Forensic Instrumentation

ELECTROPHORESIS
GROUP 7
INTRODUCTION
Electrophoresis is a laboratory technique used to separate
DNA, RNA, or protein molecules based on their size and Electrophoresis
electrical charge.

The electrophoresis phenomenon of electrophoresis was

observed for the first time in 1807 by Russian professors
Peter Ivanovich Strahov and Ferdinand Frederic Reuss at
Moscow Univerity, who noticed that the application of a
constant electric field caused clay particles dispersed in water
to migrate.
Ability to separate proteins & other biological components
mean that electrophoresis is popular in the filed of forensics
particularly in the Analysis of unknown sample, used to
separate & Analyze protein in blood for DNA fingerprint &
DNA testing.
 PRINCIPLE:

Electrophoresis is a general term that describes the migration and

separation of charged particles (ions) under the influence of an
electrical field. An electrophoretic
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system consists of two
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electrodes of opposite charge (anode, cathode), connected by a

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conducting medium called an electrolyte. The separation here. effect on
the ionic particles results from differences in their velocity (v),
which is the product of the particle's mobility (m) and the field
strenght.
 WORKING MECHANISM:
• The working principle of Electrophoresis is that it causes the separation of the
molecules, ions or colloidal particles that suspends in the matrix under the
force of an electrical field.
• The electrical field allows, the migration of the positively charged molecule
towards the anode and the migration of negatively charged molecule towards
the cathode.
• Therefore, electrophoresis is the electrokinetic phenomenon where the motion
molecules occurs under an electrical field.
DNA extraction - is the isolation and purification procedure of DNA. DNA
can be isolated from blood, frozen tissue samples or paraffin tissue blocks.
The three steps of DNA extraction are cell lysis, isolation of DNA, and
precipitation. During cell lysis, cell membrane barriers such as cell
membrane and the membranes of the nucleus break open in order to expose
DNA. The next step is the removal of membrane lipids from the sample.
Finally, the precipitation of DNA involves the removal of DNA-associated
proteins by protease and the removal of RNA by RNase.
 Phenol-chloroform extraction can also be used to separate DNA from proteins.
Here, phenol denatures the proteins in the sample, and the DNA remains in the
aqueous phase at the end of the extraction. And, in in the organic phase, you can
find the denatured proteins. Another method to extract DNA is the minicolumn
purification. Here, binding of DNA into the column relies on the pH and the salt
concentration of the buffer.
 Usage of electrophoresis in forensic:

Electrophoresis analysis is used in forensics to compare DNA, in

medical laboratories to do genetic testing, and in microbiology labs to
identify microorganisms. In addition to analyzing proteins or DNA,
electrophoresis is also used to create purified samples of proteins.

Gel electrophoresis is used to create DNA fingerprints from crime

scene and suspect samples. A match between samples suggests which
suspect committed the crime.
 3 types of electrophoresis:

1. Gel electrophoresis
2. Capillary electrophoresis
3. Matrix-Assisted Laser Desorption/Ionization Time-of-
Flight Mass Spectrometry
GEL ELECTROPHORESIS - IS
A LABORATORY METHOD
USED TO SEPARATE
MIXTURES OF DNA, RNA, OR
PROTEINS ACCORDING TO
MOLECULAR SIZE. IN GEL
ELECTROPHORESIS, THE
MOLECULES TO BE
SEPARATED ARE PUSHED BY
AN ELECTRICAL FIELD
THROUGH A GEL THAT
CONTAINS SMALL PORES.
Gel electrophoresis
 CAPILLARY
ELECTROPHORESIS - IS AN
ANALYTICAL TECHNIQUE
THAT SEPARATES IONS
BASED ON THEIR
ELECTROPHORETIC
MOBILITY WITH THE USE
OF AN APPLIED VOLTAGE.
 MATRIX-ASSISTED LASER
DESORPTION/IONIZATION TIME-
OF-FLIGHT MASS
SPECTROMETRY - IS A HIGH
THROUGHPUT TECHNOLOGY
BASED ON THE COMPARISON OF
THE PROTEIN FINGERPRINT
OBTAINED BY MICROBIAL CELLS
WITH A DATABASE OF
REFERENCE SPECTRA BY MEANS
OF THE USE OF VARIOUS
ALGORITHMS INTEGRATED IN
SYSTEMS RECENTLY MADE
COMMERCIALLY AVAILABLE.
DNA TESTING - IS USED TO IDENTIFY CHANGES IN DNA
SEQUENCE OR CHROMOSOME STRUCTURE. GENETIC
TESTING CAN ALSO INCLUDE MEASURING THE
RESULTS OF GENETIC CHANGES, SUCH AS RNA
ANALYSIS AS AN OUTPUT OF GENE EXPRESSION, OR
THROUGH BIOCHEMICAL ANALYSIS TO MEASURE
SPECIFIC PROTEIN OUTPUT.
 DNA FINGERPRINTING- IS A LABORATORY
TECHNIQUE USED TO DETERMINE THE PROBABLE
IDENTITY OF A PERSON BASED ON THE
NUCLEOTIDE SEQUENCES OF CERTAIN REGIONS OF
HUMAN DNA THAT ARE UNIQUE TO INDIVIDUALS.
Different Technique are:

-high & low voltage electrophoresis

-Immunoelectrophoresis
 High voltage electrophoresis:

-To obtain voltage gradients up to 100 Vcm, high voltage

and current supplying.
-Using cooling plates for heat dissipation generated by high
voltage.
-Less than of 1h analysis time.

-Working best with small ions deriving from small peptides and
acids.
HIGH VOLTAGE ELECTROPHORESIS
• The apparatus commonly used for high voltage paper
electrophoresis is of two types:

• Enclosed Strip
• Immersed Strip
 ENCLOSED STRIP
• In principle, paper electrophoresis is simpler than the paper
chromagtography.
• There are two features of an ion which are most pertinent to its
electrophoretic behavior; the size of the charge it carries.
• These features oppose each other; the bigger the size the slower
the ion will travel; the bigger the charge the faster it will go.
 IMMERSED STRIP METHOD
WORKING:

• In this method the sample is applied to a strip of filter paper maintained

with buffer solution and the strip is immersed in a large volume of buffer-
immiscible, electrically insulating and volative cooling liquid.
• Apparatus can also be designed to accumulative cooling liquids which have a
higher density than that of buffer solution.
 Low voltage electrophoresis:

-Two compartments to hold the buffer and

electrophoredes
-A suitable carrier for support medium ending in contact
with the buffer medium
-To provide voltage gradient -5 Vcm, a power pack
supplying up to 500 V even 1000 V and 0-150 ma.
LOW VOLTAGE ELECTROPHORESIS

The apparatus commonly used for low voltage paper

electrophoresis is of two type:

• Horizontal Strip
• Vertical Strip
APPLICATION OF LOW VOLTAGE
ELECTROPHORESIS

• In examination of biological and clinical specimen for amino

acids and proteins.
• Used for seperation of proteins and other high molecular
weight substances.
 IMMUNOELECTROPHORESIS

When an electric current is applied to a slide filled with a layer of gel, a mixture
of antigens present in the wells is separated into individual antigens based on the
size and charge. Then, the separated molecules are allowed to react with specific
antisera placed in troughs, placed parallel to electrophoretic migration and
diffusion takes place. Antisera in the troughs migrate towards antigens, react with
them, forming separate precipitin lines, which is indicative of the reaction of
individual proteins with antibodies.
 Murder of Sara Lynn Wineski, 2005 : Solved through DNA evidence.

Sadly enough, when the death of a homeless or transient person occurs, not as much attention might be
given to solving the crime. This was not the case with transient Sara Lynn Wineski, who was found
strangled and raped outside a then-Ronald McDonald House in St. Petersburg, Fla., in 2005. Screams
were heard about 11 p.m. on May 21, 2005, reported 10-News, but it was not until the following afternoon
that her body was found under a deck behind the house. DNA evidence was collected at the scene, but
progress had never been made until this year when it was linked to Raymond Samuels, aged 31 as of this
writing (November, 2013). He has been in prison since 2006 in Ohio for attempted murder and
kidnapping and will be extradited to Florida. Investigators indicate that Samuels, also a transient, was in
the area at the time of Wineski’s death. According to investigators, it was DNA that was imperative to
solving this crime, and, as a result, Samuels now faces first-degree murder charges.