|



 
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 Introduction

a

 
an analytical process used to examine the
movement of charged molecules through a
support medium under the influence of an
electric field
 Introduction
a
 
a



a  electric field in volts/cm,
 net charge on the molecule,

 frictional coefficient which depends on the mass and
shape of the molecule and
  velocity of the molecule
 Introduction
º Under 

  

  
, the movement of a
charged molecule is dependent on the ratio of /
.

º ˜he
of molecules with similar conformation 

  



.

º ˜hese conditions make equation 1 highly dependent on

the charge ( [ and mass dependence of
which means
that molecules migrate in an electric field at a rate
proportional to their charge-to-mass ratio.
 Introduction
a
 

  
 
 Introduction
º Equation 2 does not adequately describe the
electrophoretic process since important factors
such as interaction of migrating molecules with
the support medium and shielding of the
molecules by the buffer ions are not accounted in
the equation.

º ˜his means that electrophoresis is not useful for

describing specific details about the shape of a
molecule but it is applied in the analysis of purity
and size of macromolecules.
 Introduction
Basis for the analysis and separation of
electrophoretic methods:




 
 









 




    



 Introduction
Uses of electrophoretic techniques:


   of amino acids, peptides, proteins,
nucleotides, nucleic acids and other charged
molecules
 ˜ypes of Electrophoresis
Based on their support medium:
1. Cellulose
2. Gel
 Cellulose
º used as a support medium for low-molecular
weight biomolecules such as amino acids and
carbohydrates
 Gel
º widely used as support for larger molecules

º can either be    or 

º polyacrylamide - used for shorter fragments

º agarose - used for larger fragments

 Gel Electrophoresis
º no void volume in the matrix
º continuous network of pores throughout the
gel
º comparable to a single bead in gel filtration
º small molecules are eluted first before the
large ones
º usually carried out at basic pH, where most
biological molecules are anionic hence they
move toward the anode
º ˜wo commonly used electrophoretic
techniques for DNA analysis:

1. polyacrylamide gel electrophoresis (PAGE[

2. agarose gel electrophoresis (AGE[
 ·
a
a
 


 Î ʹ 1000 70 ʹ 80 000




 
 20% - 3% acrylamide 3% - 0.01% agarose
 ·
a
º prepared by the free radical polymerization of
acrylamide and the cross-linking agent @ @
-
methylene-bis-acrylamide
º enhanced resolution of sample components
because the separation is based on both
molecular sieving and electrophoretic mobility
º standard gel for protein separation is 7.5%
polyacrylamide  molecular size range of 10,000
to 1,000,000 daltons (best resolution is in the
range of 30,000 to 300,000 daltons[
 ·
a
º resolving power and molecular size range of a
gel depend on the concentrations of
acrylamide and bis-acrylamide
º M  gels have larger pores which allows
analysis of higher-molecular-weight
biomolecules
º M  gels with smaller pores which allow
analysis of lower-molecular-weight
biomolecules
 ·
a
Advantages of acrylamide polymerization in
electrophoresis:

º high resolving power for small and moderately

sized proteins and nucleic acids (up to
approximately 1x10Î daltons[
º acceptance of relatively large sample sizes
º minimal interactions of the migrating molecules
with the matrix
º physical stability of the matrix

a
º used to characterize RNA and DNA in the
range of 200 to 50,000 base pairs (50 kilobase[
º agarose concentrations of 0.3 to 2.0% are the
most effective for nucleic acid separation
º gels with less than 0.5% agarose are rather
fragile and must be used in a horizontal
configuration
 

º a linear polymer of galactopyranose
derivatives
º a natural polysaccharide that is extracted and
purified from seaweed
 ·

 

º form inert matrix
º high sulfate content
º high gel strength
º specific gel point
Ëtructure of Agarose
 ·
a vs
a
·

 ·
a
a
Ëupport medium Polyacrylamide Agarose
Mode of gelling Polymerization Non-covalent interactions
Gel preparation More difficult Easier
Visualization reagent Coomassie brilliant blue Ethidium bromide
Migration anode anode
Ëeparation range Ëmall fragments Moderate to larged size
Resolving power Higher Lower
DNA purity Higher Lower
 aa
º acronym for Electroendosmosis
º drift of a fluid through an aqueous gel towards
an electrode during electrophoresis
º drift occurs when electrically neutral, or nearly
neutral, molecules are present in a sample to
be electrophoresed and the gel medium
carries a charge
 aa
º when agarose is the medium, anionic residues
such as ester sulfate and pyruvate groups are
present and impart a net negative charge to the
gel
º although the gel itself can't move anodally, the
water sphere around it is pulled or distorted
toward the cathode by hydrated cations
associated with the bound anions
º as a result, neutral molecules in the sample are
gradually pulled towards the cathode with the
water
 aa
º results in blurred zones and drying of the gel
in the anodal area of flatbed gels
º normally seen as a negative effect, yet a few
methods take advantage of this effect to
achieve separation or detection results (MEKC
in capillary electrophoresis and counter
immunoelectrophoresis[
Preparation of gels:

º High quality low-melting agarose (which melts

at Î5°C and sets at 30°C[
º low EEO

DNA molecules remain double stranded

 a
 


OBJEC˜IVEË
º ˜o characterize the extracted DNA (maggot,
cockroach, liver[ using AGE
º ˜o describe the resulting band profile
º ˜o identify the factors affecting the quality of
the resulting band profile
 
 
º Buffer Preparation
º Gel Preparation
º Ëample Preparation and Loading
º Running the Gel
º Viewing the Gel
 á
·
 

  a  
áa

˜ris base ʹ 242 g ˜ris base ʹ 108 g ˜ris base ʹ 108 g
Glacial HOAc ʹ 57.1 mL 85% H3PO4 ʹ 15.5 mL H3BO3 ʹ 55 g
0.5 M ED˜A (pH 8[ ʹ 100 mL 0.5 M ED˜A (pH 8[ ʹ 40 mL Na2ED˜A 2H2O ʹ 9.3 g
Diluted to 1 L Diluted to 1 L Diluted to 1 mL

 a!"
#  
 
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 áa!"
#  

#  

˜ris acetate ʹ 0.04 M ˜ris-phosphate ʹ 0.08 M ˜ris-borate (pH 8.3[ ʹ 0.089

M
ED˜A ʹ 0.001 M ED˜A ʹ 0.002 M Disodium ED˜A ʹ 0.025 M
 á

º ˜ris-Acetate-ED˜A (˜AE[
º ˜ris-Borate-ED˜A (˜BE[
º ˜ris-phosphate
  

º good buffering capacity
º but phosphate will co-precipitate if DNA is
ethanol precipitated
 áa
º better resolution than ˜AE
º but it interacts with the nucleic acids resulting
to a lower recovery of DNA in gels
º more expensive than ˜AE
  a
º faster running time compared to ˜BE
º can be used for preparative work
º low buffering capacity and cannot be used for
long running times
  a
Composed of:
º ˜ris-base
º glacial acetic acid
º ED˜A
  a
º Glacial acetic acid increases the ionic strength
of the solution.
º ED˜A is used as a chelating agent for the
divalent cations that promote the
intermolecular interaction of the DNA with
denaturing enzymes like nucleases and
restriction enzymes.
º ˜AE is the buffer used in this experiment
because linear double stranded DNA tends to
run faster in it.


·
 

º weighed 0.25 g gel powder and dissolved in

12.5 mL of 1x ˜AE buffer
º agarose mixture was heated and was strirred
occasionally
º mixture was not boiled and the resulting
solution was homogenous, transparent and
fluid


·
 

º gel was allowed to cool since hot gels (˜ >

Î0°C[ could deform the gel tray (+ 150 µL EtBr[
º gel was poured carefully so as not to introduce
air bubbles in the mold
º comb was placed in the gel and in one swift
fluid motion the comb was removed after the
gel has solidified
º wells were then flushed with buffer before
loading the sample
º 1.0% agarose gel was prepared because it
coincides with the range from 0.3-2.0% of
which the nucleic acid separation is more
effective
Gel Ëtructure of Agarose
 #
·
 

$

Loading buffer preparation:
º 30% Ficoll solution + marker dyes (0.25%
bromphenol blue or 0.25% cyanole[ + 0.2 M
ED˜A (pH 8[
º dissolved in 10 volumes of buffer
 #
·
 

$

%&'    

º increases density of the solution

º preferred over glycerol and sucrose because it
minimizes tailing at the edges
 (&

 ) (&


º used as tracking dyes
a*
º disables restriction enzymes and nucleases during
the run
 #
·
 

$

º Î0 µL loading buffer + 140 µL DNA sample
º draw the ͞spot͟ up and down using a pipette
tip to mix
º load 20 µL of the mixture on the well
 ´





º Fill the gel chamber with running buffer.
º Ëet and maintain the power supply to 100V.
º ˜urn the apparatus off after 30 ʹ 35 minutes.
 V





º gel was removed from the set-up and was
placed in a transparent flat bottomed
container
º 0.5-1 drop of ethidium bromide solution was
then added over the surface of the gel
º (the addition of ethidium bromide is done in
this part if in-gel staining is not performed[
º the gel was then immediately viewed under a
UV light
 V





º bands were seen as orange under UV light
º ˜he visualization and picture taking of the gel
should be performed shortly after running the
gel because DNA will diffuse over time and the
bands will become blurry.
 aá
º a fluorescent dye that intercalates or inserts
between the bases of a double-stranded DNA
º forms an EtBr-nucleic acid complex with the
sample which exhibits a more intense
fluorescence than a free EtBr in the UV region
º absorbs at 2Î0 nm to 300 nm and has a
maximum emission at 540 nm
 In-gel Ëtaining
| 
º uses fewer glassware
º UV light can be used to monitor the run
`  
º the binding of ethidium bromide alters a
DNA͛s mass and rigidity, and therefore its
mobility
 ‰
 

º supercoiled
º linear
º nicked circles
 ‰
 

º ˜he supercoiled conformation is small, compact
and usually has the greatest mobility followed by
the rodlike, linear form of molecules.
º ˜he nicked circles or extended circular form
molecules migrate more slowly.
º ˜he relative electrophoretic mobility of the three
forms depends on experimental conditions such
as agarose concentration and ionic strength.
‰
 

º ˜he addition of ethidium bromide affects the
electrophoretic mobility of the three
conformations.
º If ethidium bromide is added to the
supercoiled form, the dye intercalates
between the stacked DNA bases causing
unwinding of some of the negative supercoils.
º As the concentration of ethidium bromide is
increased, more and more of the negative
supercoils are removed until no more are
present in the DNA.
º ˜he conformational change of the DNA
supercoil can be monitored by electrophoresis
because the mobility decreases with each
unwinding step.
º ˜he linear and nicked circles forms, under the
same conditions of increasing ethidium
bromide concentration, will also show a
gradual decrease in electrophoretic mobility.

a#

 ‰



a#

º pow
pply- introduces an applied electric
field in the gel system
º l 
o  - where the separation of charge
occurs
º l 
opho
  h
- where the melted
agarose assumes it shape when it solidifies
º pl o - where the melted agarose is
poured to form sample wells in the gel
 ‰



a#

º
 


- provides the ions in the
electrophoretic system to support conductivity
º lo 


- migrates in the gel and allows
visual monitoring or the extent of electrophoresis
º h  
o  - a fluorescent dye used for
staining nucleic acids
º 
 ll o
 - used to visualize ethidium
bromide-stained DNA in gels
u
º ˜he separation of molecules in AGE is
achieved by moving negatively charged
nucleic acid molecules through an agarose
matrix under the influence of an electric field.
º ˜he negative charge of DNA in neutral pH is
due to the presence of its negatively charged
phosphate backbone.
u
º Nucleic acids migrate in an agarose matrix at a
rate that is inversely proportional to their size.
º A linear relationship exists between mobility
and the logarithm of kilobase pairs (or
molecular weight[ of a DNA fragment.
º A standard curve may be prepared by
including on the gel a sample containing DNA
fragments of known molecular weights.
 ' 


a
º agarose concentration
º voltage
º running buffer
º ethidium bromide
 
‰


 

º Improper agarose concentration will lead to

poorly resolved bands.
º High agarose concentration is used in the
separation of smaller DNA.
º Low agarose concentration is for the
separation of larger DNA.
 V 

º As voltage is increased, larger fragments
migrate faster than small fragments.
º ˜his is because DNA becomes rigid under high
voltage condition and so, DNA travels faster.
º Low voltage means better resolution but
longer running time.
 ´

á

º provides ions to support conductivity
º absence of buffer would cause the DNA
samples not to migrate toward the anode
º the criteria in choosing the appropriate buffer
system is the length of running time and if
AGE will be used for the sample preparation
 aá
º ˜he binding of ethidium bromide to DNA will
change the charge, weight, conformation and
flexibility of the DNA molecule.
º ˜he mobility of DNA decreases as ethidium
bromide binds to it due to stiffening.
au
º reagents used
º sample preparation