DNA Fingerprinting Using RFLP Gel

Electrophoresis
Overview
Gel Electrophoresis is a procedure used in molecular biology to separate and
identify molecules (such as DNA and RNA) by size. The separation of these
molecules is achieved by placing them in a gel made up of small pores and setting an
electric field across the gel.
The molecules will move based on their inherent electric charge (i.e., negatively
charged molecules move away from the negative pole) and smaller molecules will move
faster than larger molecules; thus, a size separation is achieved within the pool of
molecules running through the gel. The gel works in a similar manner to a sieve
separating particles by size; the electrophoresis works to move the particles, using their
inherent electric charge, through the sieve.

DNA Fingerprinting Using RFLP Gel Electrophoresis

Agarose powder is isolated from the seaweed genera Gelidium and Gracilaria. It9s mixed
with a buffer and heated in a microwave, then left to cool down before pouring in the
cast. A comb is added at a specific site to form the wells required for sample upload.
Then gel is left to solidify.

The concentration of gel = weight of agarose/volume of buffer (g/ml).

For a standard agarose gel electrophoresis, 0.8% gel gives good separation of large
5–10kb DNA fragments, while 2% gel gives good resolution for small 0.2-1 Kb DNA
fragments.

During gelation, agarose polymers associate non-covalently and form a network whose
pore sizes determine a gel's molecular sieving properties.

The phosphate in the backbone of DNA is negatively charged, therefore DNA fragments
will migrate to the positively charged anode. DNA has a uniform mass/charge ratio,
therefore DNA molecules are separated by size within an agarose gel in a pattern such
that the distance traveled is inversely proportional to the log of its molecular weight.
The rate of migration of a DNA molecule through a gel is determined by the following:
1) size of DNA band (the heavier, the slower)
2) agarose gel concentration (usually 0.8%)
3) DNA conformation (linear/plasmid/etc..)
4) Voltage
5) Electrophoresis buffer.
6) Ethidium bromide: EtBr is positively charged, thus; reduces the DNA migration rate
by 15%. Other stains for DNA in agarose gels include SYBR Gold, SYBR green,
Crystal Violet and Methyl Blue.
UV light activates electrons in the aromatic ring of ethidium bromide releasing light as
electrons return to ground state. EtBr intercalates itself in the DNA molecule in a
concentration dependent manner. So higher intensity means higher amount of DNA.

DNA fingerprinting allows the comparison of DNA from different organisms and the
identification of a particular individual. Basically, DNA is extracted and cut into fragments
using restriction enzymes. The fragments form a pattern on agarose gel electrophoresis
according to their length. The longer the DNA fragments are generated, the larger their
molecular weight and the shorter they travel in an electrophoresis setting. This pattern
looks a lot like the barcode on products in the supermarket and it resembles the
individual9s DNA fingerprint.

To interpret the pattern formed, one must realize that restriction enzymes cut the DNA at
specific base-pair sequences called recognition sequences. There are hundreds of
restriction enzymes, each having a specific recognition sequence made of four to twelve
base pairs. The lengths of the fragments generated by a restriction enzyme digest of an
extracted DNA sample depend upon the number of cuts made and their locations. This
depends on the number of recognition sequences of the enzyme used on the extracted
DNA and their locations. Thus, everyone9s DNA is cut by restriction enzymes into
distinctive different sized fragments that appear on the electrophoresis gel in the form of
bands.

Restriction enzymes are present in bacteria. They are named according to the bacteria
from which they are extracted. They protect bacteria by digesting foreign DNA at specific
sequences.
Restriction enzymes such as (EcoRI) and (HindIII) are used in this experiment to Cut the
extracted DNA. EcoRI recognizes the 6 bp sequence 59 GAATTC 39 and makes a
staggered cut between the G and A creating sticky ends. HindIII recognizes the 6 bp
sequence 59 AAGCTT 39 and makes a staggered cut between the A and A creating sticky
ends. Both enzymes cut best at 37°C.

Ecori:
5’ G^AATTC 3’
3’ CTTAA^G 5’

HindIII:
5' A ↓AGCTT 3'
3' TTCGA ↑A 5'
Uses for DNA fingerprinting:
Crime scenes: identification of any evidence that contains DNA such as hair, saliva, and
blood in the crime scene or on the victim.
Paternity cases
Unidentified bodies
Animal business such as breeding of endangered species.

Interpretation of resulting gel bands:

I. Interpretation of paternity cases:
the DNA fingerprint of the mother, the child and the alleged fathers are run on an
agarose gel electrophoresis. Some of the bands of the child are inherited from the
mother and align with her bands. The remainder of the bands must have been inherited
from the father and should align with his bands.
II. Interpretation of crime scene cases:
The DNA fingerprints extracted from the crime scene are run on an agarose gel
electrophoresis along with DNA fingerprints of the suspects and the victim. Victim9s DNA
fingerprint may match one of the crime scene DNA samples. If one of the suspects had
the same DNA fingerprint as one of the crime scene DNA, this might connect that
suspect to the crime.

Learning objectives
● List the steps required for the preparation of an optimum agarose gel.
● Evaluate the role and value of chemicals and reagents used in the experiment.
● Illustrate the set-up required for gel electrophoresis.
● Enlist factors that lead to successful sample upload into the gel.
● Visualize DNA fragments.
● Identify and distinguish DNA molecules that have been processed by a previous
method such as PCR and enzymatic digestion.
● Interpret results on a gel UV transilluminator and solve medicolegal cases.
● Identify the characteristics of restriction sites in a DNA sequence.
Aim
In this virtual lab you will:
● Use DNA samples extracted from a crime scene and from suspects to identify the
criminal using DNA fingerprinting. In your laboratory, you will use restriction
enzymes to digest the isolated DNA samples at specific sites. You will run the
digested DNA in agarose gel electrophoresis to be able to see the DNA bands
resulting from digestion and compare those from the suspects to those extracted
from the crime scene. (In case of scenario 1)

● Examine extracted DNA samples for a paternity case. (In Case of scenario two)

Personal Protective Equipment

● Wear a long-sleeved lab coat, safety goggles, nitrile gloves, long pants, and
closed-toe shoes.
● Wear appropriate skin and eye protection when working with UV radiation and
ethidium bromide.
Materials and Devices Required
- Agarose powder.
- 1x TAE buffer.
- 400 ml graduated container.
- Microwave oven.
- Ethidium bromide dye.
- Pipettes.
- Gel tray.
- Casting gates.
- Electronic balance.
- Comb.
- Gel tank.
- PCR products (DNA samples).
- 6x Gel loading buffer.
- DNA pre stained ladder.
- Power supply.
- UV transilluminator.
Steps

Scenario One
1. Weigh 2 gm of agarose powder in a graduated 400 ml container, you need to drag
the container into the scale, then tare the scale and add the powder. shut the
scale after using it.
2. Add 200 ml of 1x TAE buffer into the graduated container and shake it.
3. It seems we will use the microwave oven. Click on the microwave to open its
door.Loosely cap the gel container loosely and heat it in the microwave for 1:30
min at medium heat. To ensure equal heating of the container contents, pause the
microwave at 00:30 and shake it.

Note: When the container is taken out of the microwave, check if the
agarose is completely molten.
The solution should be clear.
Let the agarose cool down to 60 °C.

4. Add 3 μl of ethidium bromide dye to the molten agarose gel using a P10 pipette.
5. Close the sides of the gel tray using the casting gates, then insert the comb.
6. Pour the gel solution into the gel tray slowly to avoid the formation of air bubbles.
Let the gel solidify for 20 minutes. Finally, remove the casting gates.

Note: Before pouring all of your gel, pour some of it at the sides of the tray.
Just enough to line the margins.
This helps prevent leakage of the gel outside of the tray.
You can use a new pipette tip to pop up any bubble formed while pouring
the gel before it solidifies.

7. Put the gel tray in the gel tank with the wells placed towards the –ve electrode
(black electrode), then add 1x TAE buffer solution to each end of the gel tank.
keep adding the buffer until it covers the gel and exceeds by about 2 mm.
Finally, gently pull the comb straight up out of the gel.

A. Sample Loading into the gel:

I. Digestion of DNA sample using restriction enzyme digestion 1 (EcoRI) and 2
(HindIII).
II. Application of the samples into the wells created in the gel by the comb.

Note: Your isolated DNA has been retrieved in order to be digested by

restriction enzyme digestion 1 (EcoRI) and 2 (Hind III).
 8. You will need 15 sterile microfuge tubes.
Label as follows:
1. <Suspect DNA 1 C=.
2. <Suspect DNA 1 E=.
3. <Suspect DNA 1 H=.
4. <Suspect DNA 2 C=.
5. <Suspect DNA 2 E=.
6. <Suspect DNA 2 H=.
7. <Crime scene DNA 1 C=
8. <Crime scene DNA 1 E=.
9. <Crime scene DNA 1 H=.
10. <Crime scene DNA 2 C=.
11. <Crime scene DNA 2 E=.
12. <Crime scene DNA 2 H=.
13. <Victim DNA C=
14. <Victim DNA E=
15. <Victim DNA H=

9. Mix well the 10X FastDigest Buffer tube by vortexing for 15 seconds.
10. Add 10 μl 10X FastDigest Buffer to all tubes.
11. Add 15 μl suspect DNA 1 to tubes (1), (2) and (3).
Add 15 μl suspect DNA 2 to tubes (4), (5) and (6).
Add 15 μl crime scene DNA 1 to tubes (7), (8) and (9).
Add 15 μl crime scene DNA 2 to tubes (10), (11) and (12).
Add 15 μl victim DNA to tubes (13), (14) and (15).
12. Add 15 μl of diluted FastDigest EcoRI to tubes (2), (5), (8), (11), (14).
13. Add 15 μl of diluted FastDigest HindIII to tubes (3), (6), (9), (12), (15).
14. Mix gently by tapping. Note: Total volume = 45 μl.
15. Spin briefly in a microcentrifuge for 30 seconds at 1000 RPM and 22 Celsius.
16. Incubate in a water bath at 37 degrees Celsius for 8 minutes.
17. Inactivate the enzyme by placing the tube in the heat block set at 80 Celsius for 5
minutes.
18. Place the tube on ice.
Note: Your tube can be stored at -20 degrees Celsius if it’s not going to be
used in the same laboratory setting.
19. Add 5 μl gel loading solution to all tubes. The loading solution allows the sample
to lay down in the well due to the presence of glycerol, and to be visualized.

B. Sample loading

20. Load 15 μl of DNA Size Marker (labelled 8M9) in the reference lane. Be careful not
to pierce the well from the bottom. This will appear as leakage into the depth of
the gel.
21. Load 15 μl of samples into the wells in the following order:
Lane 1: M (DNA ladder)
Lane 2: <Suspect DNA 1 C=.
Lane 3: <Suspect DNA 1 E=.
Lane 4: <Suspect DNA 1 H=.
Lane 5: <Suspect DNA 2 C=.
Lane6<Suspect DNA 2 E=.
Lane7<Suspect DNA 2 H=.
Lane8<Crime scene DNA 1 C=
Lane9<Crime scene DNA 1 E=.
Lane10<Crime scene DNA 1 H=.
Lane 11<Crime scene DNA 2 C=.
Lane12<Crime scene DNA 2 E=.
Lane13<Crime scene DNA 2 H=.
Lane14<Victim DNA C=
Lane15<Victim DNA E=
Lane16<Victim DNA H=

22. Attach the lid of the tank properly (black with black and red with red).
Start the electrophoresis run with these settings: 100 volts and 40 minutes. Don9t
forget to add the blue coloured patch beneath the gel tank to add colour to the
background. Stop the device if the bands reach the opposite end of the gel. take
the gel tray out of the tank and add the gel to the UV transilluminator machine.

Note: The gel was put into the UV transilluminator to take a digital photo.

This is the expected band pattern and sizes of the DNA size marker used
(GeneRulerTM 1 kb DNA ladder purchased from Fermentas). Some bands
are brighter/thicker than others because they have more DNA. This can
occur when the marker sample has multiple bands of approximately the
same size.
C. Visualize the Bands.

Case 1

Case 2

Case 3
Scenario Two
1. Weigh 2 gm of agarose powder in a graduated 400 ml container, you need to drag
the container into the scale, then tare the scale and add the powder. shut the
scale after using it.
2. Add 200 ml of 1x TAE buffer into the graduated container and shake it.
3. It seems we will use the microwave oven. Click on the microwave to open its
door.Loosely cap the gel container loosely and heat it in the microwave for 1:30
min at medium heat. To ensure equal heating of the container contents, pause the
microwave at 00:30 and shake it.

Note: When the container is taken out of the microwave, check if the
agarose is completely molten.
The solution should be clear.
Let the agarose cool down to 60 °C.

4. Add 3 μl of ethidium bromide dye to the molten agarose gel using a P10 pipette.
5. Close the sides of the gel tray using the casting gates, then insert the comb.
6. Pour the gel solution into the gel tray slowly to avoid the formation of air bubbles.
Let the gel solidify for 20 minutes. Finally, remove the casting gates.

Note: Before pouring all of your gel, pour some of it at the sides of the tray.
Just enough to line the margins.
This helps prevent leakage of the gel outside of the tray.
You can use a new pipette tip to pop up any bubble formed while pouring
the gel before it solidifies.

7. Put the gel tray in the gel tank with the wells placed towards the –ve electrode
(black electrode), then add 1x TAE buffer solution to each end of the gel tank.
keep adding the buffer until it covers the gel and exceeds by about 2 mm.
Finally, gently pull the comb straight up out of the gel.

A. Sample Loading into the gel:

I. Digestion of DNA sample using restriction enzyme digestion 1 (EcoRI) and 2
(HindIII).
II. Application of the samples into the wells created in the gel by the comb.

Note: Your isolated DNA has been retrieved in order to be digested by

restriction enzyme digestion 1 (EcoRI) and 2 (Hind III).
 8. You will need 12 sterile microfuge tubes. Label the tubes as follows:
Mother DNA C
Mother DNA E
Mother DNA H
Father 1 DNA C
Father 1 DNA E
Father 1 DNA H
Father 2 DNA C
Father 2 DNA E
Father 2 DNA H
Child1 DNA C
Child1 DNA E
Child1 DNA H
Child2 DNA C
Child2 DNA E
Child3 DNA H
9. Mix well the 10X FastDigest Buffer tube by vortexing for 15 seconds.
10. Add 10 μl 10X FastDigest Buffer to all tubes.
11. Add 15 μl 8mother DNA9 to tubes (1), (2) and (3).
Add 15 μl 8father 1 DNA9 to tubes (4), (5) and (6).
Add 15 μl 8father 2 DNA9 to tubes (7), (8) and (9).
Add 15 μl 8child 1 DNA9 to tubes (10), (11) and (12).
Add 15 μl 8child 2 DNA9 to tubes (13), (14) and (15).
12. Add 15 μl of diluted FastDigest EcoRI to tubes (2), (5), (8), (11), (14).
13. Add 15 μl of diluted FastDigest HindIII to tubes (3), (6), (9), (12), (15).
14. Mix gently by tapping. Note: Total volume = 45 μl.
15. Spin briefly in a microcentrifuge for 30 seconds at 1000 RPM and 22 Celsius.
16. Incubate in a water bath at 37 degrees Celsius for 8 minutes.
17. Inactivate the enzyme by placing the tube in the heat block set at 80 Celsius for 5
minutes.
18. Place the tube on ice.
Note: Your tube can be stored at -20 degrees Celsius if it’s not going to be
used in the same laboratory setting.
19. Add 5 μl gel loading solution to all tubes. The loading solution allows the sample
to lay down in the well due to the presence of glycerol, and to be visualized.

B. Sample loading

20. Load 15 μl of DNA Size Marker (labelled 8M9) in the reference lane. Be careful not
to pierce the well from the bottom. This will appear as leakage into the depth of
the gel.
21. Load 15 μl of samples into the wells in the following order:
Lane 1: M (DNA ladder)
Lane 2: <Mother DNA C=.
Lane 3: <Mother DNA E=.
Lane 4: <Mother DNA H=.
Lane 5: <Father 1 DNA C=.
Lane6<Father 1 DNA E=.
Lane7<Father 1 DNA H=.
Lane8<Father 2 DNA C=
Lane9<Father 2 DNA E=.
Lane10<Father 2 DNA H=.
Lane 11<Child 1 DNA C=.
Lane12<Child 1 DNA E=.
Lane13<Child 1 DNA H=.
Lane 14 8child 2 DNA C9.
Lane 15 8child 2 DNA E9.
Lane 16 8child 2 DNA H9.

22. Attach the lid of the tank properly (black with black and red with red).
Start the electrophoresis run with these settings: 100 volts and 40 minutes. Don9t
forget to add the blue coloured patch beneath the gel tank to add colour to the
background. Stop the device if the bands reach the opposite end of the gel. take
the gel tray out of the tank and add the gel to the UV transilluminator machine.
Note: The gel was put into the UV transilluminator to take a digital photo.

This is the expected band pattern and sizes of the DNA size marker used
(GeneRulerTM 1 kb DNA ladder purchased from Fermentas). Some bands
are brighter/thicker than others because they have more DNA. This can
occur when the marker sample has multiple bands of approximately the
same size.
C. Visualize the Bands

Case 1

Case 2

Case 3

Case 4