INTRODUCTION TO ELECTROPHORESIS
When
a potential difference is applied between the 2 electrodes in a colloidal
solution, it has been observed that the colloidal particles are carried to
either the positive or negative electrode, solution under the influence of an
electrical field. The rate of travel of the particle depends upon the following
factors:
·
Characteristics
of the particle
·
Properties
of the electric field
·
Temperature
·
Nature
of the suspending medium
Electrophoresis
is the motion of dispersed particles relative to a fluid under the influence of
an electric field. Electrophoresis is the most known electrokinetic phenomena.
It was by Reuss in 1809. He observed that clay particles dispersed in water
migrate under influence of an applied electric field. Electrophoresis occurs
because particles dispersed in a fluid almost always carry an electric surface
charge. An electric field exerts electrostatic Coulomb force on the particles
through these charges.
Types of Electrophoresis
There
are two main types of electrophoretic methods, depending upon whether the
separation is carried out in the absence or presence of a supporting or
stabilizing medium. When the separation is carried out in the absence of the
stabilizing medium, the method is called free solution method, and when it is
carried out in the presence of a stabilizing medium, such as paper the
technique is known as electro chromatography or zone electrophoresis.
Free solution method
This
method was first proposed by Picton and Lindex (1892), but was not fully
developed until 1937. Tiselins described the apparatus and methodology for
which he was awarded Nobel Prize. In free solution electrophoresis, the sample
solution is introduced at the bottom of a U tube that has been filled with
unstabilized buffer solution. The samples are usually injected into the bottom
of the U tube through a capillary tube side arm. An electrical field is applied
by means of electrodes located at the ends of the tube. The differential
movement of the charged particles towards one or the other electrode is then
observed. Separation takes place as a result of differences in mobilities.The
mobility of a particle is approximately proportional to its charge to mass
ratios. The free solution method was perfected by Tiselins. He applied this
method for the separation of proteins.
Zone electrophoresis or Electro chromatography
Many
of the experimental difficulties in free solution electrophoresis are avoided,
if the separations are carried out in a stabilizing medium, such as paper. Such
separations are made possible by using a supporting medium to keep convection
currents from distorting the electrophoretic pattern. The separations depend
mainly upon the properties of medium and may result primarily from the
electrophoretic effect or from a combination of electrophoresis, and
adsorption, ion exchange or other distribution equilibria.
Paper Electrophoresis
1. The apparatus
should be set up on level surface and the electrode chambers must be filled
with buffer.
2. Provision is
made for adjusting the electrolyte (buffer) in the electrode chambers to equal levels
so that siphoning action does not occur through the bed, because siphoning
action across the bed will displace and distort the electrophoretic pattern.
Types of Supporting or stabilizing medium
The
solid supporting media in electro chromatography are as numerous and varied as
found in the other chromatographic methods.
Examples of
solid supporting medium are as follows.
Filter paper,
cellulose acetate strips, starch powder, cellulose powder, starch gel, agar
gel, synthetic gel ion exchange resins and membranes, asbestos paper, rayon
acetate cloth, glass fibre paper, silica powder, Kieselguhr, glass powder,
silica gel, agarose gel etc.
Gel electrophoresis
Gel
electrophoresis is an application of electrophoresis in molecular biology.
Biological macromolecules
– usually proteins, DNA, or RNA – are loaded on a gel and separated on the
basis of their electrophoretic mobility. (The gel greatly retards the mobility
of all molecules present.)
Electrophoretic fingerprinting
Electrophoresis
is also used in the process of DNA fingerprinting. Certain DNA segments that
vary vastly among humans are cut at recognition sites by restriction enzymes (
restriction endonuclease). After the resulting DNA fragments are run through
electrophoresis, the distance between bands are measured and recorded as the
DNA “fingerprint.”
Electrophoretic deposition
Coatings,
such as paint or ceramics, can be applied by electrophoretic deposition. The
technique can even be used for 3-D printing
ELECTROPHORESIS
Definition
The movement of charged particles in
an electric field resulting in their migration towards the oppositely charged
electrode is known as electrophoresis.
Molecules with a net positive charge (cations)
move towards the negative cathode while those with net negative charge (anions)
migrate towards positive anode.
Electrophoresis
is a widely used analytical technique for the separation of biological
molecules such as separation of biological molecules such as plasma proteins
lipoproteins and immunoglobulins.
The usual
purpose for carrying out electrophoretic experiments are
§ to determine the
number, amount, and mobility of components in a given sample or to separate
them,
§ to obtain
information about the electrical double layers surroundings the particles,
§ determination of
molecular weight of biomolecules, and DNA sequencing on the other.
In 1937,
Tiselius describing his moving boundary apparatus was instrumental in
popularizing the utility of electrophoresis to the biochemist. Upon suspension
in an aqueous solvent, almost all particles eg: (RBC, bacteria, etc) & many
important biomolecular (eg: nucleic acids, amino acids, proteins, etc) acquire
either positive or negative charges. The acquisition of such charges depends
upon the nature of the particle/molecules & the nature of the solvent.
Thus if the
acidity of the solvent is increased (H+ ions) the molecule will tend to become
more positive & vice versa.
Principle
Any substance suspended in water
dissociates into charged particles. When these charged particles are subjected
to an electric field, all positively charged ions would move towards the
cathode or negative electrode and negatively charged ions towards the anode or
positive electrode as shown. This is the basic principle involved in
electrophoresis.
The Sample
Charge/mass ratio of the sample
dictates its electrophoretic mobility. The mass consists of not only the size
but also the shape of the molecule.
(i)
Charge-
the higher the charge, greater is the electrophoretic mobility. The charge,
however, is dependent on pH of the medium.
(ii)
Size-
the bigger the molecule, greater is the frictional and electrostatic forces
exerted upon it by the medium of suspension. Consequently, larger particles
have a smaller electrophoretic mobility compared to the smaller particles.
(iii)
Shape-
rounded contours elicit lesser frictional and electrostatic retardation
compared to sharp contours. As an example consider the case of globular and
fibrous proteins. Given the same size the globular protein will migrate faster
than the fibrous protein.
The Electric Field
The rate of migration under unit
potential gradient is referred to as mobility of the ion. An increase in
potential gradient increases the rate of migration. The current in the solution
placed between two electrodes is carried mainly by the buffer ions, only a
small proportion being carried by the sample ions. An increase in the potential
difference therefore increases the current.
On the other
hand, resistance plays an important role in the separation of particles. For instance,
the electric current increases when the resistance is decreased and so the
separation is faster. Moreover, the buffer ions carrying more charge than the
ions of the sample would result in slower separation. Therefore, a constant
current is to be maintained by using power packs during electrophoresis.
The Medium
An inert supporting medium is chosen
for electrophoresis. But even this inert medium can exert adsorption and/or
molecular sieving effects on the particle thereby influencing its rate of
migration. The medium may also give rise to electro-osmosis, which may also
influence the rate of sample migration.
The Buffer
Commonly used buffers are formate,
citrate, phosphate, EDTA, acetate, pyridine, Tris, and barbitone, etc. the
choice of buffer depends upon the type of sample being electrophoresced. The
buffer can affect electrophoretic mobility if it is able to bind to components
of the sample being separated.
pH
Since pH determines the degree of
ionization of organic compounds, it can also affect the rate of migration of
these compounds. Increase in pH increases ionization of organic acids and a
decrease in pH increases the ionization of organic bases.
Ionic Strength
Substances with less ionic strength
exhibit a faster separation whereas those with increased ionic strength show a
slower separation.
Electrodes
In electrophoretic studies, platinum,
carbon or Ag/AgCl electrode are used. Of these, platinum electrodes are mostly
preferred. Though, the use of carbon electrode is inexpensive, they are easily
polarized and require frequent replacement. The silver electrodes are to be
coated periodically. At each electrophoretic run, the polarity of the
electrodes is to be reversed in order to prolong the life of electrodes and
buffer solution.
Supporting Media In Electrophoresis
Various type of supporting media is
used in electrophoretic separation of substances and is as follows.
Paper
Paper containing nearly 95% of
cellulose with very low adsorption capacity can be used as a stabilizing medium
in electrophoresis.
Gels
Gels are porous in nature and so the
size of the pores in relation to size of the molecule determines the mobility
of substances. As a result, the separation depends on the charge and size of
the molecules when the gels are used as the supporting medium. For
electrophoretic separation of components, the following types of gels are used.
Starch
Though the resolving power of starch
gel is very high its pore size cannot be controlled.
Agar
Agar is soluble in aqueous buffer solutions
and it forms a gel having a large pore size but without molecular sieving.
Hence it is used to separate proteins and nucleic acids.
Polyacrylamide
Polyacrylamide gel is prepared from
components namely N,N’-methylene bisacrylamide (bis), ammonium persulpahte and
tetramethylene diamine (TEMED). It has low adsorption capacity but has no power
of electrosmosis. Using differential concentration of the reagents can control
the pore size of this gel.
Agarose-acrylamide
It is a mixed form of gel obtained by
mixing acrylamide with agarose. Here acrylamide provides sieve action while the
agarose gives physical support to the gel. Therefore this is useful to separate
compounds of very high molecular weight.
Other gels
In addition to the above types of
gels, substance like pectin, sephadex, gypsum, polyvinyl chloride, polyvinyl
acetate, etc., are also used in electrophoresis.
AGAROSE GEL ELECTROPHORESIS
Introduction
Gel
electrophoresis is routinely used analytical technique for the separation/purification
of specific DNA fragments.The gel is composed of either polyacrylamide or
agarose. Polyacrylamide gel electrophoresis (PAGE) is used for the separation
of smaller DNA fragments while agarose electrophoresis is convenient for the
separation of DNA fragments ranging in size from 100 base pairs to 20 kb pairs.
Gel electrophoresis can also be used for the separation of RNA
molecules.Electrophoresis through agarose gels is the standard method for the
separation, identification, and purification of DNA and RNA fragments ranging
in size from a few hundred to 20 kb.The technique of agarose gel
electrophoresis is simple, rapid to perform, and capable of resolving DNA
fragments that cannot be separated adequately by other procedures.The location
of DNA within the gel can be determine directly by staining with low
concentrations of intercalating fluorescent ethidium bromide dye under
ultraviolet light. If necessary, these bands of DNA can be recovered from the
gel and used for a variety of cloning purposes.
Principle
Agarose
is a polysaccharide derived from seaweeds. It forms a solid gel when dissolved
in aqueous solution at concentrations between 0.5 and 2.0% (w/v). It may be
noted here that the agarose used for electrophoresis is more purified form of
agar when compared to that used for culture purpose.Agarose (average relative
molecular mass about 12000) is made up of the basic repeat unit agarobiose,
which comparises alternating units of galactose and 3,6-anhydrogalactose.
The rate of
migration of DNA depends on a number of parameters
(1) molecular
sizes of DNA
(2) agarose
concentration
(3) conformation
of DNA and
(4) composition
of electrophoresis buffer.
Larger
molecules migrate more slowly than the smaller molecules do because they have
to find their way through the pores of the gel.Hence, gel electrophoresis can
be conveniently used for the separation of a mixture of DNA fragments, based on
their size.By using gels of different concentrations it is possible to resolve
a wide range of DNA molecules. The electrophoretic mobility of DNA is also
affected by the composition and ionic strength of electrophoresis buffer. In
the absence of ions, electrical conductivity is minimal and DNA migrates very
slowly. In buffers of high ionic strength, electrical conductance is very
efficient.Agarose forms gels with pore size ranging from 100 to 300 nm in
diameter. The actual pore size depends on the concentration of the agarose. The
size of the pores determines the range of DNA fragments that can be separated
on electrophoresis. For instance, a 0.3% agarose is used for the separation of
DNA fragments between 5 and 50 kb, while a 5% agarose can separate 100-500 bp
molecules. Several different buffers are available, viz TAE (Tris- acetae), TBE
(Tris-borate), TPE (Tris-phosphate) and alkaline buffer, at a pH range of 7.5-7.8. Electrophoresis is normally carried
out at room temperature.
Procedure
v
Agarose
gels are cast by melting the agarose in the presence of desired buffer until a
clear transparent solution is obtained. The melted solution is poured into a
rig provided in the apparatus.
v
The
gel is allowed to harden.
v
On
hardening, the agarose forms a matrix, the density of which is determined by
the concentration of agarose.
v
The
gelling properties are attributed to both inter and intramolecular hydrogen
bonding within and between the long agarose chains.
v
The
whole rig is transferred to a rectangular container which has electrodes fitted
to it at the two ends.
v
The
required buffer is then poured over the gel till the buffer level is
sufficiently high to dip the electrodes. While setting the gel a comb shaped
jig is embedded in the still hot gel upon cooling when the comb is taken out
sample wells get etched out into the gel. The sample is loaded while the gel is
submerged under the buffer.
v
Electrophoresis
can be started by connecting the electrodes to the power pack and switching on
the current. The DNA samples are placed in the wells of the gel surface and the
power supply is switched on
v
When
an electric field is applied across the gel, DNA which negatively charged at
neutral pH, migrates towards the anode
v
The
migration of DNA fragments during the course of electrophoresis can be
monitored by using dyes with known migration rates. These dyes are added to the
DNA samples before loading.
v
Gels
are stained with intercalating fluorescent ethidium bromide dye and as little
as 0.05μg of DNA in one band can be detected as visible fluorescence when
activate by ultraviolet light.
v
DNA
base pairs in association with ethidium bromide emit orange fluorescence. And
in this way the DNA fragments separated in agarose electrophoresis can be
identified.
Note: ethidium bromide is a powerful mutagen
and toxic and therefore proper care should always be taken while handling the
solution and the staining solution should always be decontaminated after its
use.
TWO DIMENSIONAL GEL ELECTROPHORESIS (2-D GEL
ELECTROPHORESIS)
It
is a powerful tool & is designed by combining the resolving power of
isoelectric focusing with SDS-PAGE to obtain very high-resolution separations
by a procedure known as two-dimensional gel electrophoresis.
As
the molecular weight & isoelectric point of a macromolecule are not related
with each other, this technique makes use of these two properties to separate
the molecules with great resolution power. By this method, a mixture of large
number of proteins can be resolved into individual fractions.
In
this technique, the protein sample is first subjected to isoelectric focusing
in a narrow strip of gel containing polyampholytes.
Isoelectric Focusing
In isoelectric focusing (IEF),
proteins are separated by electrophoresis in a pH gradient in a gel. They
separate on the basis of their relative content of positively and negatively
charged groups. Each protein migrates through the gel until it reaches the
point where it has no net charge this is its isoelectric point (pl): here, the
protein’s net charge is zero and hence it does not move in an electric field.
In IEF, a polyacrylamide gel is used that has large pores so as not to impede
protein migration and contains a mixture of polyampholytes. If an electric
field is applied to the gel, the polyampholytes migrates and produce a pH
gradient. To separate proteins by IEF, they are electrophoresed through the gel
until it reaches a position at which the pH is equal to its pl. if a protein
diffuses away from this position, its net charge will change as it moves into a
region of different pH and the resulting electrophoretic forces will move it
back to its isoelectric position. In this way each protein is focused into a
narrow band (as thin 0.01 pH unit) about its pl.
This gel strip is then placed on top
of an SDS polyacrylamide gel and electrophoresed to produce a two-dimensional
(2D) pattern of spots in which the proteins have been separated in the
horizontal direction on the basis of their pl, and in the vertical direction on
the basis of their mass. The overall result is that proteins are separated on
the basis of their size and charge. Thus two proteins that have very similar or
identical pls and produce a single band by isoelectric focusing will produce
two spots by 2D gel electrophoresis. Similarly, proteins with similar or
identical molecular masses, which would produce a single band by SDS-PAGE, also
produce two spots because of the initial separation by isoelectric focusing.
This 2D gel electrophoresis has enormous use in proteomics study.
Staining Of Proteins
The most commonly used protein stain
is the dye Coomassie brilliant blue. After electrophoresis, the gel containing
separated proteins is immersed in an acidic alcoholic solution of the dye. This
denatures the proteins, fixes them in the gel so that they do not wash out, and
allows the dye to bind to them. After washing away the excess dye, the protein
bands are visible as discreet blue bands. A more sensitive stain is soaking the
gel in a silver salt solution.
SDS-PAGE (SODIUM DODECYL SULPHATE- POLYACRYLAMIDE
GEL ELECTROPHORESIS)
Introduction
In
native polyacryamide gel electrophoresis (PAGE), proteins are applied to a porous
polyacryamide gel & separated in an electric field. When proteins are
placed in an electric field, molecules with a net charge such as proteins, will
move toward one electrode or the other, a phenomenon known as electrophoresis. SDS-PAGE is the
most widely used for qualitatively analyzing any protein mixture, monitoring
protein purity and to determine their molecular weights. It is based on the
separation of proteins according to their size and then locating them by
binding to a dye. SDS can be used to dissociate proteins into their individual
polypeptide chains, and thus the charge on the SDS-protein complex is almost
entirely due to the exposed sulfate ions.
Principle
SDS
or sodium dodecyl sulphate is an anionic detergent that binds strongly to proteins,
causing their denaturation. In the presence of excess SDS, about 1.4 g of the
detergent binds to each gram of protein, giving the protein a constant negative
charge per unit mass. As a result, protein-SDS complex move towards the anode
during electrophoresis and owing to molecular sieving properties of the
polyacrylamide gel, get separated based on their molecular weights. Since, the
principle of this technique is separation of proteins based on size
differences, by running standard proteins of known molecular weights on the
same gel as unknown protein, molecular weight of the unknown protein can be
determined.
In SDS-PAGE, SDS incorporated to the
gel is used to separate individual polypeptide chains from oligometric
proteins. The gels which have the property of molecular sieving exhibit a
linear relationship between the electrophoretic mobility of protein,
incorporated to SDS & the molecular weight of proteins. Therefore molecular
weight of proteins can also be determined by this method.
This method is used for the study of
the subunits of oligomeric proteins. To separate the subunits of these
proteins, the solubilizing agents called solubilizers denature their structure.
Urea, sodium dodecyl sulphate and β-mercaptoethanol are mostly used as solubilizers.
In concentrated urea, the hydrogen bonds readily dissociate. Sodium dodecyl
sulphate (SDS), an anionic detergent, disrupts hydrophobic interactions and
provides negative charge to the denatured polypeptide. This disulphide bonds
are broken by mercaptoethanol.
In SDS-PAGE, the protein sample is
treated with a reducing agent such as 2-mercaptoethanol or dithiothreitol to
break all disulfide bonds. It is then denatured with SDS, a strong anionic
detergent, which disrupts nearly all the noncovalent interactions & covers them with an
overall negative charge. Approximately one molecule of SDS binds via its
hydrophobic alkyl chain to the polypeptide backbone for every two aminoacid
residues, which gives the denatured protein a large net negative charge that is
proportional to its mass.
The protein mixture is then mixed with
the bromophenol blue dye & applied in to the sample wells, &
electrophoresis is performed. As all the proteins now have an identical charge
to mass ratio, they are separated on the basis of their mass. The smallest
proteins move farthest. Thus, if proteins of known molecular mass are
electrophoresed alongside the samples, the mass of unknown proteins can be
determined. SDS-PAGE is a rapid, sensitive, & widely used technique from which
one can determine the degree of purity of a protein sample, the molecular mass
of unknown sample, & the number of polypeptide subunits with a protein. The
molecules separate in an electric field on the basis of their net charge &
size of the protein, since the electrophoresis separation is carried out in a
gel, which serves as a molecular sieve. Small molecules move faster through the
pores as compared to large molecules.
The gels are made of polyacrylamide,
which is chemically inert. It is readily formed by the polymerization of
acrylamide. Choosing an appropriate concentration of acrylamide and the cross
linking agent, methylene bisacrylamide, can control the pore sizes in the gel.
The higher the concentration of acrylamide, the smaller is the pore size of the
gel. The gel is usually cast between the two glass plates of 7-20 cm2 separated
by a distance of 0.5-1.0 mm. the protein sample is added to the wells in the
top of the gel, which are formed by placing a plastic or Teflon comb in the gel
solution before it sets.
A
bromophenol blue dye is mixed with the protein sample to aid its loading on to
the gel. Because the bromophenol blue dye is a small molecule, it also migrates
quickly through the gel during electrophoresis, thus indicating the progress of
the electrophoresis. In the PAGE, buffer is same in upper and lower reservoirs
and in the gel with a pH of approximately 9, such that most proteins have net
negative charges and migrate towards the anode in the lower reservoir. An
electric current is applied across the gel from top to bottom for a period to
move the protein through the gel. When blue indicator of bromophenol dye
reaches at the bottom of the gel, electric current is switched off and the gel
is removed from the electrophoresis apparatus.
Procedure
§ Assemble the
plates for casting gel as shown below.
§ Clamp the
assembly of plates. Ensure the assembly is leak proof by filling water between
the plates. Silicon grease can be applied to spacers or 1% agarose can be used
for sealing to make it leak proof.
§ Add 50 μl of
ammonium persulpahte to 5 ml of separating gel mix and mix thoroughly.
§ Pour the gel
solution between the plates till the level is about 2cm below the top edge of
notched plate.
§ Add 200 to 250
μl of water to make the surface even.
§ After the gel is
set, wash the top of the separating gel with distilled water and drain off the
water completely.
§ Add 20 μl of APS
solution to 2 ml of stacking gel mix, mix thoroughly and pour directly onto the
polymerized separating gel.
§ Insert the comb
into the gel solution carefully without trapping any air bubbles, approximately
1cm above the separating gel. The stacking gel will set in about 10 minutes.
§ Pipette out 25
μl of protein samples (A,B and C) and 10 μl of protein maker into individual
vials. Label them appropriately. To each of these vials add 15 μl of sample
loading buffer.
§ Place the vials
in a boiling water bath for 5 minutes.
§ After the
stacking gel has set, carefully remove the comb and the bottom spacer. Wash the
wells immediately with distilled water to remove non-polymerized acrylamide.
§ Fill the bottom
reservoir with 1X reservoir buffer.
§ Carefully fix
the plate to the PAGE apparatus without trapping any air bubbles between the
buffer and the bottom of the gel, with the notched plate facing the top
reservoir.
§ Fill the top
reservoir with 1X reservoir buffer.
§ Load samples
into the wells; rinse the micropipette tip in the bottom reservoir buffer
between each load. Note down the order in which the samples have been loaded.
§ Connect the
cords to the power supply, according to the convention.
§ Set voltage at
100 V and switch on the power supply.
§ When the dye
front reaches 0.5cm above the bottom of the gel, turn off the power.
§ Remove the gel
plates and gently pry the plates apart. Use a spatula or similar tool to
separate the plates. (not at the notch).
§ Transfer the gel
to a tray containing water, wash the gel for 5 minutes. Discard the water.
§ Add 20ml of Ezee
blue & stain the gel for 30-60 minutes. Continue staining the gel overnight
if the bands appear light.
§ Destain the gel
with water, if the background is not clear.
§ NOTE: for
uniform staining & washing, place the tray on a rocker or shake
intermittently every 10 to 15 minutes.
§ Place the gel
between two sheets of plastic wrap & place it on a piece of white paper.
§ Use a ruler to
measure the distance migrated by each band of the protein marker from the start
of separating gel.
§ Similarly,
measure the migration distance of protein sample C & the tracking dye from
the start of separating gel.
§ Calculate the
relative mobilities of the proteins ( marker & sample C) as follow:
Distance
migrated by protein
Distance migrated by solvent
BLOTTING TECHNIQUES
Blotting is the technique in which nucleic acids or proteins are
immobilized onto a solid support generally nylon or nitrocellulose membranes.
Blotting of nucleic acid is the central technique for hybridization studies.
Nucleic acid labelling and hybridization on membranes have formed the basis for
a range of experimental techniques involving understanding of gene expression,
organization, etc.
|
Applications
1. Southern blotting technique is widely used to find specific nucleic acid sequence present in different animals including man. For example if we want to know whether there is a gene like insulin in sea anemone, then DNA of sea anemone is mobilized on membrane and blotted by using insulin probes against it. 2. Northern blotting technique is widely used to find gene expression and regulation of specific genes. For example if we find human insulin like gene in oyster, then by isolating and immobilizing RNA and blotting it with insulin probe we call tell whether the gene is expressing or not. 3. By using blotting technique we can identify infectious agents present in the sample. 4. We can identify inherited disease. 5. It can be applied to mapping restriction sites in single copy gene. |
SOUTHERN BLOTTING TECHNIQUE
A method, developed by a molecular
biologist E.M.Southern (1975) for analysing the related genes in a DNA
restriction fragment is called as Southern blotting technique. Southern blots
can easily provide a physical map of restriction sites within a gene located
normally on a chromosome, and reveal the number of copies of the gene in the
genome, and the degree of similarity of the gene when compared with the other
complementary genes.
§ The procedure
starts with digestion of DNA population
by one or many restriction enzymes. Consequently, DNA fragments unequal length
are produced.
§ This preparation
is passed through agarose gel electrophoresis which results in separation of
DNA molecules based on their size.
§ DNA restriction
fragments present in gel are denatured by alkali treatment. Gel is then put on
the top of the buffer satured filter paper.
§ Upper surface of
the gel is covered with nitrocellulose filter and overlaid with dry filter
paper. The dry filter paper draws the buffer through the gel. Buffer contains
single stranded DNA. Nitrocellulose filter binds DNA fragments strongly when
come in contact of it.
§ After baking at
80°C, DNA fragments are permanently fixed to the nitrocellulose filter. Then
the filter is placed in a solution containing radio-labelled RNA or denatured
DNA probe of known sequences. These are complementary in sequence to the
blot-transferred DNA.
§ The
radiolabelled nucleic acid probe hybridizes the complementary DNA on
nitrocellulose filter. The filter is thoroughly washed to remove the probe.
§ The hybridized
regions are detected autoradiographically by placing the nitrocellulose filter
in contact with a photographic flim.
§ The images show
the hybridized DNA molecules. Thus the sequences of DNA are recognized
following the sequences of nucleic acid probe.
NORTHERN BLOTTING TECHNIQUE
Southern
blotting technique could not be applied directly to the blot transfer of mRNA
separated by gel electrophoresis, because RNA was found not be bind with
nitrocellulose filter. Alwine et al
(1979) devised a technique in which RNA band are transferred from the gel onto
chemically reactive paper. An aminobenzyloxymethyl cellulose paper, prepared
from Whatman filter paper No.540 after a series of uncomplicated reactions, is
diazotised and rendered into the reactive paper and, therefore, becomes available
for hybridiztion with radiolabelled DNA probes. The hybridized bands are found
out by radiography. Thus, Alwine’s method extends that of Southern’s method and
this reason it has been given the jargon term ‘Northern blotting’. There is
nothing northern or western like Southern.
These
blot transfers are reusable because of the firm covalent bonding of RNA to the
reactive paper. The chemically reactive paper is equally effective in binding
the denatured DNA as well.Small fragments of DNA can more effectively be
transferred to the diazotised paper derivative than to nitrocellulose. These
techniques were more being advanced and more recently have been demonstrated
that mRNA bands can also be blotted directly on nitrocellulose paper under
appropriate condition (Thomas, 1980).
§ In this method
preparation of reactive paper is not required. The mRNA is isolated from the
transformed cells and electrophoresed under such conditions that do not permit
the development of secondary structures.
§ The mRNA
separated on the gel are transferred onto nitrocellulose filter which are then
hybridized by single stranded probe (RNA or DNA).
§ Thereafter,
hybrids are treated with SI nuclease and Rnase which digests the single
stranded RNA/DNA probe.
§ It does not
affect the double stranded nucleic acid formed due to hybridization of RNA by
the complementary sequences of nucleic acid probe. Structure of mRNA is
revealed to the extent to which mRNA protects the nucleic acid probe.
WESTERN BLOTTING TECHNIQUE
Towbin
et al (1979) developed the western
blotting technique to find out the newly encoded protein by a transformed cell.
Its working principle lies on antigen antibodies reaction; hence, it is an
immuno detection technique. In this method radiolabelled nucleic acid probes
are not used.
This technique
follows the following steps:
§ Extraction of
protein from transformed cells.
§ Separation of
protein by using SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel
electrophoresis) where SDS acts as solvent for electrophoresis.
§ Transfer of
electrophoresed gel in a buffer at low temperature(40°C) for half an hour.
§ Blotting of
proteins onto nitrocellulose filter paper.
§ Soaking of
nitrocellulose filter, Whatman filter and coarse filter in transfer buffer.
§ Placing of
whatman filter paper on a cathode plate followed by stack of coarse filter,
whatman filter, electrophoresed gel, nitrocellulose filter, Whatman filter
paper, coarse filter stack, whatman
filter and anode plate.
§ Putting the
complete setup in transfer tank containing sufficient transfer buffer.
§ Application of
an electric field (30 V overnight for 5 hours) to cause the migration of
proteins from the gel to nitrocellulose filter and binding on its surface. The
nitrocellulose filter has exact image of pattern of proteins as present in the
gel. This type of blotting is called western blotting.
§ Hybridization of
proteins by using radiolabelled antibodies of known structure.
§ Washing of
nitrocellulose filter with a wash solution (Tris-buffered saline + Tween 20) to
facilitate the removal of unhybridised antibodies.
§ Detection of
hybridized sequences by autoradiography. The dots of diagram shows the presence
of desired protein.
DOT BLOT TECHNIQUE
In
molecular biology southern and northern blotting techniques are most often
utilised. The whole process in time consuming and requires careful purification of samples containing
nucleic acids. It is expensive also but it may be simple if only detection and
quantificationof any sequence is to be done.
§ The procedure is
made simple by excluding purification steps, electrophoresis and blotting of
gel. Therefore, the desired purified sample of nucleic acid is dotted with a
pipette onto the surface of nitrocellulose filter paper.
§ Dotting may also
done by using an apparatus making as circular or oblong slot. Since slot or
dot on the filter paper is done,
therefore this method is also called ‘dot or slot blot’.
§ The filter paper
is put in 0.4M NaOH solution to ensure the binding of denatured DNA on
nitrocellulose filter. This technique rapidly detects the nucleic acid
sequences and determine the relative amount of RNA or RNA of the sample.
§ A densitometer
is used which scans the autoradiographic signals and quantifies the intensity
of a hybridised nucleic acid in the sample. By using this method one can
compare the amount of DNA or RNA sequence present in a large number of samples.
COLONY BLOTTING TECHNIQUES
Hybridization
probing can be used to identify recombinant DNA molecules contained in either
in bacterial colonies or bacteriophage plaques.
§ First the
colonies or plaques are transferred to nitrocellulose or nylon membrane, and
then treated to remove all contaminating material, leaving just DNA.
§ Usually this
treatment also results in denaturation of the DNA molecules, so that the
hydrogen bonds between individual strands in the double helix are broken.
§ These single
stranded molecules can then be bound tightly to the membrane by a short heat
treatment or by ultra violet irradiation.
§ The molecules
become attached to athe membrane through the sugar phosphate backbones, so the
bases are free to pair with complementary nuclic acid molecules.
§ The probe must
now be labelled, denatured by heating and applied to the membrane in a solution
of chemicals that promote nucleic acid hybridization.
§ After a period
to allow hybridization to take place, the membrane is washed to remove unbound
probe molecules and dried, and the positions of the bound probes are detected.
§ Traditionally
the probe is labelled with radioactive phosphorous, 32P, and the
position of the hybridization signals are visualized by autoradiography, in
which a sheet of X-ray sensitive flim is placedover the membrane.
§ The problem with
radioactive labelling is that it presents a health hazard to the person doing
the experiment, so a number of methods for labelling with a non radioactive
markers have now been developed.
§ In one methd the
probe DNA is complexed with the enzymehorseradish peroxidase, which can be
detected throughits ability to degrade a special substrate (luminol) with the
emission of chemiluminescence. The signal can be recorded on normal
photographic flim in a manner similar to autoradiography.
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