Biology

Project Report
On

“GEL-ELECTROPHORESIS”
Session: 2023-24

Submitted to:- Submitted by

Ms. Avinash Kaur Mayank
Lecturer in Biology Roll No.
Class XII (Medical)

SAHARA COMPREHENSIVE
SCHOOL, KURUKSHETRA
 CERTIFICATE

This is certified that Mayank, Roll No………………… student of XII

(Medical) has completed the project “Gel-Electrophoresis”. The work done in
the project is the result of candidate’s own efforts. The project is considered
as a part of fulfillment of Biology-Practical Examination of 10+2 class.

Ms. Avinash Kaur

Lecturer in Biology
 ACKNOWLEDGEMENT

I have great pleasure in recording my profound gratitude to Ms. Avinash

Kaur, for her invaluable guidance, constant encouragement and immense help
given at each step for persuing this work which reveals her vast knowledge in
the field of Biology.

Mayank
Class – XII, (Medical)
Roll No.
 CONTENTS

 Introduction
 History of GM Foods
 Process
 Tests
 Issues
 Benefits and Controversies
 Bibliography
 Gel-Electrophoresis and Its Applications

1. Introduction

Positive or negative electrical charges are frequently associated with biomolecules. When
placed in an electric field, charged biomolecules move towards the electrode of opposite charge
due to the phenomenon of electrostatic attraction. Electrophoresis is the separation of charged
molecules in an applied electric field. The relative mobility of individual molecules depends on
several factors. The most important of which are net charge, charge/mass ratio, molecular shape
and the temperature, porosity and viscosity of the matrix through which the molecule migrates.
Complex mixtures can be separated to very high resolution by this process (Sheehan, D.; 2000).

2. Principle of Electrophoresis

If a mixture of electrically charged biomolecules is placed in an electric field of field

strength E, they will freely move towards the electrode of opposite charge. However, different
molecules will move at quite different and individual rates depending on the physical
characteristics of the molecule and on experimental system used. The velocity of movement, ν,
of a charged molecule in an electric field depends on variables described by

v=E . g /f

where f is the frictional coefficient and q is the net charge on the molecule (Adamson, N. j. &
Reynolds, E. C.; 1997). The frictional coefficient describes frictional resistance to mobility and
depends on a number of factors such as mass of the molecule, its degree of compactness, buffer
viscosity and the porosity of the matrix in which the experiment is performed. The net charge is
determined by the number of positive and negative charges in the molecule. Charges are
conferred on proteins by amino acid side chains as well as by groups arising from post
translational modifications such as deamidation, acylation or phosphorylation. DNA has a
particularly uniform charge distribution since a phosphate group confers a single negative charge
per nucleotide. Equation 1 means that, in general molecules will move faster as their net charge
increases, the electric field strengthens and as f decreases (which is a function of molecular
mass/shape). Molecules of similar net charge separate due to differences in frictional coefficient
while molecules of similar mass/shape may differ widely from each other in net charge.
Consequently, it is often possible to achieve very high resolution separation by electrophoresis.

3. Gel Electrophoresis

Hydrated gel networks have many desirable properties for electrophoresis. They allow a
wide variety of mechanically stable experimental formats such as horizontal/vertical
electrophoresis in slab gels or electrophoresis in tubes or capillaries. The mechanical stability
also facilitates post electrophoretic manipulation making further experimentation possible such
as blotting, electro-elution or MS identification /finger printing of intact proteins or of proteins
digested in gel slices. Since gels used in biochemistry are chemically rather unreactive, they
interact minimally with biomolecules during electrophoresis allowing separation based on
physical rather than chemical differences between sample components (Adamson, N. j. &
Reynolds, E. C.; 1997).

3.1 Gel types

In general the macromolecules solution is electrophoresed through some kind of matrix.

The matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size.
The kind of supporting matrix used depends on the type of molecules to be separated and on the
desired basis for separation: charge, molecular weight or both (Dolnik, V.; 1997). The most
commonly used materials for the separation of nucleic acids and proteins are agarose and
acrylamide.

Medium Conditions Principal Uses

Starch Cast in tubes or slabs Proteins
Agarose gel Cast in tubes or slabs Very large proteins, nucleic
No cross-linking acids, nucleoproteins etc
Acrylamide gel Cast in tubes or slabs Proteins and nucleic acids
Cross-linking
 Agarose: The most widely used polysaccharide gel matrix nowadays is that formed with
agarose. This is a polymer composed of a repeating disaccharide unit called agarobiose
which consists of galactose and 3,6-anhydrogalactose (Fig. 1). Agarose gives a more
 uniform degree of porosity than starch and this may be varied by altering the starting
concentration of the suspension (low concentrations give large pores while high
concentrations give smaller pores). This gel has found wide spread use especially in the
separation of DNA molecules (although it may also be used in some electrophoretic
procedures involving protein samples such as immuno- electrophoresis). Because of the
uniform charge distribution in nucleic acids, it is possible accurately to determine DNA
molecular masses based on mobility in agarose gels. However the limited mechanical
stability of agarose, while sufficient to form a stable horizontal gel, compromises the
possibilities for post-electrophoretic manipulation.
 Acrylamide: A far stronger gel suitable for electrophoretic separation of both proteins
and nucleic acids may be formed by the polymerization of acrylamide. The inclusion of a
small amount of acrylamide cross linked by a methylene bridge (N,N′ methylene
bisacrylamide) allows formation of a cross linked gel with a highly-controlled porosity
which is also mechanically strong and chemically inert. For separation of proteins, the
ratio of acrylamide : N,N′ methylene bisacrylamide is usually 40:1 while for DNA
separation it is 19:1. Such gels are suitable for high-resolution separation of DNA and
proteins across a large mass range.
 Stain Use Detection limita
(ng)
Amido black Proteins 400
Coomassie blue Proteins 200
Ponceau red Proteins (reversible) 200
Bis-1-anilino-8-Naphthalene sulphonate Proteins 150
Nile red Proteins (reversible) 20
SYPRO orange Proteins 10
Fluorescamine (protein treated
prior to electrophoresis) Proteins 1
Silver chloride Proteins/DNA 1
SYPRO red Proteins 0.5
Ethidium bromide DNA/RNA 10

3.2 Staining of Gel

One of the most important aspects of gel electrophoresis technique is staining. Once
sample molecules have separated in the gel matrix it is necessary to visualize their position. This
is achieved by staining with an agent appropriate for the sample. Some of the more common
staining methods used in biochemistry are listed in Table

3.3 Preparation and running of Standard Agarose Gels

 The equipment and supplies necessary for conducting agarose gel electrophoresis are
relatively simple and include.
 An electrophoresis chamber and power supply
  Gel casting trays, which are available in a variety of sizes and composed of UV-
transparent plastic. The open ends of the trays are closed with tape while the gel is being
cast, then removed prior to electrophoresis.
 Sample combs, around which molten medium is poured to form sample wells in the gel.
 Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
 Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to
"fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and
allow visual monitoring or how far the electrophoresis has proceeded.
 Staining: DNA molecules are easily visualized under an ultraviolet lamp when
electrphoresed in the presence of the extrinsic fluor ethidium bromide. Alternatively,
nucleic acids can be stained after electrophoretic separation by soaking the gel in a
solution of ethidium bromide. When intercalated into double- stranded DNA,
fluorescence of this molecule increases greatly. It is also possible to detect DNA with
the extrinsic fluor 1-anilino 8-naphthalene sulphonate. NOTE: Ethidium bromide is a
known mutagen and should be handled as a hazardous chemical - wear gloves while
handling.
 Transilluminator (an ultraviolet light box), which is used to visualize stained DNA in
gels. NOTE: always wear protective eyewear when observing DNA on a
Transilluminator to prevent damage to the eyes from UV light.

To prepare gel, agarose powder is mixed with electrophoresis buffer to the desired
concentration, and heated in a microwave oven to melt it. Ethidium bromide is added to the gel
(final concentration 0.5 ug/ml) to facilitate visualization of DNA after electrophoresis. After

cooling the solution to about 60 oC, it is poured into a casting tray containing a sample comb and
allowed to solidify at room temperature.
 After the gel has solidified, the comb is removed, taking care not to rip the bottom of the
wells. The gel, still in plastic tray, is inserted horizontally into the electrophoresis chamber and is
covered with buffer. Samples containing DNA mixed with loading buffer are then pipetted into
the sample wells, the lid and power leads are placed on the apparatus (Fig. 2), and a current is
applied. The current flow can be confirmed by observing bubbles coming off the electrodes.
DNA will migrate towards the positive electrode, which is usually colored red, in view of its
negative charge

The distance DNA has migrated in the gel can be judged by visually monitoring
migration of the tracking dyes like bromophenol blue and xylene cyanol dyes.

3.4 Preparation and running of polyacrylamide gels

3.4.1 Preparation of polyacrylamide gel

The listed protocol is for the preparation of a polyacrylamide with the dimensions of
15.5 cm wide by 24.4 cm long by 0.6 mm thick.

Unpolymerized acrylamide is a neurotoxin and a suspected carcinogen; avoid inhalation

and contact with skin. Always wear gloves when working with acrylamide powder or solutions.
Methacryloxypropyltrimethoxysilane (bind silane) is toxic and should be used in a chemical
fume hood.

One glass plate will be treated with Gel Slick to prevent the gel from sticking and the
shorter glass plate will be treated with bind silane to bind the gel. The two plates must be kept
apart at all times to prevent cross-contamination.

To remove the glass plate treatments (Gel Slick or bind silane), immerse the plates in 10%
NaOH solution for one hour. Thoroughly rinse the plates with deionized water and clean with a
detergent.

The gel may be stored overnight on a paper towel saturated with deionized water and
plastic wrap are placed around the well end of the gel to prevent the gel from drying out.
3.4.2 Sample loading and electrophoresis

Protein is usually stained with the dye coomassie blue. Less sensitive protein dyes
include ponceau red and amido black. Ponceau red has the advantage that it stains reversibly
and may be removed from the protein to allow subsequent analysis (e.g. immunostaining.

Silver Staining: The most sensitive staining for protein is silver staining. This involves

soaking the gel in Ag NO3 which results in precipitation of metallic silver (Ag 0) at the location
of protein or DNA forming a black deposit in a process similar to that used in black- and-white
photography.

 Steps involving formaldehyde solutions should be performed in the fume hood.

 Chill the developer solution to 4˚C. Prepare the developer fresh before each use.
 Be sure to save the fix/stop solution from the first step in the silver staining to add to the
developer solution once the bands are visible.
 The 10 second deionized water rinse must not exceed this time frame. If it does, the
deposited silver may be rinsed away and the staining must be done again.

3.5 Agarose Gel Electrophoresis of DNA

3.5.1 Migration of DNA fragments in agarose

Fragments of linear DNA migrate through agarose gels with a mobility that is inversely
proportional to the log10 of their molecular weight. In other words, if you plot the distance from
the well that DNA fragments have migrated against the log 10 of either their molecular weights or
number of base pairs, a roughly straight line will appear.

Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the
same mass. Typically, uncut plasmids will appear to migrate more rapidly than the same
plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two
topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles
(Brody, J. R. & Kern, S. E.; 2004). The image to the right shows an ethidium-stained gel with
uncut plasmid in the left lane and the same plasmid linearized at a single site in the right lane.

Several additional factors have important effects on the mobility of DNA fragments in
agarose gels, and can be used to your advantage in optimizing separation of DNA fragments.
Chief among these factors are:

Agarose Concentration: By using gels with different concentrations of agarose, one

can resolve different sizes of DNA fragments. Higher concentrations of agarose facilitate
separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs.

The image to the right shows migration of a set of DNA fragments in three
concentrations of agarose, all of which were in the same gel tray and electrophoresed at the same
voltage and for identical times. Notice how the larger fragments are much better resolved in the
0.7% gel, while the small fragments separated best in 1.5% agarose. The 1000 bp fragment is
indicated in each lane.

Voltage: As the voltage applied to a gel is increased, larger fragments migrate

proportionally faster those small fragments. For that reason, the best resolution of fragments
larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel (the cm
value is the distance between the two electrodes, not the length of the gel).

Electrophoresis Buffer: Several different buffers have been recommended for

electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-
EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates
in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but
provide ions to support conductivity. If you mistakenly use water instead of buffer, there will be
essentially no migration of DNA in the gel! Conversely, if you use concentrated buffer (e.g. a
10X stock solution), enough heat may be generated in the gel to melt it.

Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates

between bases of nucleic acids and allows very convenient detection of DNA fragments in gels,
as shown by all the images on this page. As described above, it can be incorporated into agarose
gels, or added to samples of DNA before loading to enable visualization of the fragments within
the gel. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity,
and therefore its mobility.

3.6 Applications of gel electrophoresis

Agarose gel electrophoresis technique was extensively used for investigating the DNA
cleavage efficiency of small molecules and as a useful method to investigate various binding
modes of small molecules to supercoiled DNA (Song, Y.M.; Wu, Q.; Yang, P.J.; Luan, N.N.;
Wang, L.F. & Liu, Y.M.; 2006., Tan, C.P.; Liu, J.; Chen. L –M.; Shi, S.; Ji, L–N.; 2008., Zuber,
; Quada, J.C. Jr.; Hecht, S.M.; 1998., Wang, H.F.; Shen, R.; Tang, N.; 2009., Katsarou, M.E.
et. al 2008., Skyrinou, K.C. et al, 2009., Ray, A.; Rosair, G.M.; Kadam, R.; Mitra, S.;
2009., Wang, Q.; Li, W.; Gao, F.; Li, S.; Ni, J.; Zheng, Z.; 2010., Li, Y.; Yang, Z.; 2009.,
Reddy, P.A.N.; Nethaji, M. & Chakravarty, A.R.; 2004). This was mainly due to the importance
of DNA cleavage in drug designing. Natural derived plasmid DNA mainly has a closed circle
supercoiled form (SC), as well as nicked circular form (NC) and linear form as small fractions.
Relaxation of supercoiled (SC) pUC19 DNA into nicked circular (NC) and linear (LC)
conformation can be used to quantify the relative cleavage efficiency of complexes by agarose
gel electrophoresis technique. It is also a useful method to investigate various binding modes of
small molecules to supercoiled DNA. Intercalation of small molecules to plasmid DNA can
loosen or cleave the SC DNA form, which decreases its mobility rate and can be separately
visualized by agarose gel electrophoresis method, whereas simple electrostatic interaction of
small molecules to DNA does not significantly influence the SC form of plasmid DNA, thus the
mobility of supercoiled DNA does not change (Chen, Z-F.; 2011).
 We have been using this technique for some time in the development of new
metallonucleases as small molecular models for DNA cleavage at physiological conditions
(Reddy, P. R. et.al, 2004-2011). Since DNA cleavage is a biological necessity, these small
molecular models have provided much of our most accurate information about nucleic acid
binding specificity.

The DNA cleavage could occur by two major pathways, i.e., hydrolytic and oxidative:

a. Hydrolytic DNA cleavage involves cleavage of phosphodiester bond to generate

fragments which could be subsequently religated. Hydrolytic cleavage active species
mimic restriction enzymes.
b. Oxidative DNA cleavage involves either oxidation of the deoxyribose moiety by
abstraction of sugar hydrogen or oxidation of nucleobases. The purine base guanine is
most susceptible for oxidation among the four nucleobases.

Oxidative cleavage of DNA occurs in the presence of additives or photoinduced DNA cleavage
agents (Cowan, J. A.; 1998., Hegg, E. L. & Burstyn, J. N.; 1998). Photocleavers require the
presence of a photosensitizer that can be activated on irradiation with UV or visible light. The
redox active ‘chemical nucleases’ are effective cleavers of DNA in the presence of a reducing
agent or H2O2 as an additive (Pogozelski, W. K. & Tullius, T. D.; 1998., Armitage, B.; 1998).

Oxidative cleavage agents require the addition of an external agent (e.g. light or H 2O2) to
initiate cleavage and are thus limited to in vitro applications. Since these processes are radical
based (Pratveil, G.; Duarte, V.; Bernaudou, J. & Meunier, J.; 1993) and deliver products lacking
3' or 5' phosphate groups that are not amenable to further enzymatic manipulation, the use of
these reagents has been limited in the field of molecular biology and their full therapeutic
potential has not been realized. Hydrolytic cleavage agents do not suffer from these drawbacks.
They do not require co-reactants and, therefore, could be more useful in drug design. Also, they
produce fragments that may be religated enzymatically. The metal complexes that catalyze DNA
hydrolytic cleavage could be useful not only in gene manipulation but also in mimicking and
elucidating the important roles of metal ions in metalloenzyme catalysis (Liu, C. et.al, 2002).

Keeping this in view, we report here few of the several metallonucleases which were
designed, isolated, characterized, structures established and their DNA cleavage properties
investigated. The emphasis was on biomolecules which have relevance to in-vivo systems. Here
we describe in detail the DNA cleavage abilities of the following copper-amino acid/dipeptide
containing complexes.

[Cu(II)(hist)(tyr)]+ (1)
[Cu(II)(hist)(trp)]+ (2)
[Cu(II)(ala)(phen)(H2O)] ClO4 (3)
[Cu(II)(ala)(bpy)(H2O)] ClO4 (4)
[Cu(II)(phen)(his-leu)]+ (5)
[Cu(II)(phen)(his-ser)]+ (6)
[Cu(II)(trp-phe)(phen)(H2O)] ClO4 (7)
[Cu(II)(trp-phe)(bpy)(H2O)] ClO4 (8)

The cleavage reaction on supercoiled plasmid DNA (SC DNA) was monitored by agarose
gel electrophoresis. When SC DNA was subjected to electrophoresis, relatively fast migration
was observed for the intact SC DNA. If scission occurs on one strand (nicking), the SC form will
relax to generate a slower moving nicked circular (NC) form. If both strands are cleaved, a linear
form (LF) that migrates between SC form and NC form will be generated.

System I: Copper-histamine-tyrosine (1)/tryptophan (2).

The conversion of SC DNA to NC form was observed with increase in the concentrations
of complexes 1 and 2 (Fig. 3a and b). The DNA cleavage activity is continuously increases with
increasing concentration of the complexes, at 625 M they converts more than 50% of SC DNA
to NC form.
System II: Copper-alanine-phenanthroline (3) / bipyridine (4).

When the DNA was incubated with increasing concentrations of complexes, SC pUC19
DNA was degraded to NC form (Fig. 4). At 250 µM of 3 (Fig. 4a), a complete conversion
(100%) of SC DNA in to NC form was achieved while complex 4 (Fig. 4b) could convert only
52%. This may be due to the effective stacking interaction of phen compared to bpy which is
known to enhance the cleavage activity.

System III: Copper-phenanthroline-histidylleucine (5) /histidylserine (6).

Upon the addition of increasing amounts of the complexes 5 or 6, we observed the

conversion of SC form to NC form (Fig. 5) with continuous increase with respective to
concentration. A complete conversion is observed at a concentration of 500 µM for both the
complexes. A possible rationalization for the degradation of DNA is the formation of a three
centered H-bond involving the NH2 group of guanine, the electron lone pair of the imidazole

ring, and the COO - group of either histidylleucine or histidylserine.

System IV: Copper-tryptophan-phenylalanine-phenanthroline (7)/bipyridine (8).

In the case of 7 and 8, when DNA was incubated with increasing concentrations of
complexes SC DNA was degraded to NC form. The catalytic activities of 7 and 8 are depicted
in Fig. 6. The complex 7 show a complete conversion of supercoiled plasmid DNA into the
nicked circular form at 50 M and at 100 M the DNA was completely smeared (Fig. 6a). In
contrast only 40% cleavage was achieved by 8 (Fig. 6b). This may be due to the efficient
binding of 7 with DNA compared to 8 and may also be due to the generation of stable

[Cu(phen)2]+ species which could be related to the presence of an indole ring of tryptophan-
phenylalanine moiety which is known to stabilize the radical species.

The gel-electrophoresis technique was also utilized for obtaining kinetic data for the
above systems. From these kinetic plots the rate of hydrolysis of phosphodiester bond was
determined.

The time dependent DNA cleavage reaction in the presence and absence of the
complexes was also studied to calculate rate of hydrolysis. Fig. 7-10 shows the extent of
decrease and increase of SC and NC forms, respectively
System – I

System - II
System - III

System - IV

The conversion versus time follows the pseudo-first-order kinetics and both the forms
fitted well to a single exponential curve. From these curve fits, the DNA hydrolysis rates were

determined as 0.91 h-1 (R=0.971), 0.79 h-1 (R=0.971), 1.35 h-1 (R=0.983), 0.56h-1 (R=0.959),

1.32 h-1 (R = 0.971), 1.40 h-1 (R = 0.971 ), 1.74 h-1 (R=0.985), 0.65h-1(R=0.963) for 1-8
respectively. The enhancement of DNA hydrolysis rate constant by metal complexes in the
range of 0.09- 0.25 h-1 was considered impressive (Rammo, J. et al, 1996., Roigk, A.; Hettich,
R.; Schneider, J.; 1998). The above rate constants of the complexes (1-8) amounts to (1.5 – 4.6)

x107 h-1 fold rate enhancement compared to uncatalyzed double stranded DNA (3.6 x 10 -8 h-
1) (Sreedhra, A.; Freed, J. D.; Cowan, J. A.; 2000) is impressive considering the type of ligands
and experimental conditions employed

Conclusion

The rates of DNA hydrolysis of complexes (1-8) were impressive compared to uncatalyzed
double stranded DNA considering the type of ligands and experimental conditions involved.
These studies have proved that this technique has provided an insight into the type of cleavage,
percentage of cleavage and its utility in the drug design. It is obvious from the above examples
that the gel electrophoresis technique is not only useful in studying the pattern of DNA cleavage
but also to evaluate the catalytic efficiency of the metallonucleases.