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The Basis of DNA and Gel Electrophoresis

Tarek Zieneldien

11/04/2017

BSC 2010L Section 027

Mackenzie Ebert & Melanie Aviles


Gel electrophoresis is a commonly used method in forensics when aiming to identify

criminals. This experiment used gel electrophoresis to match the DNA found in a crime scene with

the DNA of one of the two possible suspects. The DNA sample in the crime scene, and the DNA

samples of both suspects were cut with enzymes EcoRI and HindIII. The crime scene samples

were already prepared in this experiment. However, the samples of the suspects needed to be

prepared with test tubes. Then, a 0.8% agarose gel was prepared and stained with ethidium bromide

because DNA is transparent. While handling ethidium bromide, it was crucial to wear gloves

because it could potentially cause cancer. Thereafter, 15 microliters of each sample were loaded

onto the device along with two markers at each end. The gel was then run for 40 minutes at 150

volts and 150 milliamperes. Finally, the gel was placed under ultraviolet light in order to see the

image. When the produced gel image was examined, it was determined that suspect 2 was the

possible criminal. Suspect 2 matched both crime scene samples when cut with either enzyme. On

the other hand, suspect 1 only matched the sample that was cut with Restriction Enzyme 1.
Introduction

Deoxyribonucleic acid (DNA) is a heritable double-stranded molecule that contains the

genetic information necessary to produce proteins (Berkowitz, 2017). It is a polynucleotide that

is composed of a deoxyribose, a nitrogenous base, and a negatively charged phosphate group (Urry

et al, 2016). The nitrogenous bases are either pyrimidines which have one ring, or purines which

are double-ringed. (Upadhyaya, 2017). While pyrimidines could be either Cytosine (C) or

Thymine (T), purines could be either Adenine (A) or Guanine (G) (Urry et al, 2016). In DNA,

Adenine must pair with Thymine, and Guanine must pair with Cytosine (Upadhyaya, 2017). In an

organism, particular characteristics are determined by the heritable genetic units called genes

which hold a specific array of DNA in a locus, or particular location, in a chromosome

(Upadhyaya, 2017). Alleles, which are varied versions of genes, are different in the sequence of

their bases. Thus, they result in different expressions of the gene, which lead the characteristics of

an organism to be either recessive or dominant (Upadhyaya, 2017). Alleles are related to genes

because they are different versions of the gene which lead to the particular traits displayed by the

organism (Urry et al, 2016). These genes store genetic information in distinct code words

composed of three nucleotides, which allow for 64 unique code words to exist (Upadhyaya, 2017).

In regards to humans, their genome is comprised of 20,000 – 25,000 genes (Upadhyaya, 2017).

As such, the unique code words that compose genes, and the different variations of such genes,

cannot ever be equal for two particular individuals since it is so abundant. Also, there are possible

errors that could occur while DNA is replicating, which make individuals more unlikely to have

the same DNA, such as mutation from ultraviolet light, or even deletion (Upadhyaya, 2017).

DNA fingerprinting is a reliable and explicit source of identifying samples of DNA which

is generally more advantageous than other identification methods because it does not just omit a
particular match (Upadhyaya, 2017). DNA fingerprinting analyzes DNA samples of various

lengths or molecular weights by separating the samples with an electric field (Upadhyaya, 2017).

Although electrophoresis uses an electric field, porous gels are used in gel electrophoresis in order

to provide friction (Upadhyaya, 2017). The use of porous gels is crucial in gel electrophoresis

because the process aims to isolate the molecule based on the molecular weight, or length

(Upadhyaya, 2017). This isolation occurs because the movement of large molecules is slowed

down by friction, while smaller molecules are not affected as much. The porous gels that are used

for gel electrophoresis are typically polyacrylamide and agarose (Upadhyaya, 2017). The porous

gels in electrophoresis can also be manipulated to receive better results for the particular DNA

sample. The lower the concentration, the larger the pores are in the agarose. Smaller pores are able

to isolate small DNA pieces more effectively than larger pores and vice-versa (Upadhyaya, 2017).

Likewise, in order to generate finer resolution results, one should use elongated run times with

reduced voltages (Upadhyaya, 2017). Gel electrophoresis relates to DNA fingerprinting because

it is the economical and relatively quick process used in forensics to conduct DNA fingerprinting

(Upadhyaya, 2017).

Restriction enzymes are enzymes that aid in deducing the dissimilarity of DNA sequences

among varying individuals. Restriction enzymes function by linking to DNA at specific locations

called recognition sites. This binding leads to a cleavage between the DNA strands at the

recognition sites, and these sites tend to be 4 – 8 base pairs and symmetrical (Upadhyaya, 2017).

These restriction enzymes cut DNA in irregular manners which result in strands that are referred

to as "sticky" ends (Urry et al, 2016). This experiment made use of the restriction enzymes HindIII

and EcoRI which tend to cause sticky ends (Upadhyaya, 2017).


Materials and Methods
1.) Preparing the samples – restriction enzyme digestion

The DNA Fingerprinting experiment began by preparing the samples for the restriction

enzyme digestion (Upadhyaya, 2017). The tubes were labelled 1-4 to differentiate the digesting of

DNA with the different restriction enzymes, and the substances were added to the respective tubes

as shown on Table A (Respective Volumes Added to Test Tubes) (Upadhyaya, 2017).

Table A Test Reaction S DNA S DNA Enzyme Enzyme Final


Tube Buffer 1 2 (μl) 1 (μl) 2 (μl) Volume
(μl) (μl) (μl)
Suspect 1 10 15 0 15 0 40
1
Suspect 2 10 15 0 0 15 40
1
Suspect 3 10 0 15 15 0 40
2
Suspect 4 10 0 15 0 15 40
2
The data on Table A refers to the required volumes needed in the respective test tubes

The tubes labelled 1-4 were covered with the cap to avoid spillage, and gently tapped on

the table to mix and settle the solution at the bottom of the tube (Upadhyaya, 2017). Afterwards,

the tubes were incubated for approximately 45 minutes at 37 degrees Celsius. When this time

period was finished, the tubes were handed to the teaching assistant in order to refrigerate the

samples for gel electrophoresis (Upadhyaya, 2017).

2.) Loading and casting agarose as the porous gel for electrophoresis

The concentration needed of agarose was 0.8% which resulted to be 0.4 grams of agarose

per 50 milliliters of solution. To attain the desired amount of agarose, the agarose was weighted

with a scale. The agarose was then added to a 250 milliliter flask, along with 50 milliliters of l x

TBE buffer (Upadhyaya, 2017). The solution was then mixed thoroughly in order to disseminate
the agarose powder (Upadhyaya, 2017). The flask was then covered with a plastic wrap to reduce

the evaporation, and placed in a microwave for one minute to dissolve the agarose powder. The

flask was carefully removed from the microwave, and appeared to be transparent. Soon after, the

flask was placed on the table until the temperature lowered. While the flask was set aside, the gel

combs needed to be placed at the end of the tray. Afterwards, the tray was rigidly implanted in the

gel box as shown by the teaching assistant (Upadhyaya, 2017). When the flask became warm and

able to be touched, the teaching assistant added 2.5 microliters of ethidium bromide (EtBr) to DNA

in order to see the sample in ultraviolet light (Upadhyaya, 2017). Ethidium bromide demanded

proper equipment such as gloves when handling it because it has carcinogenic properties

(Upadhyaya, 2017). The agarose solution was then carefully poured into the tray, and it was crucial

not to pour the solution when it was scalding because it could have damaged the tray (Upadhyaya,

2017). Once poured, the agarose gel solution was allowed to settle in the tray for about 20 minutes

where it became nontransparent when it settled. Subsequently, the gel cooled down and had

become firm. The tray and gel were then detached from the gel box and repositioned. This step

was necessary because it aligned the wells with the negatively charged pole. Since DNA contains

a phosphate group, and is negatively charged, it will move towards the positive pole (Upadhyaya,

2017). The electrophoresis apparatus was then filled with an estimated 250 milliliters of 1X TBE

buffer which submerged the gel, and the gel comb was removed. The tubes labelled 1-4 then

received 5 microliters of 10x gel loading solution, and were gently blended. Then, 15 microliters

of every sample were loaded onto the electrophoresis device. Finally, the electric wires were

connected, and the gel was run at 150V and 150mA for 40 minutes. When the time was over, the

gel was removed and placed on the ultraviolet illuminator while being covered with a plastic wrap.
Results

Electrophoresis Gel Sample of Crime Scene and Possible Suspects

The results shown in the image above were attained through the use of gel

electrophoresis. Agarose gel was prepared, loaded, and casted in order to be run. The

samples were derived from the crime scene, and two possible suspects. The cutting

enzymes Restriction Enzyme 1 and Restriction Enzyme 2 were both used to cut each

sample because some organisms could have the same pattern for one enzyme.

In this experiment, it was difficult to read the produced image of the gel. This

occurred because of a mishap loading the gel which caused the bands to look uneven. When
viewing the crime scene sample that was cut with Restriction Enzyme 1, both suspects

matched the pattern caused by the cleavage of Restriction Enzyme 1. On the other hand,

the crime scene sample that was cut with Restriction Enzyme 2 only matched one of the

suspects. The suspect 2 sample that was cut with Restriction Enzyme 2 seemed to match

the crime scene sample that was cut with Restriction Enzyme 2. The suspect 1 sample that

was cut with Restriction Enzyme 2 did not match the crime scene sample cut with

Restriction Enzyme 2. Thus, it could be expected that suspect 2 was the possible criminal.

Suspect 2 matched both samples of the crime scene when they were cut with the respective

restriction enzymes. However, suspect 1 only matched one of the samples.


Discussion
When analyzing the image of the gel, it was concluded that suspect 2 was the

probable criminal. The suspect 2 sample matched the DNA from the crime scene when cut

with both restriction enzymes. However, this individual cannot be considered guilty based

on this evidence. The DNA matched from the scene could have been contaminated by an

unfortunate civilian who might have made contact with the sample, or even shed DNA

(Oleiwi et al., 2015). Nevertheless, the DNA samples collected from the crime scene could

have also been contaminated because of negligence when collecting the sample (Oleiwi et

al., 2015). While the produced image of the gel was able to match suspect 2 as the possible

criminal, some errors occurred during the experiment. While preparing the tubes labelled

1-4, incorrect measurements were used. The suspects samples contained 1.5 microliters

instead of 15 microliters of their respective DNA (Upadhyaya, 2017). Additionally, suspect

1 and 2 also had 1.5 microliters of the restriction enzymes used to cut them instead of the

required 15 microliters. Hence, the final volume of the suspects' samples were about 13

microliters instead of 40 microliters. This error arose because the micropipette was set to

1.5 microliters instead of 15 microliters. Nevertheless, the samples were still able to be

read efficiently. This experiment was crucial because DNA fingerprinting has a plethora

of uses. DNA fingerprinting is currently used to develop a DNA database, clarifying the

identification of specimens, and short tandem repeat analysis (Saad, 2005). The use of a

DNA database could help match a criminal's DNA with that of a crime scene if they are

registered in the database, and it could also confirm legal documents such as passports by

matching them with a fingerprint (Saad, 2005). Likewise, it could also be used to clarify

the identification of specimens that are shipped to a medical facility (Saad, 2005). The short

tandem repeat analysis allows scientists to compare samples of two individuals which is
useful during transplantations (Saad, 2005). Finally, DNA fingerprinting could even be

used for paternity testing, and even checking for hereditary diseases (Upadhyaya, 2017).

Nonetheless, fingerprinting has some weaknesses. While viewing the produced image of

the gel, it was evident that both suspects matched the crime scene when their samples were

cut with Restriction Enzyme 1. This issue is problematic because it does not allow for the

identification of one suspect. In order to clarify the results, using a second enzyme was

required. The second enzyme expanded the results, and increased the validity of the

experiment. When the suspects samples were cut with Restriction Enzyme 2, there was

only one match for the crime scene that was cut with Restriction Enzyme 2. Using two

restriction enzymes showed that the probable suspect was suspect 2. If Restriction Enzyme

1 was the only one used, it would have been impossible to identify the suspects. However,

because two enzymes were used, the results became tangible. The use of multiple

restriction enzymes is important for DNA fingerprinting because some samples could be

similar when cut with only one restriction enzyme. To clarify the results, the samples

should be able to match the model sample with various restriction enzymes. If not, it would

be difficult to assess which sample is the desired one when they both matched the model

sample. In this experiment, the model sample was the crime scene. In forensics, the use of

multiple restriction enzymes avoids incarcerating innocent bystanders because it increases

the accuracy of the results (Urry et al, 2016).


References

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Oleiwi, A., Morris, M., Schmerer, W., & Sutton, R. 2015. The relative DNA-shedding

proponsity of the palm and finger surfaces. Science & Justice. 55(5):329-334.

Saad, R. (2005). Discovery, development, and current applications of DNA identity

testing. The National Center for Biotechnology Information. [Online]. Available:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1200713/. [October 29, 2017].

Upadhyaya, A. 2017. Cellular Processes (BSC 2010L) Laboratory Manual. University of

South Florida Department of Biology, Tampa, Florida, 81 pps.

Urry, L., Cain, M., Wasserman, S., Minorsky, P., Reese, J. 2016. Campbell Biology, 11th

edition. Pearson, New York City, NY, 1488 pps.