Introduction Enzymes are proteins, they act as catalysts to speed up biochemical reactions that take place within cells. They are crucial components of life on earth and serve a range of functions in the human body such as adding with metabolism and digestion (Live Science). The enzyme amylase is produced by salivary glands and pancreases of Homo Sapiens. Salivary amylase is the first amylase enzyme involved in the breakdown and digestion of starch in the oral cavity.
The enzyme amylase was examined along with the amylase, alpha 1A (salivary) gene (AMY1A) that produces this enzyme. The amylase produced by the pancreas and salivary glands is also referred to as alpha-amylase as it is able to break down the alpha glyosidic linkages in polysaccharides, such as starch and glycogen, which are broken though the process of hydrolyzation (Tracey 2018). Starch is a photosynthesized polysaccharide that is found in green plants which is an energy source for most living organisms. There are two components of starch, amylose and amylopectin. Amylose is a polysaccharide composed of alpha-D-glucose units bonded to each other through alpha (1 ? 4) glycosidic linkages. Amylopectin is a larger molecule in which is composed of ?-1,4-linked glucans connect through a ?-1,6 linkage to form a highly branched structure.
The crystalline structure of amylopectin is a result of the branching which occurs regularly at different regions of the molecule. Moreover, glycogen is an alpha-glucan which is found in animals and microorganism. It is composed of ?-1,4-linked glucans which is connected by the ?-1,6 linkage. The linkages occur frequently and are distributed throughout the glycogen molecule, making it soluble in water (Science Direct). The aim of this experiment was to determine if there is an association between the enzyme production, gene copy number and gene evolution. Hence, the purpose of this experiment is to determine if the variation in the gene copy number and enzyme production is in response to the quantity of starch that one’s ancestors consumed in their diets. Due to gene duplication, extra copies of the gene are added to the genome, which results in a variation of the number AMY1A gene that an individual possesses.
The selective pressures that may have acted on the individuals from populations or ancestry that consume high starch diets resulted in them having a higher average number of repeats than individuals from populations or ancestry that consume a low starch diet (Tracey 2018). According to the findings in the “Diet and the evolution of human amylase gene copy number variation” article by Perry et al. in 2007. The AMY1A gene shows a great amount of variation amongst different populations. It was concluded that the AMY1A gene copy number is positively correlated to the salivary amylase concentration. Individuals from populations with a high starch diets have more AMY1A copy genes than individuals from populations with a low starch diets (Perry et al. 2007). The saliva of the test subjects was diluted through a process called serial dilution; a technique where series of 10-fold dilutions of the original undiluted solution are produced.
This is done in order to find the optimal dilution of the results where the concentration can be calculated (Tracey 2018). The gene copy number was found using the polymerase chain reaction (PCR) which was used to amplify the target DNA, the AMY1A copy gene, in this experiment. The results of PCR were visualised through gel electrophoresis, a process in which the DNA fragments are pulled through the agarose gel, separating the DNA fragments according to their size.
A picture of the gel was taken using ultraviolet lighting. The DNA fragments size was determined using a DNA base pair ladder (Morris et al. 2016). It was hypothesized that a high starch ancestral diet would then result in a high number of AMY1A gene repeats leading to a high amount of salivary amylase produced due to their positive correlation. Moreover, the two main variables being tested in this experiment were concentration of the salivary amylase, which is dependent on the ancestral diet. The intermediate variable between the two was the AMY1A gene copy number. The concentration of salivary amylase being dependent on number of gene copies and the number of gene copies being dependent on ancestral diet.
MethodRefer to the Biology 1A03 2018 lab manual. There were four changes made while carrying out the experiments in the Biology 1A03 lab manual. During lab three, the set two tubes were not placed on the ice after the dilution. Also, during lab four, a similar change was made where the set two test tubes were not placed on the ice after the dilution.
Third, during lab four part A the 1ml of saliva in the microcentrifuge tube was centrifuged for 60 seconds instead of five seconds. Lastly, in lab seven part B, five microliters of the ladder were loaded into the first well on the left side instead of ten microliters. Results A calibration curve was created during lab 3 to determine the concentration of the unknown salivary amylase. By using a spectrometer, the absorbance of the different known concentrations of the salivary amylase were obtained. Using excel, the calibration curve of the amylase concentration and absorbance was formed (Figure 2).
The equation of the line of best fit was used to calculate unknown salivary amylase concentration. Moreover, the serial dilution technique was used to create a dilution series of the test subjects in order to find the optimal dilution results to create a concentration that can be measured. The dilution series was created with the following dilutions: 10X, 100X, 1000X etc. The line of best fit equation obtained from lab 3 was used to determine the unknown salivary amylase concentration. The absorbance (620nm) of the solution was inputted into the equation to determine the unknown concentration of the salivary amylase. (Figure 3). The dilution factor of the solution was accounted for in the calculations (1000x), therefore the concentration was multiplied by a 1000.
Standard error bars were used to determine the relationship between the amount of starch in ancestral diets and the mean amylase concentration (Figure 4). The range error bars were also graphed to determine the spread of data amongst the different ancestral diet groups (Figure 5). The PCR was performed on the cheek epithelium cells that were extracted from buccal cavity of the test subjects. The results of PCR were visualised through gel electrophoresis, a process in which the DNA fragments are pulled through the agarose gel, separating the DNA fragments according to their size.
A picture of the gel was taken using ultraviolet lighting (Figure 6). The adjusted volumes of the actin and amylase, 398,619 and 502,554 respectively, were found using the gel the photo (Table 1). The ratio of the volumes was used to obtain the gene copy number of AMY1A gene. The graph (Figure 6) of the amylase concentration and gene copy number was created, the relationship had a R2 value of 0.
0258. Discussion The purpose of this experiment was to determine if there is an association between the enzyme production, gene copy number, and gene evolution. Homo sapiens evolution had significant shifts in diet due to the development of new technology and the realization of potential nutrition in plants and animals. Over time the polysaccharide starch had become an essential part of the Homo sapiens diet, especially in agricultural societies. It was hypothesized that a high starch ancestral diet would then lead to a high number of AMY1A gene repeats resulting in a high amount of salivary amylase produced, due to their positive correlation. Figure 1 exemplifies a graph with moderate correlation between the salivary amylase concentration (R2 = 0.3097).
This graph represents the hypothesized results and depicts a distinct positive correlation amongst the two variables. During lab three, a calibration curve (Figure 2) was created using the relationship between the absorbance (620nm), allowing the concentration of salivary amylase to be found. The two then variables show a positive correlation (R2 = 9573). The absorbance is inversely proportional to the intensity of the colour measured, therefore it is inversely proportional to the concentration of the starch, as it is the coloured molecule. As the concentration of amylase was increased the colour of the solution gradually became lighter since more starch was broken down, the absorbance (620nm) decreased.
The inversely proportional relationship is portrayed on the graph (Figure 2) (Tracey 2018). The line of best fit equation was obtained from the calibration curve. During lab four saliva samples from the test subjects were taken and the samples were serially diluted. The respective absorbance (620nm) the salivary solution was found. Using the equation from lab 3, the salivary amylase concentration was found Figure 3, (2.
43 units), which was hypothesized as a high concentration. In Figure 4, a significant difference can be seen between the different data groups as the error bars do not overlap. Thus, the mean amylase concentration varies significantly for the different amounts of starch in ancestral diets. From the error range bars, Figure 5, it could be concluded that the individuals with a moderate amount of starch in their diets have a higher mean amylase concentration and the individuals with a very high amount of starch have a lower amylase concentration. Hence, the amount of starch in ancestral diet and the mean amylase concentration have an inversely proportional relationship. The PCR was used to amplify the DNA fragments of the AMY1A copy gene. Cheek epithelium cells were extracted from the buccal cavity of the test subjects using a swab and was placed in a culture tube. After extracting the subject’s DNA, NaOH was added and the solution was heated to 95?C, which helped breakdown the hydrogen bonding of the nitrogenous base pairs.
The Tris buffer was then added to the solution to help resist the pH changes. The results of PCR were visualised through gel electrophoresis and a picture of the gel was taken using ultraviolet lighting. The DNA fragments size can be determined with a DNA base pair ladder placed as a reference point, Figure 6. The adjusted volumes were found using the gel the photo, Figure 6 (Tracey 2018). The volumes in Table 1 were used to obtain the gene copy number of AMY1A gene. The ratio of the adjusted volume of the amylase and actin was used to determine the number of AMY1A copies for each subject. According to the graph of the number of AMY1A gene copies and salivary amylase concentration, Figure 7, it was found that the there was no correlation between the two variables (R2 value = 0.
0258), thereby, the hypothesis was refuted. Therefore, the amount of starch in ancestral diet of an individual had no effect on the current amylase concentration of the individual. The results concluded from this experiment did not reflect the results found in the article “Diet and the evolution of human amylase gene copy number variation” by Perry et al.
It was determined that there is no correlation between the AMY1A gene copy number and the concentration of amylase, so therefore there is no relationship between them. Whereas the article concluded that there was a positive correlation between the AMY1A gene copy number and the concentration of amylase, thus claiming that the concentration of salivary amylase of an individual is dependent on their ancestral diet. However, there were many different variables that were not considered for the duration of the experiment causing the results to have no correlation between them. For further improvement in the future, the individuals tested in the experiment could have a more controlled sampling of their saliva. The saliva sampled from the individuals could have had a higher or lower concentration of the starch based on their previous meals.
The different variables such as acidity of their previous meal could have caused the level of salivary amylase concentration to rise or drop. As proteins such as salivary amylase would denature under acidic conditions. Therefore, individual participants in the test must not consume food for a certain length of time prior to the experiment as this will lead to more accurate results. Another source of improvement that could be applied to this experiment could be done during lab four, part A. In part A, the subject’s 1 ml of salivary amylase could be centrifuged for a longer period of time at a greater gravitational force. The saliva of each individual is composed of different sources. According to the theory of centrifugation, a greater force is needed to centrifuge smaller cellular components. Centrifugation at a greater gravitational force and for a longer period of time can allow for a greater elimination of impurities and cellular packing in the salivary solution.
This could lead more accurate results for the salivary amylase concentration (Baskoro et al. 2017). Additionally, the sample of population used was on a smaller scale, this did not allow for a great range of diversity in the test sample. A greater diversity would allow for larger range of ancestral diets resulting a larger range of the amylase concentrations. A more diverse data set allow for more insight of the relationship between the salivary amylase concentration and the ancestral diet. Actin is a housekeeping gene that is involved in cell structure, mobility and intercellular signal in Homo sapiens. Of the six different isoforms of the actin gene, beta-Actin was used in the PCR reaction tube to normalize the expression of the AMY1A gene. In the process of preparing the PCR reaction tube, to amplify the amylase, an error was made.
The 5ul of the ACTBL2 Primer mix containing the beta-Actin was not added for one of the subject’s samples used for the gel photo, Figure 6. This can be seen in Figure 6, as the first band in the 2nd and 3rd lane is missing. Hence, the gel electrophoresis machine was able to use the beta-Actin as control in order to measure the copies of the AMY1A gene, which represents the absence of the beta-Actin gene. Therefore, another subject’s gel electrophoresis sample was used to obtain the adjusted volume values seen in Table 1 and the semi-log graph in Figure 8. Another source of error could have taken place during lab seven, gel electrophoresis. During the lab, an accurate pipetting of the DNA sample from the PCR reaction tube is difficult to obtain. This could have caused cross contamination amongst solutions in adjacent wells. This could have led to inaccurate results hence giving inaccurate gene copy numbers for their respective salivary amylase concentrations.
Furthermore, the concentration of the agarose gel could have resulted in a source of error. If the concentration is higher or lower than the fragments of the DNA, it will migrate towards the cathode too slowly or too quickly, resulting in inaccurate base pair size measurements and volume adjusted values. This would have led to inaccurate number of AMY1A gene copies resulting in inaccurate results.
