Running Header: ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS

Analysis of Restriction Enzymes Using

Electrophoresis
Emily Corcoran, Jennifer Herrera, Allen Hong, Erin Murphey,
Jamie Navarro, Melissa Restrepo, and Julianna Saez
Block 1
25 October 2018
ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​1

Introduction (JS, JN, MR)

Restriction enzymes, also referred to as restriction endonucleases, are essential in bacteria

cells, especially when it comes to the process of bacterial transformation. Bacterial

transformation is the altering of genetics as a result of the introduction of foreign DNA in a cell

(Griffiths ​et al.,​ 2000). This procedure was discovered in 1928 by Frederick Griffith through his

testing ​Streptococcus pneumoniae​. From his observations and the results of the experiment,

Griffith deemed the presence of a “transforming principle” (Shuttleworth, 2008). Later in 1944,

DNA was discovered to be the “transforming principle” behind the transformation of bacteria

(Cobb, 2014). Restriction enzymes recognize certain DNA sequences from a foreign source, thus

allowing the DNA to be pasted into a plasmid, a small circle of DNA present in bacteria that can

be carried into another cell. After this, the process where plasmid is placed into bacteria, known

as transformation, is ready to commence (“Bacterial Transformation,” 2007).

The specific restriction enzymes utilized in this study for electrophoresis were:​ Bam​HI,

​ I, and ​Hind​III. Each restriction enzyme recognizes a different base sequence that is used to
EcoR

determine the cut site for the DNA strands, thus causing every enzyme to cut the DNA at

different locations (“Restriction Enzyme Digestion,” 2018). This process can also be referred to

as restriction mapping. ​Bam​HI, from ​Bacillus amyloli​, is a type II restriction endonuclease that

recognizes six base pairs of DNA. This enzyme binds at the sequence of 5’-GGATCC-3’ and

cuts after the 5’-guanine, creating sticky ends of four base pairs (Silva, Leslie, and Marcey,

2001). Upon binding to DNA, many ​Bam​HI subunits make a connection with phosphates of the

DNA half-sites; base pairs make connections with the ​Bam​HI subunit and nitrogenous bases. The

bases are then bound with the protein through hydrogen bonds (Silva, Leslie, and Marcey, 2001).
ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​2

​ I is another enzyme that binds to the sequence 5’-GAATTC-3’ and cuts between the G and
EcoR

the A (“Restriction Enzymes”). It then creates sticky ends of four base pairs. ​Hind​III, from

Haemophilus influenzae,​ is another type II restriction enzyme that binds to 5’-AAGCTT-3’

sequence and cuts after the first A (Nasri and Thomas, 1986).

Gel electrophoresis is used to separate mixtures of DNA, RNA, and protein according to

their size (“Gel Electrophoresis,” 2007). The charged molecules travel through a gel when an

electric current is applied. The electricity causes one end of the apparatus, the anode to be

positively charged, while the other end, the cathode is negatively charged. When the electric

field is activated, the charged molecules within each sample migrate through the agarose gel

towards the opposite charge. Since DNA is negatively charged, the electric current causes the

DNA to migrate towards the positively charged end (“What is Gel Electrophoresis?” 2006).

During this process, shorter fragments of DNA move faster, therefore travelling a longer

distance. Larger fragments move slower through the gel matrix, resulting in the fragments

traveling a shorter distance. The gel most commonly used for this procedure is a gelatinous

substance called agarose, which is derived from seaweed (Cheriyedath, 2018). The agarose gel is

formed with tiny wells that house the samples. The process of gel electrophoresis is used for

DNA fingerprinting in forensic sciences and paternity testing (“Gel Electrophoresis,” 2007).

Other applications of gel electrophoresis include analyzing genes associated with specific

diseases and antibiotic resistance, profiling for taxonomy studies to distinguish various species,

and studying the structure and function of proteins (Cheriyedath, 2018).

In this study, gel electrophoresis focused on comparing the sizes of restriction enzyme

cuts on DNA. The restriction enzymes that were used were: ​Bam​HI, ​EcoR
​ I, and ​Hind​III. It has
ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​3

been hypothesized that as the distance increases, the length of the base pairs will decrease in the

ideal model. ​The purpose of this study is to analyze the base pair lengths cut by the restriction

enzymes ​in the ​DNA fragments following electrophoresis and compare them to an ideal model.

Methodology (EC)

Procedure:

This study was completed between Monday, October 15 and Thursday, October 18, 2018,

in the Marine Academy of Technology and Environmental Science (MATES) Biotechnology

laboratory classroom. TBE buffer was diluted using 25 mL of buffer concentrate mixed with 475

mL of distilled water in a 1 L beaker, and 60 mL of this solution was set aside to be used in the

agarose solution. The remaining 440 mL of TBE buffer solution was dyed using 24 drops of

stain. To create the 0.8% agarose solution, 0.48 grams of powdered agarose was added to the 60

mL of buffer in a 100 mL Erlenmeyer flask. This mixture was then microwaved for two

45-second intervals. It was cooled for ten minutes, until the temperature of the flask was safe to

touch. Afterward, 2 drops of stain

were added to the solution. The

solution was poured into the casting

tray until it was up to about 1 mm

below the notches on the sides of the

casting tray. The well-forming comb

was inserted quickly after the solution was poured (​Figure 1​). The gel was left to set for about

20 minutes, at which point the rubber edges of the casting tray and the comb were removed, and

the gel plate was gently slid out of the casting tray into a weigh boat containing a small amount
ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​4

of the TBE buffer solution. The weigh boat and the beaker were then both covered with parafilm,

labeled, and placed in the refrigerator.

The procedure was continued on Tuesday, October 16, 2018, with the restriction digest

process. The enzymes used for the restriction enzyme were

Bam​HI, ​Eco​RI, and ​HindI​ II with a control with no enzyme.

Four 1.5 mL tubes were labeled according to the restriction

enzyme. A micropipette was used to add 4μ of DNA and 5

μ of buffer into each of the four tubes, using a new tip for

the two substances (​Figure 2​). The tubes were spun in a

centrifuge for about 1 second to ensure that all contents

were pooled at the bottom. Then, 1μ of each enzyme were

dropped into their respective tube. Afterward, 1μ of

distilled water was dropped into the tube labeled –, using a

new tip for each reagent. The reagents in each tube were

then mixed by tapping the sealed tubes against the

lab table for 1 to 2 minutes. All tubes were then

placed in a 37 ̊C running water bath for 2 hours and 6

minutes to incubate (​Figure 3​). Afterwards, they

were frozen in a test tube rack overnight.

On Wednesday, October 17, 2018, the gel

was loaded and run in the electrophoresis chamber.

The gel plate was placed in the tray, positioned with

ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​5

the wells on top of the black anode mark, and set the tray into the electrophoresis chamber. The

chamber was filled with the TBE buffer solution until the solution covered the gel, and no

bubbles appeared on top of the wells (​Figure 4​). Next, a micropipette was used to add 1 μL of

loading dye to each reaction tube. The dye was mixed

with the digested DNA by tapping the tubes on lab

tables and pulsing them in the microcentrifuge for 1

second. The sample in each reaction tube were then

loaded into a separate well in the gel using a

micropipette and a different tip for each tube. To load

the samples, the micropipette was steadied over the

well, and air in the tip end was expelled. The pipet tip

was dipped through the surface of the buffer and

positioned over the well. The mixture was slowly

ejected into the well (​Figure 5​). After these steps were completed, the top of the electrophoresis

chamber was closed with the electrical leads

connecting to a power supply, anode to anode and

cathode to cathode. The power supply was turned on,

and the voltage was set to approximately 125 volts.

The DNA was allowed to electrophorese for 33

minutes and 19 seconds, at which point the power

supply was turned off, the leads were disconnected

from the inputs, and the top of electrophoresis

ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​6

chamber was removed. The casting tray was then carefully

removed from the chamber, and the gel was slid into a

weigh boat to be stained with Carolina BLU™ Final Stain.

The stain was poured into the weigh boat until it covered

the gel plate, and the boat was agitated for 5 minutes until

the excess dye was poured back into the bottle using a

funnel. The gel was then rinsed with a light stream of tap

water for two minutes (​Figure 6​).

On Thursday, October 18, 2018, the gel was stained again, viewed on the lightbox, and

observations about it were made. The gel was flooded with

Carolina BLU™ Final Stain in the weigh boat and was

allowed to stain for 15 minutes before the excess dye was

poured back into the container and the gel was rinsed in a

light flow of tap water. Afterward, the gel was viewed on a

lightbox. Photographs of the gel were taken to record the

appearance of the bands of DNA that formed due to the

electrophoresis (​Figure 7​). Once completed, the gel plate

was disposed of, and the additional buffer solution was

poured into a waste container.

Data Analysis:

The data was analyzed using the ideal model provided in the “DNA Restriction Analysis

Kit Instructor’s Manual” (1995). The band distance (mm) was measured from the ideal model
ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​7

and compared to the provided base pair lengths (kbp). An exponential trendline was derived

from the comparison of the ideal band distance and base pair lengths (​Figure 8​). The formula for

the tread line was used to determine the base pair lengths of the experimental DNA.

Data (AH, EM)

Table 1: ​The restriction enzyme ​HindI​ II with ideal distance (mm) and total base pairs (kbp) in

comparison to the experimental values following electrophoresis.

Table 2: ​The restriction enzyme ​Eco​RI with ideal distance (mm) and total base pairs (kbp) in

comparison to the experimental values following electrophoresis.

ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​8

Table 3: ​The restriction enzyme ​Bam​HI with ideal distance (mm) and total base pairs (kbp) in

comparison to the experimental values following electrophoresis.

Figure 8: ​A comparison of the band distance (mm) to the base pair length (kbp) of each type of

restriction enzyme of the ideal model on a logarithmic scale (n = 21). Base pair lengths were

provided from the “DNA Restriction Analysis Kit Instructor’s Manual” (1995).
ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​9

Figure 9: ​A comparison of the experimental band distance (mm) to the calculated base pair

length (kbp) of each restriction enzyme on a logarithmic scale (n = 9). The base pair lengths were

derived from the formula given in ​Figure 8​. The red and blue points represent the same base pair

length for both the ​Eco​RI sample and the ​HindI​ II enzyme sample.

Results (EC, JN)

The experimental data shows that the ​Hind​III enzyme cut three different band lengths

with band distances (mm) between 13 and 17 mm, making them less than the seven ideal bands

with distances between 24 and 72 (​Table 1​). The ​Hind​III total base pairs lengths (kbp) were

between 37.2 and 77.1 kbp. This makes them greater than the ideal base pairs lengths between

27.5 and 2.0 kbp (​Table 1​). The experimental data band distances for the four ​Eco​RI bands were
ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​10

between 11 and 17 mm, while the seven ideal distances for this restriction enzyme were between

​ I were between 41.6

25 and 55 mm (​Table 2​). The experimental data base pair lengths for ​EcoR

and 77.1 kbp, while the ideal distances for this restriction enzyme were between 24.8 and 3.5 kbp

​ I bands are 9 and 20, while

(​Table 2​). The experimental data band distances for the two ​BamH

the seven ideal distances for this restriction enzyme were between 29 and 44 mm (​Table 3​). The

​ I were 46.9 and 25.2 kbp, while the ideal distances

experimental data base pair lengths for ​EcoR

for this restriction enzyme were between 16.8 and 5.5 kbp (​Table 3​). For all three restriction

enzymes, there were less values recorded for experimental data (n = 9) than the ideal model (n =

21) because there were fewer bands. There was a negative exponential correlation between the

band distance and base pair lengths in the ideal model (​Figure 8​). The ideal model was used to

determine the base pair lengths of the experimental data by deriving the formula given in ​Figure

8​ (​Figure 9​).

Discussion (AH, EM, JH, JS)

The results of the restriction enzymes following electrophoresis suggested that greater

distance from the wells resulted in DNA fragments with fewer base pairs. Based on the ideal

model, there is a negative exponential correlation between band distance and base pair length

(​Figure 1​). This was the expected result, as shape and size determine the rate the fragments

move through the gel. Other studies suggest that fragments with fewer base pairs will move at a

faster rate through the gel which results in more distance between the wells and the fragment

(“Restriction Enzyme Digestion,” 2018). Larger DNA fragments with more base pairs will travel

at a slower rate and will be closer to the wells than the smaller fragments.
ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​11

The broadest range in fragment size cuts were found in both ​Bam​HI and ​Eco​RI restriction

enzymes. This was the largest range in fragment size cuts shown for all three enzymes.

Furthermore, the restriction enzyme ​Hind​III was observed to have the smallest size range among

all three enzymes. This contradicts the ideal model because the ideal data suggests that ​HindI​ II

​ I would have the smallest size range

would have the largest fragment size range, while ​BamH

(​Tables 1, 3​). A possible cause for observing this disparity in the data was limited time for the

DNA fragments to separate into smaller fragments as it moved farther into the gel (“Gel

Electrophoresis” 2016). By doing this, the fragments stick together and their size range is

minimized.

Using the results of the analysis of the ideal model, the base pair length of the

experimental data was able to be predicted (​Figures 8, 9​). The base pair length was predicted

using the formula of trendline of the ideal model. All three enzymes were used to create the

formula to provide the greatest sample size. A 0.8% agarose solution was used in the ideal

model. The formula derived was l = 78.493e−0.056d where ​d i​ s the band distance (mm) and ​l​ is

the base pair length (kbp). This formula was used to predict the base pair lengths of the

experimental data. A formula derived from a DNA ladder using various concentrations of

agarose solution can be used to create formulas to determine base pair length (​Table 4​). The base

of the ideal model formula which used a 0.8% agarose solution, lies between the bases of the

ladders that used a 0.7% and 1.0% agarose solutions. The base, in this case 78.493, causes the

base pair lengths to increase at a greater rate (Larson, 2014). This shows the effect of the agarose

concentration on the movement of the bands. A solution with a higher concentration of agarose
ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​12

will slow down the movement and decrease the separation of the bands (Douches and Song,

2009).

Overall, there are similarities and differences between the experimental results and the

​ I enzyme cut the DNA into the

results from other studies. Other studies suggest that the ​BamH

least amount of fragments. This coincides with the experimental data because it also suggests

that BamHI cut the DNA into the least amount of visible fragments (​Table 3​). The other study

also found that of the tested restriction enzymes, ​Hind​III should cut the DNA into the most

fragments (Kopchak, 2016). However, the experimental data seen here contradicts those results

because ​Eco​RI cut the DNA into the most visible fragments (​Table 2​). In another study, the

fragments cut by the ​Hind​III enzyme had the most base pairs which meant they were the longest

(Clark, 2016). The experimental data suggests a similarity to this study because ​Hind​III did cut

the longest fragment which consisted of 77,100 base pairs each. However, the experimental data

​ I also cut a fragment of equal size (​Tables 2, 3​).

differed from this study because ​EcoR

​ I cut the smallest fragments (Clark, 2016).

Furthermore, the other study also found that ​EcoR

However, this does not coincide with the experimental data. According to the data, ​Bam​HI cut

the smallest fragment which consisted of only 25,200 base pairs (​Table 1​).

In the experimental gel, there were less visible bands observed when compared to the

ideal gel. In the experimental gel, electrophoresis only occurred for 33 minutes and 19 seconds at

about 125 volts of electricity. These time constraints limited the movement of the bands because

ideally, electrophoresis would take place at a low voltage over many hours. The voltage was also

set higher than usual to try to compensate for the limited amount of time that was available for

​ I enzyme
the lab. However, this caused bleeding to occur in the first well, containing the ​BamH
ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​13

and could have consequently affected the results. In addition, more bands are visible when the

amount of the sample of each respective restriction enzyme is maximized in the well. ​It is also

possible that the DNA fragments were damaged during shipping prior to the lab. Upon arrival,

the package containing the DNA was no longer fully frozen. This may have resulted in the DNA

breaking into fewer fragments than shown in the ideal model. Furthermore, after the DNA was

found slightly thawed, it was left in the freezer to return to a solid state.

It is recommended for future studies to perform electrophoresis for a longer period of

time so the DNA fragments would have more time to migrate through the gel. Furthermore, there

could be better management of the DNA fragments, ensuring that it remains frozen during

transport. The results of the analysis of the ideal model supported the hypothesis that there is a

negative correlation between the band distance and the base pair length. The base pair length of

the experimental model was able to be predicted using the ideal model.

Table 4: ​Two formulas were created from DNA ladders by two different manufacturers. These

two different ladders were created for electrophoresis in different agarose concentrations.

Conclusion (JH, MR)

In conclusion, the data suggests that the shape and size of the DNA fragments are linked

to the rate that the fragments travel through the gel. This implies that fewer number of base pairs

resulted in greater distance from the wells. Therefore, there was a strong negative correlation

between fragment size and distance. Further studies should include gel electrophoresis occurring
ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​14

over a longer period of time at a lower voltage for the visibility of more bands. DNA fragments

should remain fully frozen before use to limit possible damage to the DNA. Retaining the DNA

fragments at a solid state is crucial to avoid physically altering the restriction enzymes.

Acknowledgements (MR, AH)

Thank you to Mr. Sprague for the guidance provided on the procedure, as well as

providing us with the opportunity to conduct this lab. In addition, our thanks also go out to

MATES for permission to utilize the equipment necessary to conduct this study. Lastly, we

would like to thank our peers for their feedback and for taking the pictures used in this study.
ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​15

References (MR)

Bacterial transformation. (2007, November 16). Retrieved October 21, 2018, from

https://www.sciencelearn.org.nz/resources/2032-bacterial-transformation

Cheriyedath, S. (2018, August 23). What Does Gel Electrophoresis Involve? Retrieved

October 23, 2018, from

https://www.news-medical.net/life-sciences/What-Does-Gel-Electrophoresis-Involve.a

spx

Clark, N. (2016, February 25). Lambda DNA Lab Report. Retrieved October 20, 2018, from

https://noahsbiologyblog.wordpress.com/2016/02/15/lambda-dna-lab-report/

Cobb, M. (2014, January 20). Oswald Avery, DNA, and the transformation of biology.

Retrieved October 20, 2018, from

https://www.sciencedirect.com/science/article/pii/S0960982213015157

DNA Restriction Analysis Kit Instructor’s Manual. (1995). Retrieved October 20, 2018, from

https://drive.google.com/file/d/1efmEUJQiCqefn-gMKgvOT9BdhqzG7sap/view

Gel electrophoresis. (2007, November 20). Retrieved October 21, 2018, from

https://www.sciencelearn.org.nz/resources/2029-gel-electrophoresis

Griffiths, A. J., Miller, J. H., Suzuki, D. T., Lewontin, R. C., & Gelbart, W. M.​ (2008).

Bacterial transformation. Retrieved October 20, 2018, from

https://www.ncbi.nlm.nih.gov/books/NBK21993/

Kopchak, R. (2016, May 14). DNA Restriction Analysis Lab. Retrieved October 20, 2018,

from https://www.scribd.com/doc/314354682/dna-restriction-analysis-lab-report

Larson, R. (2014). Precalculus with limits (3rd ed.). Boston: Brooks Cole.
ANALYSIS OF RESTRICTION ENZYMES USING ELECTROPHORESIS ​16

Nasri, M., & Thomas, D. (1986, January 24). Relaxation of recognition sequence of specific

endonuclease HindIII. Retrieved October 23, 2018, from

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC339466/

Restriction Enzyme digestion - How does it work? Why is it useful? (2018). Retrieved

October 20, 2018, from

https://www.apsnet.org/edcenter/K-12/TeachersGuide/PlantBiotechnology/Pages/Acti

vity3.aspx

Restriction enzymes. (n.d.). Retrieved October 23, 2018, from

http://parts.igem.org/Help:Restriction_enzymes

Shuttleworth, M. (2008). Transforming Principle. Retrieved October 23, 2018, from

https://explorable.com/transforming-principle

Silva, N., Leslie, K. A., & Marcey, D. (2001). BamHI Complexed with B-DNA. Retrieved

October 21, 2018, from

http://earth.callutheran.edu/Academic_Programs/Departments/BioDev/omm/bamh1/m

olmast.htm

Song, G., & Douches, D. (2009). Agarose Gel Electrophoresis. Retrieved October 20, 2018,

from https://msu.edu/course/css/451/Lecture/PT-electrophoresis (2009).pdf

What is Gel Electrophoresis? (2016, January 25). Retrieved October 20, 2018, from

https://www.yourgenome.org/facts/what-is-gel-electrophoresis

Final Edit (EM)