DNA Fingerprinting GeNeiTM DNA Fingerprinting GeNeiTM

GeNeiTM DNA
Fingerprinting
Teaching Kit Manual
(Using RFLP Technique)

6109400011730
Cat No.
6109400021730
Revision No.: 00050516
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DNA Fingerprinting GeNeiTM DNA Fingerprinting GeNeiTM

CONTENTS

Page No.

v Objective 1

v Principle 1

v Kit Description 2

v Materials Provided 3

v Procedure 4

v Observation & Interpretation 6

AGAROSE GEL ELECTROPHORESIS

v Introduction 7

v Principle 7

v Procedure 12

v Ordering Information 14

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Objectives:
• To learn the technique of DNA fingerprinting by
Restriction Fragment Length Polymorphism (RFLP)
method through the following experiments:
• Restriction digestion of DNA
• Analysis of the digested DNA by agarose gel
electrophoresis

Principle:
DNA Fingerprinting is a technique that is used to identify
pattern that occur in DNA. No two individual have identical
DNA so this procedure can be used to identify if a sample
DNA come from a particular individual. The technique requires
that the DNA be cutup into small fragments. Restriction
enzymes (REs) are used to perform this digestion. The
technique takes advantage of the polymorphism in the genetic
codes of individuals which result in variations in phenotype.
RFLP Methodology involves cutting a particular region of DNA
with known variability, with REs, then separating the DNA
fragments by Agarose Gel Electrophoresis (AGE) and
determining the number of fragments and their relative sizes.
RFLP is one technique used by forensic scientists in DNA
finger printing. It is also used for tracing ancestry, to study
evolution and migration of wild life and detection and diagnosis
of certain diseases.

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DNA Fingerprinting GeNeiTM DNA Fingerprinting GeNeiTM

Kit description: Materials Provided:

The kit is designed to identify the bacterial isolate that The list below provides information about the materials
has acquired a Multiple Drug Resistant (MDR) plasmid using supplied in the kit. The components should be stored as
RFLP method. Five plasmids isolated from individual isolates suggested.
(one Control and four Test Samples) are supplied. Following Quantity
restriction digestion and resolution of fragments on agarose
gel, students will compare the DNA band pattern of four Test Materials 6109400011730 6109400021730 Store
(5 Expts.) (25 Expts.)
Samples with that of the Control. On analyzing the results,
they will identify the isolate that has acquired the MDR Restriction 25 µl 125 µl -20°C
plasmid. Enzyme: Ssp I
6109400011730 : The kit is designed to carry out 5 DNA 10X Assay Buffer 125 µl 625 µl -20°C
Fingerprinting experiments.
Control DNA 50 µl 250 µl -20°C
6109400021730 : The kit is designed to carry out 25 DNA
Test Samples 50 µl 250 µl -20°C
Fingerprinting experiments.
(1, 2, 3, 4) (each)
Note: Electrophoresis equipment is required for both Low Range DNA Ruler 50 µl 250 µl -20°C
the kits. (Ready to use)

Duration of Experiment: Approximately 3 hours. Gel Loading Buffer 0.25 ml 2 x 0.25 ml -20°C
Agarose 5g 20 g RT
50X TAE 20 ml 100 ml RT
1.5 ml vials 25 Nos. 125 Nos. RT

Note: Room Temperature (RT) not to exceed 20°C-25°C.

Materials Required but not Provided:

Equipment : 37°C Dry bath, UV-transilluminator,
Electrophoresis apparatus.
Reagent : Ethidium bromide, Distilled Water
Other Requirements : Micropipettes, Microtips.

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DNA Fingerprinting GeNeiTM DNA Fingerprinting GeNeiTM

Note: 9. Prepare 1.5% agarose gel (Refer Agarose

• Read the entire procedure prior to starting the
experiment.
• Wear gloves while handling Ethidium bromide and gels
stained with Ethidium bromide (refer page 11 for
disposal).
• Add Ethidium bromide at a concentration of 0.5 µg/ml
(from 10 mg/ml stock) while preparing the gel (refer
Agarose Gel Electrophoresis page 9).
• Low Range DNA Ruler supplied is ready to use and can
be loaded directly on the agarose gel.
• Use separate microtip for each of the reagents to avoid
contamination.
Electrophoresis page 13).
10. Following restriction digestion, add 5 µl of Gel Loading
Buffer to each of the vials. Load the samples in the
wells of the agarose gel with 10 µl of Ready to Use Low
Range DNA Ruler (supplied) in one well as marker. Note
down the order in which the samples and marker are
loaded.
11. Electrophorese the samples at 100 volts for 1-2 hours
till the tracking dye (Bromophenol blue) reaches 3/4th
of the length of the gel.
12. Visualize the gel under a UV transilluminator.

Procedure:
1. Place the vial containing the Restriction Enzyme on
ice.
2. Thaw the vials containing Control DNA, Test Samples
and 10X Assay Buffer.
3. Label five 1.5 ml vials as Control, Test Sample 1, Test
Sample 2, Test Sample 3 and Test Sample 4
respectively.
4. Add 34 µl of distilled water to each of the five vials.
5. Add 10 µl each of Control and Test Samples to the
respective labeled vials.
6. Add 5 µl of 10X Assay Buffer to each vial.
7. Add 1 µl of the Restriction Enzyme to each vial.
8. Mix by tapping the vial or gently vortex and incubate
the vials at 37°C for 1 hour.

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Observation:
Compare Test plasmid DNA digests with Control MDR
plasmid DNA digest and identify the Test Plasmid DNA that
has similar DNA band pattern as that of MDR Control plasmid
DNA. Record the results.

Interpretation:
The Test plasmid DNA that has similar restriction profile
as that of MDR Control plasmid DNA, would impart multiple
drug resistance to the host. The restriction enzyme
recognizes and cuts only a particular base sequence unique
to it. Any mutation in this unique sequence, would mean that
there is a loss of a site, hence giving rise to different band
pattern. It is this specificity that helps in achieving reproducible
restriction profile on digestion of a DNA sample.
Agarose Gel Electrophoresis
Thus RFLP technique helps in identifying a particular trait of
an organism (in this case multiple drug resistance) that can
be linked to a few mutations in the restriction enzyme target
sequence.
1 2 3 4 5 6

Lanes 2-5 : Test Plasmid DNA

digested with
restriction enzymes
Lanes 1&6 : Low Range DNA
Ruler

Fig: DNA Fingerprint from Test plasmid DNA samples

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DNA Fingerprinting GeNeiTM DNA Fingerprinting GeNeiTM

Introduction:
Agarose gel electrophoresis is a procedure used to
separate DNA fragments based on their molecular weight
and is an intrinsic part of almost all routine experiments carried
out in molecular biology.

The technique consists of 3 basic steps:

• Preparation of agarose gel
• Electrophoresis of the DNA
• Visualization of DNA fragments

Principle:
Preparation of Agarose Gel:
Agarose is a linear polymer extracted from seaweeds.
Its basic structure is shown in the figure.

HO
CH2O
H
OH
HO
Fig: Basic unit structure of agarose.

Purified agarose is a powder insoluble in water or buffer

at room temperature but dissolves on boiling. Molten solution
of agarose is then poured into a mould and allowed to solidify.
As it cools, agarose undergoes polymerization i.e., sugar
polymers cross-link with each other and cause the solution
to gel, the density or pore size of which is determined by the
concentration of agarose.

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DNA Fingerprinting GeNeiTM
Electrophoresis of DNA fragments:
Electrophoresis is a technique used to separate charged
molecules. DNA is negatively charged at neutral pH and when
electric field is applied across the gel, the DNA migrates
towards the anode. Migration of DNA through the gel is
dependent upon:
1. Molecular size of DNA
2. Agarose concentration
3. Conformation of DNA and
4. Applied current
Matrix of agarose gel acts as a molecular sieve through
which DNA fragments move on application of electric current.
Higher concentration of agarose gives firmer gels, i.e., spaces
between cross-linked molecules is less and hence smaller
DNA fragments easily crawl through these spaces. As the
length of the DNA increases, it becomes harder for the DNA
to pass through the spaces. Lower concentration of agarose
helps in the movement of larger DNA fragments as the spaces
between the cross-linked molecules is more. The progress
of gel electrophoresis is monitored by observing the migration
of a visible dye (tracking dye) through the gel. Two commonly
used dyes are Xylene cyanol and Bromophenol blue that
migrate at the same speed as double stranded DNA of size
5000 bp and 300 bp respectively. These tracking dyes are
negatively charged, low molecular weight compounds that
are loaded along with each sample at the start of
electrophoresis, when the tracking dye reaches towards the
anode, electrophoresis is terminated.
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DNA Fingerprinting GeNeiTM
Visualisation of DNA fragments:
Since DNA is not naturally coloured, it will not be visible
on the gel. Hence the gel, after electrophoresis, is stained
with a dye specific to the DNA. Discrete bands are observed
when there is enough DNA present to bind to the dye and
make it visible, otherwise the band is not detected. The gel
is observed against a light background wherein DNA appears
as dark coloured bands.
Alternatively, an intercalating dye like Ethidium bromide
is added to agarose gel and bands are visualized by examining
the gel under UV light. DNA appears fluorescent.
Note:
l Ethidium bromide must be handled carefully as
it is a mutagen and a carcinogen. Wear gloves
while handling EtBr solution and gels stained with
EtBr.
l Gels and gloves with Ethidum bromide are to be
collected in separate plastic bags and shipped
to the external waste management agency for
incineration as part of hazardous solid waste.
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DNA Fingerprinting GeNeiTM
Procedure:
Preparation of 1.5% Agarose Gel
1. Prepare 1X TAE by diluting appropriate amount of
50X TAE buffer. (For one experiment, approximately
200 ml of 1X TAE is required. Dilute 4 ml of 50X TAE to
200 ml with distilled water).
2. Weigh 0.75 g of agarose and add to 50 ml of 1X TAE.
This gives 1.5% agarose.
3. Boil till agarose dissolves completely and a clear
solution is obtained.
4. Add 2 µl Ethidium bromide to molten agarose (from a
stock of 10 mg/ml in water), when temperature is around
50°C. Mix and cast the gel.
5. Meanwhile place the comb of electrophoresis set such
that it is approximately 2 cm away from the cathode.
6. Pour the agarose solution in the central part of tank,
when the temperature reaches approximately 60ºC. Do
not generate air bubbles. The thickness of the gel
should be around 0.5 to 0.9 cm. Keep the gel
undisturbed at room temperature for the agarose to
solidify.
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DNA Fingerprinting GeNeiTM
Electrophoresis
7. Pour 1X TAE buffer into the gel tank till the buffer level
stands at 0.5 to 0.8 cm above the gel surface.
8. Gently lift the combs, ensuring that wells remain intact.
9. Connect the power cord to the electrophoretic power
supply according to the convention
red: anode, black: cathode.
10. Load the samples in the wells in the desired order.
11. Set the voltage to 50 V and switch on the power supply.
12. Switch off the power when the tracking dye
(bromophenol blue) from the well reaches ¾th of the
gel. This takes approximately one hour.
13. After electrophoresis, DNA samples can be visualized
under UV-light. They appear fluorescent.
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Ordering Information
Product Size Cat #

GeNei™ DNA Fingerpriniting 1 EA 6109400011730

Teaching Kit
(Using RFLP technique)
(Consumables for 5 Experiments)

GeNei™ DNA Fingerpriniting 1 EA 6109400021730

Teaching Kit
(Using RFLP technique)
(Consumables for 25 Experiments)

Email:
Sales: sales@geneilabs.com

Customer Support: techsupport@geneilabs.com

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