Materials

 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 agarose is poured to form sample wells in the gel.
 Electrophoresis buffer, usually Tris-acetate-EDTA 9TAE) or Tris-borate-EDTA (TBE).
 Loading buffer, which contains something dense such as 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 of how far the electrophoresis has proceeded.
 Ethidium bromide, a fluorescent dye used for staining nucleic acids.
 Transilluminator (an ultraviolet lightbox), which is used to visualize ethidium bromide-
stained DNA in gels.

Gel Electrophoresis nice procedure

 In the early days of DNA manipulation, DNA fragments were laboriously separated by
gravity. In the 1970s, the powerful tool of DNA gel electrophoresis was developed. This
process uses electricity to separate DNA fragments by size as they migrate through a gel
mix.
 Gel electrophoresis can be used to separate DNA fragments. Electrophoresis used an
electric current to separate different sized molecules in a porous, sponge-like matrix.
Smaller molecules move more easily through the gel pores than larger molecules.
 While at Cold Spring Harbor Laboratory, Phil Sharp, Joe Sambrook, and Bill Sigden
developed the DNA electrophoresis technique using an agarose gel, made from a highly
purified seaweed. This could be used to separate DNA molecules ranging from several
hundred nucleotides in length to over 10,000 nucleotides.
 The gel is submersed in a tank filled with a salt solution that conducts electricity.
 Using a pipette, DNA samples are loaded into slots made in the agarose gel. The DNA
samples are colorless, but researchers add a blue “tracking” dye. This makes it easier to
load the samples, and visually track the DNA migration through the gel.
 The phosphate groups in the DNA backbone carry negatively-charged oxygens, giving
the DNA molecule an overall negative charge. In an electric current, the negatively
charged DNA moves toward the positive pole of the electrophoresis chamber. The DNA
molecules move through the gel by “reputation”, a reptile like snaking action through the
pores of the agarose matrix. Smaller DNA fragments migrate faster and further over a
given period of time than do larger fragments. This is how DNA fragments can be
separated by size in an agarose gel.

Gel Electrophoresis II general procedure and some uses

 Most of the procedures used in recombinant DNA technology rely on a researcher’s
ability to purify a DNA fragment of interest. In an important procedure called agarose gel
electrophoresis, DNA fragments are separated by size as they move through a gel matrix.
 Gel electrophoresis is one of the most useful means of separating and purifying DNA
fragments for further analysis. In this technique, a jello-like slab of material called
agarose is molded with wells, placed in a buffer solution and hooked up to positive and
 negative electrodes. The DNA solutions, to which blue dye is added, are then pipette into
the wells. A well is also reserved for the placement of DNA of known sizes, and then the
power supply is turned on.
 The blue tracking dye is negatively charged and thereby migrates toward the positive
electrode, as does the DNA. The DNA backbones contain negatively charged phosphate
groups which are attached to the positive electrode. The smallest fragments move the
fastest, being entangled less in the agarose mixture of the gel.
 When the blue dye reaches the bottom of the gel, the power is turned off. A fluorescent
dye is then used to stain the DNA fragments. Each band of DNA may contain millions of
fragments of the same size.
 In many cases, a researcher may want to determine which DNA fragment contains a
DNA sequence of interest. To do this, the researcher prepares the DNA in the gel to make
a copy, known as a blot. First, the gel is soaked in a basic solution so that the double
stranded DNA denatures into single strands.
 The gel is then transferred to a salt solution and a nylon filter is placed on top of the gel.
Absorbent towels are placed on top of the filter. The salt solution draws the DNA through
the gel toward the nylon filter, where the DNA adheres.
 The filter is treated so that the DNA adheres to it permanently, and then the filter is
placed in a solution with a radioactive probe. The probe consists of a single-stranded
DNA that is complementary and will hybridize to the band of interest.
 The filter is then washed to remove any unhybridized probe. A piece of x-ray film is
placed over the filter. The radioactive probe exposes the film, revealing the locations of
hybridization.
 Knowing which bands contain the sequence of interest, an identical agarose gel can be
created, and a band of interest can be cut out of the gel. The DNA in this band can then
be manipulated for further analysis.

Gel Electrophoresis III

 A molecule with a net charge will move in an electric field. This phenomenon,
termed electrophoresis, offers a powerful means of separating proteins and other
macromolecules, such as DNA and RNA. The velocity of migration (v) of a
protein (or any molecule) in an electric field depends on the electric field strength
(E), the net charge of the protein (z), and the frictional coefficient (f).
 The electric force Ez driving the charged molecule toward the oppositely charged
electrode is opposed by the viscous drag fv arising from friction between the
moving molecule and the medium. The frictional coefficient f depends on both
the mass and the shape of the migrating molecule and the viscosity (n) of the
medium.

Gel Electrophoresis IV
 DNA and RNA molecules are highly charged because the phosphate group in
each nucleotide contributes on negative charge. As a result, DNA and RNA
molecules move towards the positive electrode during gel electrophoresis.
Smaller molecules move through the gel matrix more readily than larger
molecules, so that molecules of different length, such as restriction fragments
separate. Because the gel matrix restricts random diffusion of the molecules,
 molecules of different lengths separate into bands whose width equals that of
the well into which the original DNA mixture was placed.
 Two methods are common for visualizing separated DNA bands on a gel.
If the DNA is not radiolabeled, the gel is incubated in a solution
containing the fluorescent dye ethidium. Ethidium is a planar molecule that
binds to dna by ((intercalating)) between the base pairs. Binding concentrates
ethidium in th DNA and also increases its general fluorescence. As a result,
when the gel is illuminated with ultraviolet light, the regions of the gel
containing DNA fluoresce much more brightly than the regions of the gel
without DNA.
o Radioactively labeled DNA can be visualized by autoradiography of
the gel. In this case, the gel is laid against a sheet of photographic film
in the dark, exposing the film at the positions where the labeled DNA
is present. When the film is developed, a photographic image of the
DNA is observed. Radiolabeled DNA bands can also be observed by
laying the gel against a ((phosphorimager)) screen, which counts beta
particles released by labeled molecules in the gel. The resulting data is
stored by a computer and can be converted into an image of the gel.

Restriction Enzymes
 The discovery of enzymes that could cut and paste DNA made genetic engineering
possible. Restriction enzymes, found naturally in bacteria, can be used to cut DNA
fragments at specific sequences, while another enzyme, DNA ligase, can attach or
rejoin DNA fragments with complementary ends.
 Restriction enzymes are sequence specific. They recognize and bind to specific DNA
sequences. EcoRI for example, binds to the sequence GAATTC.
 Once they bind to their recognition sequence, restriction enzymes cut the sugar-
phosphate backbones of the DNA strands.
 Enzymes such as HaeIII and AluI cut straight across the double heix, producing blunt
ends. EcoRI, however, as many other enzymes, cuts unevenly, in an offset fashion,
leaving overhangs of single stranded DNA known as “sticky ends.”
 The sticky ends can be reattached by another enzyme called ligase. It catalyzes the
chemical reaction that rejoins the DNA sugar-phosphate bonds. The result is a
molecule known as recombinant DNA.

BTW
 Electrophoresis separations are nearly always carried out in gels (or on solid
supports such as paper) because the gel serves as a molecular sieve that enhances
separation. Molecules that are small compared with the pores in the gel readily
move through the gel, whereas molecules much larger than the pores are almost
immobile. Intermediate-size molecules move through the gel with various degrees
of facility. Electrophoresis is performed in a thin vertical slab of polyacrylamide.
The direction flow is from top to bottom. Polyacrylamide gels, formed by the
polymerization of acrylamide and cross linked by methylenebisacrylamide, are
choice supporting media for electrophoresis because they are chemically inert and
 are readily formed. Electrophoresis is the opposite of gel filtration in that all of
the molecules, regardless of size, are forced to move through the same matrix.
The gel behaves as one bead of a gel-filtration column.
 Proteins can be separated largely on the basis of mass by electrophoresis in a
polyacrylmamide gel under denaturing conditions. The mixture of proteins is first
dissolved in a solution of sodium dodecyl sulfate, an anionic detergent that
disrupts nearly all noncovalent interactions in native proteins. Mercaptoethanol or
dithiothreitol is also added to reduce disulfide bonds. Anions of SDS bind to main
chains at a ratio of about one SDS anion for every two amino acid residues. This
complex of SDS with a denatured protein has a large net negative charge that is
roughly proportional to the mass of the protein. The negative charge acquired on
binding SDS is usually much greater than the charge on the native protein; this
native charge is thereby rendered insignificant. The SDS-Protein complexes are
then subjected to gel electrophoresis. When the electrophoresis is complete, the
proteins in the gel can be visualized by staining them with silver or a dye such as
Coomassive blue, which reveals a series of bands.
 As in DNA electrophoresis, small proteins move rapidly through the gel, whereas
large proteins stay at the top, near the point of application of the mixture.

DNA Fingerprinting
 What is DNA? : DNA is a chemical structure that forms chromosomes, which
often contain genes that code for a particular trait.
o Structurally, DNA is a double helix, or two strands of genetic material
spiraled around each other. Each strand contains a sequence of bases, also
called nucleotides. These consist of the four chemicals adenine, guanine,
cytosine, and thymine.
o The two strands of DNA are connected by hydrogen bonds at each base.
Each base will bond only with one other base, as adenine will only bond
with thymine, and guanine will only bind with cytosine.
o DNA starnds are read in aparticular direction, from the top 9called the ‘ or
five primeend) to the bottom (called the 3’ or three prime end). In a double
helix, the starnd sgo opposite ways.
 What is DNA fingerprinting?: Athought the chemical structure of everyone’s
DNA is the same, most individuals differ in the order of their base pairs. There
are so many billions fo base pairs in eachpersons DNA that every person has a
different structure.
o Using said sequenced, each individual could be identified strictly by
the sequence of their base pairs. Because there are so many millions of
base pairs, however, this task would be extremely time consuming.
Instead, scientists are able to use a shorter method in which they are able
to determine whther two DNA samples are from the same person, related
people, or nonrelatedpeople. A small number of sequenced of DNA that
are known to vary among individuals are used in these methods, and are
then analyzed to suggest the probability of a match.
 How is it done?: southern blot:
 Isolating the DNA in question from the rest of the cellular
materialin the nucleus. This