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Biology Lab Report - Dr. Kajuira

Cellular and Molecular Biology (McMaster University)

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Biology Lab Report

Alakshiya Arumuganathan
400083138

Lab Section 28; Lab TA: Natashia

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Abstract:

Amylase is an enzyme produced by the salivary glands and hydrolyzes the alpha

glycosidic linkages in polysaccharides such as starch and glycogen (Tracey 2016). The

purpose of this lab is to determine if there is a relationship between enzyme productions,

gene copy number, and gene evolution through amylase concentration, amylase gene

copy number and ancestral diet. Individuals from distinct backgrounds were examined to

help analyze the correlation between the different starch populations and their ancestral

diet. I believe that levels of starch consumption in my ancestral diet are low, which may

make my gene copy and amount of amylase being produced to be significantly lower than

the mean. It is believed that individuals with a high starch consumption can lead to a

significantly higher that the mean number of AMY1 gene copy number and amylase

concentration and vice versa for low starch consumption. By the end, the hypothesis was

invalid as the results were unreliable causing it to have no correlation between enzyme

production, gene copy number, and gene evolution. On the other hand, my hypothesis

was right as my gene copy and amount of amylase being produced was significantly

lower than the mean.

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Introduction:

Is there an association between enzyme production, gene copy number, and gene

evolution? Amylase is an enzyme that helps digest carbohydrates, which are made in the

pancreas, and glands that make saliva. In this experiment, the gene that produced amylase

is AMY1A, which is also known as alpha 1A. Enzymes are proteins that act as catalysts

during biochemical reactions. Our bodies can't effectively use the starches in our diet

unless they are first broken down into simple sugars. Amylase is the protein in our saliva

that breaks down starch in which it catalyzes the breakdown of starch into sugars in the

mouth and small intestine. Individuals from a population that consume a high starch diet

are known to have higher amounts of AMY1A gene repeats than individuals from a

population who consume a low starch diet. There is a positive correlation between the

number of AMY1A gene repeats and the amount of amylase produced (Tracey 2016).

The aspects that are investigated in this experiment are how enzyme production, gene

copy number and gene evolution are related through amylase concentration, gene copy

number and ancestral diet type.

This image shows the breakdown of the amylase enzyme.

In addition, there is a variety in the dietary starch intake among the human

population. That is, some populations having a high starch diet, which comprises of

potatoes, wheat, rice and so on. However, a small fraction of the population with low

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starch comprises of few starchy foods such as meats, fruits, milk and so on. (Perry et al.

2007)

People who live in humid and arctic like living conditions tend to eat less starch

than those living in more dry environments. This is because the amount of starch plants

being produced and thus this affects ones ancestral starch diet (Brown 2014). European

Americans, Japanese, and Hadza hunter-gatherers rely greatly on starch whereas Biaka

and Mbuti rainforest hunter-gatherers, and the Yakut, a pastoralist, fishing society were

considered the low starch diet populations (Perry et. Al 2007).

Therefore, I believe that if the level of starch in my ancestral diet is low, it may make my

gene copy to be significantly lower than the mean as well as the amount of amylase being

produced lower than the mean. In general, it is hypothesized that individuals with a copy

number of the salivary amylase gene (AMY1) is correlated positively with salivary

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amylase protein level and that individuals with high-starch diet populations tend to have

more AMY1 copies than those with traditionally low-starch diets (Perry et al. 2007).

Variables

Dependent Variable: Concentration, gene copy

Independent Variable: Hypothesized ancestral diet

Method:

Lab 2:

 Refer to the lab manual for more detailed information

Lab 3:

 Refer to lab manual for more detailed information

Lab 4:

 Refer to lab manual for more detailed information

Lab 5:

 Refer to lab manual for more detailed information

Lab 6:

 Refer to lab manual for more detailed information

 Changes made: PCR sample was put in the thermocycler for 1 minute rather than

3-5 seconds

Lab 7:

 Refer to the lab manual for more detailed information

 Changes made: During part b: loading the gel;

o 5ul of ladder were loaded into the first well on the left side rather than

10ul

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Lab 8:

 Refer to lab manual for more detailed information

iesults:

Lab 3: Amylase Assay

 In lab three, data obtained from Set 1 tubes 1-6 are plotted using excel to obtain a

standard curve (calibration curve) as well as the equation of the line. In figure 1

(seen in appendix), the scatter plot graph shows the relationship of light

absorbance (650nm) vs. Amylase Concentration (ug/ml). A linear trend line was

inserted and it gave the equation: y= -0.0674x + 1.6671. The r-squared value

determines how close and accurate the values are to the linear trend line. In this

case the value was 0.9431. This value shows that the there is a strong correlation

between the light absorbance and amylase concentration.

Lab 4: Amylase Assay and Salivary Amylase Concentration

 In lab four, the concentration of amylase in my saliva was calculated using the

equation from the previous lab. The calculation for this is in the appendix

(Calculation 1).

 Table 1: This table shows the absorbance values for the particular tube sets

Tube Set Absorbance Value (620nm)

10XI 0.04
100XI 1.2
1000XI 1.49
Lab 5: DNA Extraction and Cheek Epithelium

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 In Lab 5, I created a graph using information pertained from lab 4. The

hypothesized levels of diet and calculated amylase mg/ml are taken from each

student in the lab and the data was posted on Avenue to Learn. I created two

graphs; one that displayed standard deviation error bars and one displaying range

error bars. Figure 2 and 3 display both graphs and can be seen in the appendix.

Lab 6: PCR

 In Lab 6, the amylase is amplified through PCR. The sample went through 35

amplification cycles. Therefore, 3.4 x 1010 copies of the target sequence will be

produced. The amount of highly purified water needed for the primer is 363 uL

and this calculation is in the appendix (Calculation 2).

Lab 7: Gel Electrophoresis

 The 100bp ladder was used as a reference to measure the distance of the fragment

migrated and to help figure out the estimated size of my PCR fragment. A semi

log graph was created to help determine the sizes of the fragments of the other

bands on my gel (figure 4). At 600bp from the 100bp ladder graph, my PCR

fragment migrated 30 mm and at 900bp my fragment migrated 25mm. My first

band (600bp) was amylase and the second was actin. The gel photo is in the

appendix (image 1).

Lab 8: Gene Duplication and Data Analysis

 The adj. vol. (Int) data values for both actin and amylase DNA fragments were

collected then converted into number of amylase copies. A scatter plot was then

made to show the number of amylase copies vs. the calculated amylase mg/ml

(figure 5).

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Discussion:

The purpose of this lab is to understand the connection between enzyme productions,

gene copy number, and gene evolution. As my hypothesized level of starch diet is low, I

believe that my gene copy number and amount of amylase being produced is going to be

significantly lower than mean. In figure 1 (seen in the appendix) the graph represents the

relationship between light absorbance values verses amylase concentration ug/ml. The

information on this graph was pertained from the binding of starch solution and iodine

solution. All test tubes solutions varied in a blue color. The solution with the lowest

amylase concentration and highest absorbance value of 0ug/ml had a very intense dark

blue color. As the amylase concentration increased the absorbance value decreased,

meaning the intensity of the blue color decreased such that of when the amylase

concentration was 25ug/ml (light blue color). It can be deduced that as absorbance values

decrease, the amylase concentration increases resulting in the iodine starch solution to go

from an intense blue color to a very faint blue (figure 1). An equation and R2 value was

given when adding a linear trend line to the graph. The R2 value was 0.9431, which

indicates that there was a very strong correlation between light absorbance and amylase

concentration. The equation created is used in lab four to determine the amylase

concentration in my saliva (Calculation 1). Figure 2 in the appendix represents the mean

value of amylase concentration dependent on the hypothesized levels of starch with

standard deviation error bars and figure 3 represents the same graph as figure 2 but with

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range error bars. The standard deviation error bars show how the data is spread around

the mean. The standard deviation error bar for the highest level of starch diet is higher

than that of the moderate diet indicating that the mean and sample size is higher than the

average. Figure 3 represents a histogram graph that describes the relationship between the

mean value of amylase concentration and hypothesized levels of starch with range error

bars. The range error bars are used to describe the range of the data points from lowest to

highest. The range error bar for the high level of starch diet was very high compared to

moderate and low due to a higher variation within the set. No standard or range error bar

is created for the low starch diet as there was only one value for low diet. A forward and

reverse primer was used for the Polymerase Chain Reaction (PCR). It is necessary to

have two primers because they are needed for PCR and are also complimentary to DNA

regions at the 5’ or 3’ end of the DNA region (Tracey 2016). The DNA sample went

through 35 amplification cycles and it led to 3.4 x 1010 copies of the target sequences

being produced. The amplified DNA went through gel electrophoresis where it is used to

separate the DNA fragments based on their base pair size. In a 100bp ladder, the sample

was placed into wells in the gel. The wells were then viewed using the BioRad Gel

DocTM EZ Imager. Image 1 in the appendix shows the different bands and wells. The

first well on the left is the sample well; this was used to create a semi log graph (figure 4)

to then later find the estimated size of my amylase and actin fragment. Using the BioRad

Imager, an adj, vol. (Int). value was given and recorded to calculate the gene copy

number, which was done by the lab TA. The data given shows that majority of the

individuals had one gene copy, 2 individuals with 3 gene copies and 1 individual with 2

gene copies (figure 5). This variation may have been caused, as there was a small sample

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size. Sources of error could have led the results to be uncertain. For example, there may

have been an error in pipetting, human error and sample size may have cause the data to

have variation. There may have been an error due to pipetting, as perhaps all of the

solution may not have gone into the pipette. This can be improved by reducing the

number of pipetting steps, changing the tip of the pipette each time and pipetting larger

volumes for greater accuracy. Human error could have also occurred when putting the

saliva into the micro centrifuge tube. As the sample size was 19 individuals, the results

would not be accurate; therefore a larger sample size is needed to see more precise and

reliable results. The scope of this lab is to determine the relationship between enzyme

production, gene copy number and gene evolution. To figure out if there is a relationship,

an analysis of amylase concentration, amylase copy number and ancestral diet type was

used. It is predicted that individuals that possess a high starch ancestral diet type tend to

have a higher amylase concentration and gene copy number and vice versa for

individuals with low starch diets. This however, is false, which leads to the conclusion

that the predicted hypothesis was wrong. Individuals with one gene copy number with a

higher frequency showed almost no association of amylase concentration and ancestral

diet type. Conversely, my hypothesis regarding my diet type was correct. My gene copy

number and amylase concentration was lower than the mean value. Furthermore, Perry et

al’s assumption on the relationship between the amylase concentration and gene copy

number stated in the introduction being positively correlated does not correspond with

figure 5 where there is no correlation established (Perry et al. 2007). The gene copy

numbers gathered were from 1-3 but, it is believed that humans possess 2-20 copies of

the AMY1A gene (Tracey 2016). The results were inaccurate, therefore, the hypothesis is

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rejected and no correlation is seen between amylase concentration, gene copy number

and ancestral diet.

Appendix:

Figure 1: This graph displays the relationship between light absorbance and amylase

concentration

Lighi Absorbance vs. Amylase Conceniraiion

2
1.8
1.6 f(x) = − 0.07 x 8 1.67
Absorbance (620nm)

1.4 R² = 0.94
1.2
1
0.8
0.6
0.4
0.2
0
0 5 10 15 20 25
Amylase Conceniraiion ug/ml

Calculation 1:

Concentration of Amylase in my saliva equation derived from graph:

y= -0.0674x + 1.6671

y – 1.6671= -0.0674x

(y – 1.6671)/(-0.0674) = x

Dilution Factor

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100XI

X1= (1.2 – 1.6671)/-0.0674 = 6.93ug

Multiply by dilution factor

6.93 x 100 = 693 ug/mL

1000 XI

X2= (1.49 – 1.6671)/-0.0674 = 2.63ug

Multiply by dilution factor

2.63 x 1000= 2630ug/mL

Convert ug to mg

1ug=0.001mg

693ug/ml x 0.001mg/1ug = 0.693 mg/ml

2630ug/ml x 0.001mg/1ug = 2.63 mg/ml

Average both values out:

(0.693 + 2.63)/2 = 1.66 mg/ml

Therefore the concentration of amylase in my saliva was 1.66 mg/ml.

Figure 2: This graph represents the relationship between the mean value of salivary

amylase and the hypothesized levels of starch with standard deviation error bars

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Mean Value of Salivary Amylase vs. Hypoihesized Levels of Siarch wiih Siandard Deviaiion Error Bars
5

3
onceniraiion(mg/ml)

2
ylaseC

1
alueofSalivaryA

0
Moderate High Very High
eanV

Hypoihesized Levels of Siarch

M

Figure 3: This graph represents the relationship between the mean value of amylase

concentration and the hypothesized levels of starch with range error bars

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Mean Value of Amylase Conceniraiion vs. Hypoihesized Levels of Siarch wiih Range Error Bars
4

3
g/ml)

2
ylaseConceniraiion(m

1
alueofSalivaryA
eanV m

0
M

Moderate High Very High

Levels of Siarch

Calculation 2:

Convert nM to uM

1 uM= 1000 nM

=36.3nM x (1 uM/1000nM)

=0.0363 uM

Amount of uL highly purified water need to be added to nM of dried primer in order to

create a 100uM stock solution

(0.0363 uM)/ 100 uM/L = 0.000363 L

0.000363L x 1000ml/L = 0.363 mL

0.363 mL x (1000uL/1 mL) = 363 uL

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There are 363 ul of highly purified water in order ot create a 100uM stock solution

Image 1: Gel Photo

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Figure 4: This is an image of my semi log graph

Figure 5: This graph shows the relationship between the number of amylase copies and

calculated amylase mg/ml

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Calculaied Amylase vs. Number of Amylase Gene Copies

6

5
Number of Amylase Gene Copies

0
0.5 1 1.5 2 2.5 3
Calculaied Amylase (mg/mL)

Works Cited

Perry G, Dominy N, Claw K, Lee A, Fiegler H, Redon R, Werner J, Villanea F, Mountain

J, Misra R, and others. 2007. Diet and the evolution of human amylase gene copy number
variation. Nat. Genet. [Internet]. [cited 2016 Nov. 20]; 39(10): 1256 – 1260. Available
from: http://www.nature.com/ng/journal/v39/n10/pdf/ng2123.pdf

Elizabeth Brown. 2014. Starchy Dangers in Human Evolution [thesis]. [America]:

Harvard University

Tracey A. 2016. Biology 1A03 laboratory manual/notebook. Plymouth (MI): Hayden-

McNeil.

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BIOLOGY 1A03 FORMAL LAB REPORT GRADING SCHEME

THIS PAGE SHOULD BE INCLUDED AS THE LAST PAGE OF YOUR FORMAL REPORT

NAME:ALAKSHIYA ARUMUGANATHAN STUDENT NUMBER: 400083138

LAB SECTION: 28 TA NAME: NATASHIA

ABSTRACT /4

INTRODUCTION /5

METHODS /3

RESULTS /7

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DISCUSSION /8

LITERATURE CITED /4

FIGURES AND TABLES /8

GENERAL (title page, format, writing) /6

Grade /45

TA Initials m

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