Restriction Fragment Length Polymorphism (RFLP) Technique

Restriction Fragment Length

Polymorphism (RFLP) Technique
By Susha Cheriyedath, M.Sc.
Reviewed by Afsaneh Khetrapal, BSc

Restriction fragment length polymorphism (RFLP) is a technique invented in

1984 by the English scientist Alec Jeffreys during research into hereditary
diseases. It is used for the analysis of unique patterns in DNA fragments in
order to genetically differentiate between organisms – these patterns are called
Variable Number of Tandem Repeats (VNTRs).

Genetic polymorphism is defined as the inherited genetic differences among

individuals in over 1% of normal population. The RFLP technique exploits these
differences in DNA sequences to recognize and study both intraspecies and
interspecies variation.

Restriction Fragment Length Polymorphism (RFLP)

Principle
Restriction endonucleases are enzymes that cut lengthy DNA into short pieces.
Each restriction endonuclease targets different nucleotide sequences in a DNA
strand and therefore cuts at different sites.

P
Saved from URL: https://www.news-medical.net/life-sciences/Restriction-Fragment-Length-Polymorphism-(RFLP)-Technique.aspx
1
/4
Restriction Fragment Length Polymorphism (RFLP) Technique

The distance between the cleavage sites of a certain restriction endonuclease

differs between individuals. Hence, the length of the DNA fragments produced
by a restriction endonuclease will differ across both individual organisms and
species.

How does it Work?

RFLP is performed using a series of steps briefly outlined below:

DNA Extraction

To begin with, DNA is extracted from blood, saliva or other samples and
purified.

DNA Fragmentation

The purified DNA is digested using restriction endonucleases. The recognition

sites of these enzymes are generally 4 to 6 base pairs in length. The shorter the
sequence recognized, the greater the number of fragments generated from
digestion.

For example, if there is a short sequence of GAGC that occurs repeatedly in a

sample of DNA. The restriction endonuclease that recognizes the GAGC
sequence cuts the DNA at every repetition of the GAGC pattern.

If one sample repeats the GAGC sequence 4 times whilst another sample
repeats it 2 times, the length of the fragments generated by the enzyme for the
two samples will be different.

Gel Electrophoresis

The restriction fragments produced during DNA fragmentation are analyzed

using gel electrophoresis.

The fragments are negatively charged and can be easily separated by

electrophoresis, which separates molecules based on their size and charge. The
fragmented DNA samples are placed in the chamber containing the
electrophoretic gel and two electrodes.

When an electric field is applied, the fragments migrate towards the positive

P
Saved from URL: https://www.news-medical.net/life-sciences/Restriction-Fragment-Length-Polymorphism-(RFLP)-Technique.aspx
2
/4
Restriction Fragment Length Polymorphism (RFLP) Technique

electrode. Smaller fragments move faster through the gel leaving the larger
ones behind and thus the DNA samples are separated into distinct bands on the
gel.

Visualization of Bands

The gel is treated with luminescent dyes in order to make the DNA bands
visible.

Applications of RFLP
RFLP has been used for several genetic analysis applications since its invention.

Some of these key applications of RFLP are listed below:

To determine the status of genetic diseases such as Cystic Fibrosis in an

individual.
To determine or confirm the source of a DNA sample such as in paternity
tests or criminal investigations.
In genetic mapping to determine recombination rates that show the
genetic distance between the loci.
To identify a carrier of a disease-causing mutation in a family.

Disadvantages of RFLP
Since its invention, RFLP has been a widely used genome analysis techniques
employed in forensic science, medicine, and genetic studies. However, it has
become almost obsolete with the advent of relatively simple and less expensive
DNA profiling technologies such as the polymerase chain reaction (PCR).

The RFLP procedure requires numerous steps and takes weeks to yield results,
while techniques such as PCR can amplify target DNA sequences in a mere few
hours.

Additionally, RFLP requires a large DNA sample, the isolation of which can be a
laborious and time-consuming process. In contrast, PCR can amplify minute
amounts of DNA in a matter of hours.

Due to numerous reasons such as these, the PCR technique has largely
replaced RFLP in most applications requiring DNA sequencing such as paternity

P
Saved from URL: https://www.news-medical.net/life-sciences/Restriction-Fragment-Length-Polymorphism-(RFLP)-Technique.aspx
3
/4
Restriction Fragment Length Polymorphism (RFLP) Technique

testing or forensic sample analysis.

Furthermore, the identification of single-nucleotide polymorphisms in the

Human Genome Project has almost replaced the need for RFLP in disease status
analysis.

References
https://www.nlm.nih.gov/visibleproofs/education/dna/rflp.pdf
http://www.bio.davidson.edu/courses/genomics/method/rflp.html
http://www.kau.edu.sa/Files/0002923/Files/18591_Restriction%
20Fragment%20Length%20Polymorphism.pdf
http://cdn.intechopen.com/pdfs-wm/35104.pdf
http://www.dnaforensics.com/DNAFingerprinting.aspx

Further Reading
All Restriction Fragment Length Polymorphism Content

Last Updated: Jun 26, 2019

Written by

Susha Cheriyedath
Susha is a scientific communication professional holding a Master's degree in
Biochemistry, with expertise in Microbiology, Physiology, Biotechnology, and Nutrition.
After a two-year tenure as a lecturer from 2000 to 2002, where she mentored
undergraduates studying Biochemistry, she transitioned into editorial roles within
scientific publishing. She has accumulated nearly two decades of experience in
medical communication, assuming diverse roles in research, writing, editing, and
editorial management.

P
Saved from URL: https://www.news-medical.net/life-sciences/Restriction-Fragment-Length-Polymorphism-(RFLP)-Technique.aspx
4
/4