0.9% saline and the second spit was collected in the falcon tubes. Rising the mouth with saline will enable us to collect the buccal epithelial cells from the mouth. The cells are then transferred to the centifugation tube and centrifugation was performed to enable the cells to get settled as a pellet at the bottom from the saline. The cell pellet is then removed from the solution and strored in the Phosphate- buffered saline (PBS). PBS being neutral in pH will preserve the cell from osmosis and diffusion. The cell pellet is then lysed using the 0.1M NaOH solution. The high base solution will rupture the cell membrane and create pores in the membrane. Then 100µl of 0.1M HCl and 100µl of 1M Tris – HCl was added at a pH of 7.5. The cell organelles are present in the solution and they are separated from the solution through centrifugation. The crude cell lysate will contain the DNA. From the DNA, the required gene can be isolated using the PCR analysis. PCR amplification of PER3 gene: The forward and reverse primers were chosen for PER3 gene and they were added to the PCR tubes along with crude DNA, polymerase buffer, Taq polymerase and DNTPs. Forward and reverse primers are chosen based on the sequence of the PER3 gene. The primers must have the unique sequence region of each gene that is not present in any other gene. The chance for primer – dimer formation is high because of many reasons. The annealing temperature may not be correct or the template may not be proper or the DNA may have contaminants. To prevent the primer – dimer formation, the above points must be taken care off. DNA polymerase buffer helps to increase the dNTPs binding with the DNA template. Taq polymerase enzyme polymerizes double stranded DNA formation. It acts as the catalyst for the amplification reaction.The master mix was prepared according to the instructions. The tubes were then placed in the PCR and the amplification was performed. To check the success of PCR amplification, the amplified PCR product was run in the agarose gel electrophoresis. 1.2% agarose gel was preared and casted in the casting tray. The electrophoresis box was filled with the electrophoresis running buffer and the DNA products were loaded in the gel with the loading dye. DNA ladder was loaded in the first lane and the samples in the other lanes. The positive control was a readily avalilable one and the negative control was distilled water. The postive control enables us to make sure that the separation of the DNA bands are according to the PER 3 gene amplification or not. DNA ladder is added to the first lane because; they show the bands with the fixed molecular weight. This is used for the identification of the molecular weight of the unknown bands in the agarose gel. Positive and negative controls are added to the last two lanes to check the accuracy of the results.The DNA was allowed to run at 140V for 2 hours until the DNA reached ¾ of the gel. The agarose DNA gel was then stained with safeview nucleic acid stain and viewed under Gel documentation system. The results were recorded. AM or PM people identification: A questionnaire was prepared to determine whether the selected groups of people are morning persons or evening persons. Three options were given for each question and the marks were given as 0, 1 and 2. The person who scores above 7 is considered as evening person and those who score between 0-6 as morning person. PCR amplification is required because, the sample obtained from the individuals will have a single copy of the DNA and the PER3 gene must be extracted from the crude DNA sample for further analysis. Moreover the DNA quantity will be very less for analysis in the gel electrophoresis, so amplification of the DNA was done using the PCR techniuqe. PCR technique also enables us to select the particular gene from the crude DNA using the selective primers.the primers anneals to the respective 3’end and DNA polymerese will extend the primers based on the complementary sequence. This process will repeat in each cycle of the PCR and many primers will bind to the finished single product of previous step and amplifiy the PER3 gene. If the polymorphism is present, then the number of bands in the gel will be four or five for each sample. If the individual is homozygous, there will be a single band in the gel. Since the PER3 primers will bind to the VNTRs, if the polymorphism is present, then they will produce multiple bands. Results Figure 1a: Class Data Homozygote +/+ Heterozygote +/- Homozygote -/- Totals AM people 22 9 7 38 PM people 8 4 0 12 Totals 30 13 7 50 Figure 1b: Graphical representation of AM/PM people. The graph indicates the number of homozygotes and heterozygotes present in both AM/PM people. The + homozygote concentration is more in the both the AM and PM people and little – homozygote concentration in the AM people and no – homozygotes in the PM people. Heterozygote concentration is less in both AM and PM people. This graph signifies that most of them are AM people in both homozygote and heterozygote alleles. Figure 2: Total Numbers of Alleles: Frequency + allele - allele Total Frequency of +allele Frequency of -allele AM + PM People 73 27 100 0.73 0.27 Figure 3a: Total Numbers of Alleles: Observed data + allele - allele AM People 53 23 PM People 20 4 Figure 3b: graph - Total numbers of alleles: Observed data. The figure shows that the + allele concentration is more in both AM and PM people and less – allele concentraion in the AM and PM people. This data signifies that the concentration of + allele and – allele is high in the AM people than PM people. Figure 4: Total numbers of Alleles: Expected data + allele - allele AM People 55.48 20.52 PM People 17.52 6.48 Figure 4b: Graph -Total numer of alleles: expected data. The expected data also shows high + allele concentration and less – allele concentration in the AM people when compared to the PM people. The expected data graph signifies that AM people are more than the PM people and both the + allele and – allele concentration is high in the AM people and the results are much significant with the observed data. Figure 5: Chi square test: AM people PM people Total Observed number of + allele (o) 76 24 100 Expected number of + allele (e) 55.48 17.52 76 Deviation (d) = (o-e) 20.52 6.48 27 Deviation2(d2) 421.07 41.99 463.07 d2/e = chi2 7.59 2.4 9.99 Discussion The gel image shows that all the bands of the samples are at equal height. This indicates that there is only one thick DNA band in all the lanes. If PER3 primer was able to identify the VNTRs present in the samples, then there would be 4 or 5 bands in each lane. This indicates that there are no VNTRs in the given all samples. This indicates that there is no difference in the genotype of AM and PM people. Chi square test was performed to analyze the samples statistically. It was observed that that the chi square values lies in the significant range, that is, between 0.01 and 0.001. As the probability is less than 0.10, the deviation in the results is considered significant and the proposed hypothesis is not the valid one. There is some relationship between the PER3 genotypes in the AM and PM people. There are some allelic frequency differences between the AM and PM people. The results obtained through the statistical analysis and the gel reports vary in the conclusion. There are some errors in the gel electrophoresis. Since all the samples have band at the top of the gel, there may be some experimental errors in the electrophoresis. The hypothesis that there is no significant difference between the AM and PM people was ruled out. The statistical analysis has revealed that there is significant difference between the AM and PM people. The chi square test has shown significant difference between the results and the results are 99.99% accurate. So, this result indicates that the influence of PER3 polymorphism between AM and PM people requires further analysis. The rate for the occurrence of AM person is more than PM person if I am a homozygote or heterzygote. If I am a + homozygote or heterozyogte, the chance for being AM person is more and if I am a – homozygote, I will be an AM person. References: Archer, S. N., Robilliard, D. L., Skene, D. J., Smits, M., Willaims, A., Ardent, J & von Schantz, M. (2003). A length polymorphism in the circadian clock gene Per3 is linked to delayed sleep phase syndrome and extreme diurnal preference. Sleep, 26 (4): 413-5. Ebisawa, T., Uchiyama, M., Kajimura, N., Mishima, K., Kamei, Y., Katoh, M., Watanabe, T., Sekimoto, M., Shibui, K., Kim, K., Kudo, Y., Ozeki, Y., Sugishita, M., Toyoshima, R., Inoue, Y., Yamada, N., Nagase, T., Ozaki, N., Ohara, O., Ishidam N., Okawa, M., Takahashi, K & Yamauchi, T. (2001). Association of structural polymorphisms in the human period3 gene with delayed sleep phase syndrome, EMBO Reports, 2 (4): 342- 346. Nadkarni, N. A., Waele, M. E., von Schantz, M & Thomas, M. G. (2005). Evolution of a length polymorphism in the human PER3 gene, a component of the circadian system. Journal of Biological Rhythms, 20 (6): 490 -9. Perea, C. S., Nino, C. l., Lopez-Leon, S., Gutierrez, R., Ojeda., D., Arboleda, H., Camarqo, A., Adan, A & Forero, D. A. (2014). Study of a functional polymorphism in the PER3 gene and diurnal preference in a Colombian sample. The Open Neurology Journal, 8: 7 -10. Viola, A. U., James, L. M., Archer, S. N & Dijk, D. J. (2008). PER3 polymorphism and cardiac autonomic control: effects of sleep debt and circadian phase, American Journal of Physiology, Heart and Circulatory physiology, Vol.295, No.5, pp: H2156 – 63. Von Schantz, M. (2008). Phenotypic effects of genetic variability in human clock genes on circadian and sleep parameters. Journal of Genetics, 87 (5): 513 -9. Read More