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Title:
RFLP, forensic DNA fingerprinting
Aims:
1. To understand the use of RFLP in forensic science.
2. To use RFLP to determine paternity and establish whether a suspect’s DNA matches one from
crime scene.

Introduction:
The discovery of DNA fingerprinting has revolutionised the forensic science, allowing to identify a
suspect if a DNA sample is found at the crime scene. Our genetic code is unique to everyone (apart
from identical twins), therefore, a matching DNA sample can prove whether someone is guilty. A
gene coding for a certain trait can exist in two or more versions, called alleles. Alleles are inherited
from our parents and these can be either dominant or recessive, having two identical alleles is said to
be homozygous, whereas if they are different, they are heterozygous. The difference in alleles is
usually caused by a small difference in the nucleotide sequence of the gene, this can result in
variations in the restriction sites when the DNA is treated with restriction enzymes, resulting in
fragments of different lengths (Anthony JF Griffiths et al., 2016).

Raymond White, an American geneticist, identified regions of DNA that did not code for proteins,
however they were specific between individuals. With the use of restriction enzymes, White
separated the DNA fragments based on size, calling the variations restriction fragment length
polymorphisms (RFLP) (de Souza, 2007). The first use of RFLP in forensic science was by Alec Jeffreys,
who found that the technology could be used to develop patterns of cut up DNA that are specific to
an individual. His work resulted in the release of a wrongfully convicted man and conviction of the
true perpetrator. (Saad, 2005).

In the RFLP technique, genomic DNA is treated with one ore more restriction enzymes which cut the
DNA at unique restriction sites of specific base sequences. This generates a number of DNA
fragments of different lengths as variations in the genome, different alleles will cause unique
patterns of fragments when gel electrophoresis is carried out. If a DNA sample from a crime scene is
obtained, the unique pattern generated can then be compared to those from suspects, a match
would indicate whether the suspect was present at the scene and results in a verdict as both samples
would produce identical bands on the gels. Usually two or more restriction enzymes are used on the
DNA samples, this prevents sticky ends forming and gives better accuracy - more restriction sites will
produce more bands for comparison as often different suspects may have similar patterns.

Materials:
Double digest

• Eco RI/ Pst I enzyme mix

• Pipet tips, 2–200 µl
• Adjustable micropipette, 2–20 µl
• Microfuge tube:
• Permanent marker
• Waste container

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• Microfuge rack
• Ice
• DNA Hyperladder standard
• Baby DNA with buffer, rehydrated in reaction buffer
• Mother DNA with buffer, rehydrated in reaction buffer
• Father #1 DNA with buffer, rehydrated in reaction buffer
• Father #2 DNA with buffer, rehydrated in reaction buffer
• 37°C heat block
• Microcentrifuge
Gel electrophoresis

• Agarose gel electrophoresis system

• 60 ml molten 1% agarose in 1x TAE plus gel red dye
• 8-well comb
• Casting tray
• tape
• Ice
• Microfuge
• Tracking dye
• Tracking dye.
• TAE buffer
Method
Digest

1. The tube containing restriction enzymes was labelled ENZ and placed on ice
2. Micro test tubes were labelled as B (baby), M (mother) F1 (father 1), F2 (father 2) and placed
in the rack
3. Using a fresh pipette tip, 10 µl of each DNA sample was transferred to the corresponding
tubes, followed by 10 µl of enzyme mix into the verry bottom of each tube. Tubes were
capped, mixed by flicking using a finger and pulse spun in the centrifuge to move all the
liquid to the bottom.
4. The tubes were placed in a heating block at 37°C over night. After incubation stored at -20°C.

Gels

1. 1% agarose gel was prepared, and digested DNA samples removed from the freezer to thaw.
2. Tubes were pulse spun in the centrifuge to place all of the liquid at the bottom.
3. Using a fresh tip for each sample, 5 µl of loading dye was added. Pulse spun in a centrifuge
again.
4. Agarose gel was placed in the electrophoresis chamber and covered with approximately
275ml of TAE buffer, ensuring that the wells are near the black electrode.
5. Using fresh pipette tips, 2 µl of DNA Hyperladder and 10 µl of each sample was loaded into
the wells.
6. The chamber lid was closed, and the red and black cables were plugged into the power
supply. Power was turned on and samples were run at 80V for 2 hours.
7. The gel was then observed in the Transilluminator and photographed.

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Results

Figure 1. Gel electrophoresis at 80V for 2 hours of the samples to determine paternity. Arrows
indicating matching bands, 4 matches for Father #2 (red arrows), 1 match for Father #1 (blue),
matches between baby and mother shown by black arrows.

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Figure 2. Crime scene RFLP results. Well 1 – Lambda Hind III, Well 2 – Crime scene sample, Well 3 –
Suspect 1, Well 4 – Suspect 2, Well 5 – Suspect 3, Well 6 – Suspect 4, Well 7 – Suspect 5

Discussion
Figure 1 shows the baby and father #2 share 4 bands of equal sizes and only 1 shared band between
the baby and father #1, therefore we can say father #2 is baby’s real parent.

The crime scene results in Figure 2 show the most similarity with suspect #3 – Abigail, other suspects
have bands of smaller or bigger sizes which travelled different distances than the crime scene
sample, we can conclude that it was Abigail’s DNA found on the gun.

RFLP apart from paternity/maternity tests and crime scenes can be used to identify victims in mass
graves, whether from accidents like the World Trade Centre or from crimes against humanity where
a number of civilians was executed (World of Forensic Science, n.d.). In these cases, usually dental
records might not be available, and bodies may be too deformed, therefore, the only way to identify
the victims is through DNA testing, where the samples are compared with their parents’ DNA.

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The technique has many advantages, no prior information about the DNA sequence of the sample is
required to perform the procedure, its ease of use without any complicated steps involve makes it
highly robust and the results can be easily reproduced between laboratories. However, large
amounts of DNA are required, and it is difficult to automate, however PCR can be used to amplify
very small amounts of DNA, usually in 2-3 hours, to the levels required for RFLP analysis.

Conclusion
Over 30 years after the development of RFLP, the method, thanks to its simplicity is still used in
linking suspects to DNA evidence from crime scenes, it is also used in paternity/maternity tests, in
diagnosing inherited diseases and studying various genetic disorders.

References

Anthony JF Griffiths, Gelbart, W.M., Miller, J.H. and Lewontin, R.C. (2016). RFLP Mapping. [online] Nih.gov. Available at:
https://www.ncbi.nlm.nih.gov/books/NBK21320/ [Accessed 3 Mar. 2021].

de Souza, N. (2007). Get out the map. Nature Reviews Genetics, [online] 8(1), pp.S10–S10. Available at:
https://www.nature.com/articles/miledna08 [Accessed 26 Feb. 2021].

Saad, R. (2005). Discovery, Development, and Current Applications of Dna Identity Testing. Baylor University Medical Center
Proceedings, [online] 18(2), pp.130–133. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1200713/ [Accessed
26 Feb. 2021].

World of Forensic Science (n.d.). DNA Mixtures, Forensic Interpretation of Mass Graves | Encyclopedia.com. [online]
www.encyclopedia.com. Available at: https://www.encyclopedia.com/science/encyclopedias-almanacs-transcripts-and-
maps/dna-mixtures-forensic-interpretation-mass-graves [Accessed 4 Mar. 2021].

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