Restriction

Fragment Length
Polymorphism
What is Restriction Fragment
Length Polymorphisms (RFLPs)?:
 RFLP; the acronym is pronounced "riflip".
 Polymorphism is any difference in DNA sequence, coding or
non-coding, that can be detected between individuals.
 Is a biotechnological laboratory technique in which DNA
regions are digested using restriction endonuclease(s)
and subjected to radioactive complementary DNA probes
to compare the difference in DNA fragment lengths
between individuals Differences are noticed when the
length of fragments are not the same, telling us that the
restriction enzyme cut the DNA at two unrelated
locations.
 Genetic variations at the site where a restriction enzyme cuts
a piece of DNA, Such variations affect the size of the
resulting fragments. These sequences can be used as
markers on physical maps and linkage maps.
 Restriction fragment length polymorphism is the identification
of specific restriction enzymes that reveal a pattern difference
between the DNA fragment sizes in individual organisms,
usually results from a genetic mutation (as an insertion or
deletion) and that may be used as a genetic marker.
A Closer Look…
 Restriction fragment length
polymorphism analysis
involves the comparison of
different lengths of DNA
 The first step is to extract a
sample of DNA and subject it
to the restriction enzyme(s),
this enzyme will produce
restriction fragments
(fragments that will vary in size
depending on the DNA of the
organism) as seen in figure 1
Figure 1
• Next through a process known
as “Gel Electrophoresis”, the
fragments are separated, each
sample forms a characteristic
pattern of bands because of
the slight variation in each
organism’s DNA. The
fragments form a band as a
electrical current is passed
through the gel causing the
negatively charged DNA to
travel to the positive side of
the electrode. The whole
process is shown in figure 2.
FIGURE 2
 The amount of DNA is
usually so large and the
bands so numerous that gel
electrophoresis is unable to
resolve them. Differences in
the pattern of fragments
must be detected to
distinguish DNA from two
different sources. The gel is
subjected to a chemical that
causes the double-stranded
DNA to be de-natured into
single-stranded DNA as in Figure 3
figure 3
• These single-strands are
transferred onto special paper or
nylon membranes through
capillary action known as
“southern blotting” named after
E.M. Southern who developed it.
The single stranded DNA is
transferred to the nylon
membrane with the aid of an
electrical current. The nylon
membrane is placed on the gel
with a positive electrode behind;
since DNA is negatively charged it
will be transferred out of the gel
and “blotted” on to the nylon
membrane, where they will bind. Figure 4
• The nylon membrane containing
the single-stranded DNA is
immersed in a solution containing
radioactive complementary
nucleotide probes for specifically
chosen regions. These regions
can be a mutation in an allele that
lead to a specific disease or
variable number tandem repeat
found in non-coding region of the
DNA. Complementary base
pairing between the radio active
probes and the DNA via hydrogen
bonds will occur this is known as
“HYBRIDIZATION” as seen in
figure 5
Figure 5
• The nylon membrane is
placed against X-RAY
film. The radioactive
probes cause exposure of
the x-ray film in the area
where hybridizations took
place. This is called an
“AUTORADIOGRAM” the
differences in pattern can
be detected. Seen on the Figure 5

right of figure 5
How Is It Used?
 RFLPs have provided valuable information
in many areas of biology including:
 Systematics, Genetics and Ecology
 DNA Fingerprinting
 Screening human DNA for the presence of
potentially deleterious genes
•Systematics, Genetics and Ecology
 Phylogentics is the study of
evolutionary relationships, this is just
one field that has benefited from
RFLPs. By comparing the fragment
patterns of different species (Fig. 6)
Top, an evolutionary biologist can
gather information about the possible
relatedness of those species.
 RFLPs are not only used to compare
species, but information about
intraspecific variation can also be
obtained. Such comparisons can be
useful to a geneticist trying to
determine the genetic make-up of a
population or to an ecologist who
needs information about the
genealogy of a population (Fig 7)
Bottom.
•DNA Fingerprinting
 Sequences of DNA differ from person to person,
but every cell within the same person contains the
same sequence of DNA. So, your hair, blood, skin
and all of the other cells in your body are exactly
the same at the molecular level.This comes in very
handy when police are investigating a crime. If a
person left a strand of hair, a drop of blood or any
other cells at a crime scene, the police will know
that that person was there.
 Criminal investigation also uses RFLPs as part of
forensic analysis of the crime scene. They
compare RFLP patterns of DNA found on evidence
to those of the victim and suspect(s) (Fig. 4).
 They observe the patterns in the fragments and try
to match the suspects DNA with the DNA found at
the crime scene. As in figure 8
 DNA fingerprinting is based on variations of
satellites DNA (non-coding regions), since humans
share almost all the same DNA in the coding
regions the only variation that occurs is in the non-
coding regions. DNA fingerprinting takes
advantage of this fact.

Figure 8
• Screening human DNA for the
presence of potentially deleterious genes
 RFLPs analyses have become very
popular in the field of medicine.
Genetic counselling is founded on
RFLP analyses and the ability to
determine genotypes (Fig.9 ). RFLPs
can be used to determine the
genotype of potential parents, and
following an amniocentesis or human
chronic fluid sampling, RFLPs are
used to screen for deleterious
genotypes in the fetus.
 For instance, an RFLP has been
identified that is associated with the
genetic disease sickle cell anaemia.
Sickle cell anaemia is caused by a
mutation in the alpha-globin gene and Figure 9
results in an abnormal form of
haemoglobin.
“Works Cited”
 Restriction fragment length polymorphism Torbert R. Rocheford, assistant professor of corn breeding,
Department of Agronom, cited dec 19 2005. <
http://www.ag.uiuc.edu/~vista/html_pubs/irspsm91/fragment.html>
 U. Melcher molecular genetics-RFLP’s Last Updated: 3 September, 2001 cited December 19, 2005, <
http://opbs.okstate.edu/~melcher/MG/MGW1/MG11124.html>
 Tara T. VanToai Seed Biology:Department of Horticulture and Crop Science, The Ohio State
University, USES OF DNA TECHNOLOGY IN SEED RESEARCH, <
http://www.ag.ohio-state.edu/~seedbio/van1.html >
 Kimball JW. 1997 May. Restriction Fragment Length Polymorphisms (RFLPs). <http://
www.ultranet.com/~jkimball/BiologyPages/R/RFLPs.html>
 Neale D, Sederoff R. 1996 Sept. Genome mapping in pines takes shape. National Agricultural Library. <
http://www.nalusda.gov/pgdic/Probe/v1n3_4/genome.html>
 Stokely RD. 1997 May. DNA profiling in a first degree murder case: RFLP analysis. <
http://www.biology.arizona.edu/human_bio/activities/stokely/RFLP.html>
 Department of Biology, Davidson College, Davidson, NC 28036
 <http://www.bio.davidson.edu/Courses/Molbio/MolStudents/01anford/molecularpaper1.html >
 Lebrasseur, Nicole D.. Restriction Fragment Length Polymorphism (Rflp)." <
http://www.bookrags.com/sciences/genetics/restriction-fragment-length-polymor-wog.html >