RESTRICTION

FRAGMENT LENGTH
POLYMORPHISM
(RFLP)
PRESENTED BY: MUHAMMAD FAIZ
 Discovery
Alec Jeffreys
in 1984
Royal Society’s Royal
Medal in 1994
Wolf Prize in
Medicine in 1998
 Genetic variation
among individuals
or population
Length of DNA Fragments
generated by restriction enzyme
vary from one plant variety to
other
Variation due to VNTRs
 To perform
RFLP
technique
We have 3 Reference DNA
samples from 3 Different
Varieties & 4th Sample used
to compare with other
samples.
We have 2 restriction
endonuclease enzyme
for double digestion
DNA
Extractio
n
 Reaction Mixture
for RFLP

DNA Sample= 15 microliter

10X Assay Buffer= 3.0 microliter
Molecular Biology Grade Water= 10
microliter
ECoR1= 1.0 microliter
Pst1= 1.0 microliter
Loading 15 micro litre DNA
Sample in microfuge tube
Loading 3 micro litre 10X
Assay buffer in microfuge
tube
Adding 10 micro litre
Molecular Grade Water in
microfuge tube
Restriction
Digest
ECOR1
Restriction
Endonuclease
Enzyme
Loading 1 micro litre
ECOR1 in microfuge tube
Restriction
Digest
Pst1
Restriction
Endonuclease
Enzyme
Loading 1 micro litre
Pst1 in microfuge tube
Incubation at 37 degree for 2-3 hours
Adding 6X Gel
loading dye to
all 4 samples
Purpose of loading
DNA Ladder
DNA size marker or
molecular weight marker
A mixture of DNA
fragments of known sizes

To compare size of
DNA fragments of
experimental sample
Loading 1kb or 3
microliter DNA
Ladder in first Well
Loading 25 micro litre
DNA solution mixture to
wells
70 Volts for
about 45 Minutes
 Denaturation
Gel placed in Sodium
hydroxide (NaOH) for
Denaturation
Heating the DNA
fragments in an alkaline
solution(NaOH) which
break hydrogen bonds
Single Stranded
DNA formed
 Blotting

DNA fragments tranfer

onto nitrocellulose or
nylon membrane.
Purpose
1. To Immobilize the DNA
2. To increase Sensitivity
3. To Probe the DNA
4. Identify presence of gene of
interest from complex mixture
1_Place Sponge 2_Another Sponge
blotting paper after paper after dipping
dipping with tranfer with tranfer buffer
buffer
3_Place Filter paper 4_Place the Gel
after dipping with on filter paper
tranfer buffer accurately
5_Place Nylon 6_Place filter paper on
membrane om Gel nylon membrane
7_ Place Sponge 8_ Place another
filter paper on it Sponge filter paper on it
9_Finally
place
postive
Electrode, a
anode on it
10_Place in the 11_Add transfer buffer
tank apparatus to tank apparatus
Electro plotting transfer apparatus
connect with powerpad for 2 hours
Cross linking of
DNA onto Nylon
membrane under
UV Light for 20
Minutes
Incubation at 70
degree for 20 minutes
Add 10ml pre Incubate at 70rpm for
hybridization buffer 45 Minutes
Add 10 ml of
Hybridization buffer
at Room
Temperature
Biotinylated probe,
a short sequence of
nucleotides complementary Dip Probe in boiling
to gene of interest water bath for 5 min
Place Probes on ice 15 micro litre probe
for 5 min added in solution
Approximately on petri dish
Add 10ml Incubate for 5
wash buffer 1 min
And repeat 3
times
Add Blocking buffer
10ml for 1 hour at
Room Temperature
 Add 9 microliter of
Add 9ml of conjugate
Toeen 20 in same flask
dilution buffer in flask
 Add 10 ml wash
Add 6 microliter of streptavidine-HRP
&place 20 minutes at Room buffer III at room
Temperature temperature, 5 min
 Shake until blue
Add wash buffer 4 color produce
Now observation of hybridized
probe with gene of interest
Gene of interest is detected
finally
 APPLICATIONS
1_Genetic Mapping
2_DNA Finger Printing (In
forensic, paternity
testing, criminal
investigations)
3_Disease Diagnosis
4_Evolutionary Studies
Criminal
Investigatio
n
Paternit
y testing
Disease Diagnosis
 Advantages
1_ High Resolution
2_ Wide applicability
3_ Stable
4_ Co dominant
 Disadvantages
1_ Time Consuming
2_ Low throughput
3_ Radioactive
Labelling
4_ Limited markers
5_Interpretation
Jazakallah