Investigating the Relationship Between Level of Starch Consumption in Ancestral Diet and Number of AMY1 Gene Copies Abstract Amylase is an enzyme that is produced by the salivary glands and pancreas of humans to break the glycosidic linkages in polysaccharides such as starch (Tracey 2018). The purpose of this experiment is to investigate the relationship between ancestral starch diet and the number of AMY1A gene copies. As I have South Asian heritage, I ranked my ancestors as having moderate consumption of starch, as rice was a staple, but various other starches were less common. I believe that I have the average number of AMY1A gene copies due to my ancestors having a moderate consumption of starch. In order to determine the relationship between AMY1A gene copies are starch consumption, a series of interconnected experiments were carried out which allowed us to determine the concentration of salivary amylase, and thus calculate our gene copy number. DNA had to be extracted from cheek epithelial cells, amplified using polymerase chain reaction and then separated using gel electrophoresis. A series of calculations were carried out to determine the gene copy number. Originally, I believed that ancestral diet and starch consumption would share a strong correlation, but the results displayed a weak correlation between these two variables. However, my hypothesis was proven correct as my gene copy number was 3 which was very close to the mean of 2.66. IntroductionIn Homo sapiens, salivary amylase, or alpha amylase, is an enzyme produced by the salivary glands to break down the alpha – 1,4 – glycosidic linkages in polysaccharides, including starch, through hydrolysis reactions (Tracey 2018). This is essential, as in order for polysaccharides to be utilised as a source of energy, they must be broken down into glucose monomers, which are readily used as a source of energy in the body. Salivary amylase is coded for by the AMY1A gene. The number of copies each individual possess varies through the population (Tracey 2018). This variation is a result of gene duplication events (Tracey 2018), which were preserved in the genome, due to the selective advantage in their environment. Thus raises the question, is there a relationship between ancestral starch consumption and number of amylase gene copies. Individuals with high starch content on average, should have an above average number of copies of amylase, and individuals with low ancestral starch diets should have a lower than average number of amylase copies (Perry et al.2007).The purpose of this experiment, is to investigate the relationship between ancestral starch diet, and number of AMY1A gene copies. According to a study done by Perry et al. (2007), a positive correlation exists between salivary amylase protein level, and ancestral starch consumption. Further, salivary amylase protein level is on average higher for individuals with high ancestral starch consumption. I have South Asian heritage, I classified myself as having a moderate ancestral starch consumption, and thus believe that I will have an average amylase gene copy number, as my ancestors did not face selective pressures to maintain a very high amylase concentration.The variables investigated were level of ancestral starch consumption, which were ranked low, medium and high and gene copy number. The sample size investigated was 29.Methods The investigation took place over a series of experiments which were conducted according to the Bio 1A03 Lab Manual as described in Labs 2-8. The gel electrophoresis lab, lab 7, was modified to utilise 5ul of the 100basepair ladder rather than the outlined 10ul.    ResultsLab 3: Amylase AssayIn Lab 3, known concentrations of amylase were reacted with starch and iodine mixtures in order to generate a calibration curve. It was observed that as amylase concentration increased, the blue-purple color of the starch-iodine solution became increasingly faint. Upon analysis by the spectrophotometer, as amylase concentration increased, less absorption was measured. A calibration curve was generated (Figure 1) using the known amylase concentrations and the absorption levels, which defined a linear relationship with the equation, y=-0.0601x +1.6381. The R2 value for the trend line was 0.9204, demonstrating that the relationship between amylase concentration and absorbance is strongly linear.Lab 4: Amylase Assay and Salivary Amylase ConcentrationIn Lab 4, the salivary amylase concentration extracted from my mouth was serially diluted in order to obtain measurable results. By measuring absorbance at each dilution and plugging these values in into the equation from the previous calibration curve, y=-0.0601x +1.6381, the average concentration of salivary amylase was determined, as shown in Calculation 1. It was determined that the concentration of salivary amylase in my saliva was 2.65mg/mL.Lab 5: DNA Extraction In Lab 5, each student submitted their calculated salivary amylase concentration with their hypothesised ancestral diet type. Graphs was created to model this relationship, one with standard deviation error bars (Figure 2) and one with error bars(Figure 3).Lab 6: Polymerase Chain ReactionIn lab 6, the amylase that was previously extracted was amplified through polymerase chain reaction. It went through 35 amplification cycles in the Thermocycler, which according to the equation 2x=number of gene copies  where x=amplification cycles, producing 3.4×1010 copies of the target sequence.Lab 7: Gel ElectrophoresisIn Lab 7, our sample from polymerase chain reaction was separated using gel electrophoresis (Figure 4). Through running a 100bp ladder with the DNA sample, we were able to compare the known size of DNA fragments in the 100bp ladder, to the unknown length of DNA fragments in our DNA sample. Amylase was the smaller of the two genes that underwent gel electrophoresis. By measuring the distance that the 100bp ladder travelled, with the known sizes of the fragments, we generated a semi log graph. By drawing a line of best fit, we were able to estimate the size of the actin and the salivary amylase gene, based on distance travelled. The AMY1 gene travelled 42 mm, and the actin gene travelled 33mm. Using the line of best fit to estimate the size of each fragment, it was determined that the actin gene was approximately 440bp and the AMY1 gene was about 180bp. as identified with the gel electrophoresis image (Figure 4).Lab 8: Gene Duplication and Data AnalysisThrough data analysis of the gel electrophoresis, adj.vol (Int) were determined, which was in turn used to calculate the number of amylase gene copies. Amylase had an adj.vol (Int) of 608210 and actin had an adj.vol (Int) of 592210.  It was determined that I had 3 amylase gene copies. A graph was produced, showing the relationship between salivary amylase concentrations and number of amylase gene copies (Figure 5).Discussion  The purpose of this experiment was to investigate the relationship between ancestral level of starch consumption and the number of the AMY1 gene copies. According to a study by Perry et al. (2007) there is a positive correlation between number of AMY1 gene copies and salivary amylase concentration. Further, individuals with high ancestral starch consumption tend to have a higher salivary amylase concentration. As a result, I hypothesized that due to the moderate consumption of starch of my ancestors, that I would have the average amount of AMY1 gene copies, as it would be metabolically expensive for me to produce high amount of salivary amylase, if my diet is not high in starch. The results show an average AMY1 gene copy of 2.6 within the sample population, and as my AMY1 gene copy number was determined to be 3, I coincide with the average, supporting my hyptoehsis. However, my salivary amylase concentration of 2.65mg/mL was a well over the average for individuals with moderate ancestor starch consumption which was determined to be 1.44mg/mL. This result suggests that the relationship between salivary amylase concentration, AMY1 gene copy number, and ancestral starch consumption are not all proportionally interconnected as previously thought. The results support that ancestral starch consumption does predict AMY1 gene copy number, however, it disproves that ancestral starch consumption is an indicator of salivary amylase concentrations. In Lab 3, the reaction between various concentrations of amylase with starch and iodine were observed. At low concentrations of amylase, the reaction mixture had a distinct blue color, but as amylase concentrations increased, the color became increasingly faint. This change in color intensity at various amylase concentrations was measured by the spectrophotometer. Upon creation of a calibration curve, it was observed that a strong linear relationship was present, as determined by the R2 value of 0.9204, and that the equation y==-0.0601x +1.6381 accurately represents the relationship between absorbance and amylase concentrations.  In lab 4, serial dilutions of saliva were carried out to determine the average salivary amylase concentration. If the absorbance fell out of the range of the calibration curve, then inaccurate salivary amylase concentrations would be calculated, thus by measuring absorbance at various dilutions, outliers could be identified and neglected. Lab 5 required us to graph the relationship between ancestral diet type and salivary amylase concentrations. Figure 2 and 3 display that the mean amylase concentration was higher for individuals with high ancestral starch diet than individuals with low ancestral starch diet, supporting the theory that individuals with high ancestral amylase consumption would have higher salivary amylase concentrations. The overlap of error bars between the moderate and high starch consumption groups shows that the relationship between ancestral starch consumption and salivary amylase concentration is not distinct and clear. Individuals who have high ancestral starch consumption, have salivary amylase concentrations that better indicate a moderate starch consumption. This raises questions as to the extent to which this theory can be used to predict future relationships.Polymerase chain reaction is the process by which a DNA target sequence is copied multiple times, amplified, in order to produce large quantities of the target sequence. In order to do so, a front and back complementary primer are needed for the target sequence in order to bind to the 5’ and 3’ end of the target sequence. This process consists of denaturing, annealing, and extending( (Morris et al. 2016)This was used to produce a large quantity of the DNA target sequence for gel electrophoresis.In lab 7, the amplified DNA samples were separated using gel electrophoresis in order to determine the size of the AMY1 and actin gene fragments. The principle of gel electrophoresis is that the negatively charge phosphate groups in DNA will be attracted the positive electrode (anode), and thus travel towards it. The distance and speed of travel is dependent on the size of the fragment, larger fragments move slower than smaller fragments. A semi log graph was created to plot the distance travelled by known sizes of fragments in the 100bp ladder, and a line of best fit was drawn to interpolate the size of the AMY1 and actin genes based on distance travelled.  It was determined that AMY1 was 180bp in size and the actin was 440bp in size.  Error could arise from the fact that the line of best fit was hand drawn, and thus it might not b be completely accurate in determining the size of the actin gene or the AMY1 gene. In a modification of this lab, the graph can be constructed using excel to get an accurate line and an equation.In Lab 8, the data collected through gel electrophoresis, specifically the Adj.vol (Int) was utilised to determine the AMY1 gene copy number. A scatter plot was produced using the data collected from a sample size of 29. A graph was constructed to determine the relationship between amylase gene copy number and salivary amylase concentration and upon including a linear trendline, the R2 value was 0.0756. Further it should be noted that the correlation coefficient of the data was -0.274939 indicating a weak, negative correlation between salivary amylase concentration and AMY1A gene copy number. This contradicts the idea that there is a positive correlation between gene copies and amount of salivary amylase produced (Tracey 2018), which was the initial theory behind this investigation.There were several sources of error associated with this investigation. Primarily, the sample size of 29, is too small to accurately represent the population. In future experiments, a larger sample size should be used, perhaps the data from all the tutorials could be collected and used to determine the nature of the relationship between salivary amylase concentration and gene copy number. This would result in the results of the experiment being a better representation of the population, and allowing it to be generalized. Further, salivary amylase concentrations are not always consistent. A study by Nater et al. (2006), indicate that stress, whether it be emotional, physical, or psychological, can lead to changes in salivary amylase concentrations. These factors were not accounted for or controlled when conducting the experiment, which could result in inaccurate results and could explain the variability observed in the results. This error could be minimized by collecting multiple saliva samples over a series of days and recording the average calculated concentration of amylase.Further, salivary amylase is only a portion of the total amylase in the human body, it begins the process in the mouth, and pancreatic amylase completes it in the gut (Bonnefond et al. 2017). Since pancreatic amylase also contribute to the process of breaking down starches, it could be a further indicator of the relationship between amylase concentration and ancestral starch consumption. A further extension of this experiment could be monitoring both pancreatic and salivary amylase concentrations, which could account for all the amylase activity in the body and determining the relationship with ancestral starch consumption.AppendixFigure 1  Calculation 1: Concentration of Amylase at Each Dilution Factor     Table 1: Fragment Sizes and Distance Migrated for 100bp Ladder100bp LadderDistance Migrated (mm)Size (bp)211500251000269002780028700306003250034400373004020044100  Figure 4:  Gel Electrophoresis shown under UV light. 100bp ladder is in wells 1 and 10, and my sample is in wells 4 and 5. The salivary amylase gene is the block that is the highest, and the actin gene is directly below.      Literature Cited:Bonnefond A, Yengo L, Dechaume A, Canouil M, Castelain M, Roger E, Allegaert F, Caiazzo R, Raverdy V, Pigeyre M, et al. 2017. Relationship between salivary/pancreatic amylase and body mass index: a systems biology approach. BMC Med. [internet][cited 2018 Nov15]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322607/doi:  [10.1186/s12916-017-0784-x]2.      Morris JR, Hartl DL, Knoll AH, Lue RA, Michael M, Berry A, Beiwener AA, Farrell BD, Holbrook NM 2016. Biology How Life Works. New York: W. H. Freeman and Company. 505p.3.      Nater UM, La Marca R, Florin L, Moses A, Langhans W, Koller MM, Ehlert U. 2006. Stress-induced changes in human salivary alpha-amylase activity—associations with adrenergic activity. Psychoneuroendocrinology. [internet][cited 2018 Nov15]. 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