biotechnology-theory lec. 2 3rd student-medical analysis

Biotechnology-Theory Lec. 2 3rd Student-Medical Analysis 2020

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Electrophoresis Analysis

Electrophoresis (Electricity + Migration): is defined as a technique used in

the laboratory for the separation (migration) of charged particles (e.g. DNA, RNA,

Amino Acid, Protein, Lipoprotein, Isoenzyme, Hemoglobin) through a solution

or gel, under the influence of an electrical field. “Electro” refers to the energy of

electricity. “Phoresis,” from the Greek verb phoros, means “to carry across.”

This electrokinetic phenomenon was observed for the first time in 1807

by Reuss.

Arne Tiselius (1937) was firstly developed the moving boundary technique for the

electrophoretic separation of substances (Electrophoresis in free solution), for which, besides

his work on adsorption analysis, he received the Nobel Prize in 1948.

The rate of movement of particle depends on the following factors:

1. The charge of the particle (Anion and Cation) [Types and number of charges].

2. Molecular weight of particles.

3. Molecular shape.

4. Applied electric field (Cathode and Anode poles).

5. Temperature.

6. Nature of the suspended medium (Viscosity).

7. Concentration of buffer used and pH of buffer.

Illustration Electrophoresis

http://en.wikipedia.org/wiki/Electrokinetic_phenomena

http://en.wikipedia.org/wiki/File:Electrophoresis.gif


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Types of Electrophoresis

The various methods electrophoresis can be grouped in to the following

categories:

Electrophoresis in Free Solution.

Electrophoresis in Supporting Media: There are many types of

electrophoresis according to the supporting media;

1. Gel Electrophoresis: Variety of gel matrices is used. The gel can be as vertical

cylindrical rods or plates as well as horizontal slabs. Further, the following

different types of gel electrophoreses are used.

Agarose Gel Electrophoresis and Pulsed Field Gel Electrophoresis.

Polyacrylamide Gel Electrophoresis.

Starch Gel Electrophoresis.

2. Paper and Thin Layer Electrophoresis.

3. Cellulose Acetate Electrophoresis.

Gel Electrophoresis

Gel electrophoresis is a powerful tool for the separation of macromolecules

(DNA, RNA and Protein) on the basis of size, electric charge, and other physical

properties for example that it is used by the molecular biologists in study of different

size of DNA fragments.

Principles:

These compounds (Nucleic acid and Protein) carry an electric charge and they

migrate in the direction of electrode bearing the opposite charge, viz. cationic

(positively charged) molecules move toward cathode (-ve electrode) and anionic

(negatively charged) molecules travel towards anode (+ve electrode) e.g. DNA carry

negative charge they migrate toward anode (+ve electrode). The molecules to be

separated are maintained in aqueous phase.

The speed or the rate of migration (electrophoretic mobility) of a molecule

depends on two factors, its shape or conformation of molecules and its charge-to-


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mass ratios. Unfortunately, most DNA molecules are the same shape and all have very

similar charge-to-mass ratios. Fragments of different sizes cannot therefore be separated

by standard electrophoresis (show this figure 1).

The size of the DNA molecule does, however, become a factor if the

electrophoresis is performed in a gel. A gel, which is usually made of agarose,

polyacrylamide, or a mixture of the two, comprises a complex network of pores, through

which the DNA molecules must travel to reach the positive electrode. Gel

electrophoresis therefore separates DNA molecules according to their size (see this

figure 2).

Figure 1

Figure 2


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Agarose Gel Electrophoresis Analysis:

The standard method lies near the heart of molecular cloning and is used to separate,

identify, purify DNA fragments, analyze and characterize recombinant DNA molecules

and determine molecular weight of DNA fragments (Purposes agarose gel

electrophoresis).

The technique is simple, rapid to perform, and capable of resolving fragments of

DNA that cannot be separated adequately by other sizing procedures. Furthermore, the

location of DNA within the gel can be determined directly by staining with low

concentrations of fluorescent intercalating dyes, such as ethidium bromide or SYBR

Gold; bands containing as little as 20 pg or 1 ng of double-stranded DNA can be detected

by direct examination of the gel in UV light.

The idea of using electrophoresis through a supporting matrix to analyze DNA came

from Vin Thorne, a biochemist, /virologist who in the mid-1960s that he was working

at the Institute of Virology in Glasgow.

Agarose Gel:

Agarose gels are mostly used when large pores for the analysis of molecules over 10

nm in diameter are needed. Agarose is a polysaccharide obtained from red seaweed that

is composed of alternating residues of D- and L-galactose joined by a-(1-13) and P-

(1+4) glycosidic linkages.

By removal of the agaropectin, gels of varying electroendosmosis and degrees of

purity can be obtained. They are characterized by their melting point (35 °C to 95 °C)

and the degree of electroendosmosis (mr).

The pore size depends on the concentration of agarose: one usually refers to the

weight of agarose and the volume of water. In general gels with a pore size from150nm

at 1 %( w/v) to 500nm at 0.16% are used.

For DNA separations 1 to 10 mm thick gels are cast on UV-transparent trays, because

the bands are usually stained with fluorescent dyes: Ethidium bromide or SYBR

Green.

Chemical structure of agarose and structure of the polymers during gel formation.

3.5-anhydro L-Galactose D-Galactose


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In practice the composition of the gel determines the sizes of the DNA molecules that

can be separated. A 0.5 cm thick slab of 0.5% agarose, which has relatively large pores, would

be used for molecules in the size range 1–30 kb, allowing, for example, molecules of 10 and

12 kb to be clearly distinguished. At the other end of the scale, a very thin (0.3 mm) 40%

polyacrylamide gel, with extremely small pores, would be used to separate much smaller DNA

molecules, in the range 1–300 bp, and could distinguish molecules differing in length by just a

single nucleotide.

Factors effect on mobility of DNA fragments through Agarose Gel

Electrophoresis:

1. Agarose Concentration: Higher concentration of agarose facilitates separation of small

DNAs, while low agarose conc. allows resolution of larger DNAs.

2. Voltage: As the voltage applied to gel is increased larger fragments migrate

proportionally faster the small fragments, for that reason, the best resolution of

fragments larger than 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).

3. Electrophoresis Buffer: the most commonly used for DNA are TAE (Tris-Acetate-

EDTA), TPE (Tris-Phosphate-EDTA) and TBE (Tris-Borate-EDTA). The latter is the

one used in our Lab, because it gives good resolution and has higher buffering capacity.

Buffer not only establish pH, but also provide ions to support conductivity.

The equipment and supplies of Agarose Gel Electrophoresis Preparation:

1. An electrophoresis chamber and power supply.

2. The gel casting trays.

3. Sample gel combs.

4. Electrophoresis buffer.

5. Loading buffer: contain something dense e.g. (glycerol) to make the DNA samples

denser and allow the DNA sample to fall into the sample wells, one or two dyes e.g.

bromophenol blue which migrate in the gel and allow visual monitoring or how far

the electrophoresis has proceeded.

6. Ethidium bromide: using to stain Nucleic acids.

7. UV Transilluminator: An ultraviolet high box used to visualize ethidium bromide

stained DNA in gel.


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Visualizing DNA Molecules in an Agarose Gel

The easiest way to visualize the results of a gel electrophoresis experiment is to

stain the gel with a compound that makes the DNA visible. Ethidium bromide (EtBr) is

also used routinely to stain DNA in agarose and polyacrylamide gels. Bands showing

the positions of the different size classes of DNA fragment are clearly visible under UV

irradiation after EtBr staining, as long as sufficient DNA is present. Unfortunately, the

procedure is very hazardous because EtBr is a powerful mutagen. Moreover, EtBr

staining also has limited sensitivity, and if a band contains less than about 10 ng of DNA

then it might not be visible after staining.

For this reason, non-mutagenic dyes that stain DNA green, red or blue are now

used in many laboratories. Most of these dyes can be used either as a post-stain after

electrophoresis or, alternatively, because they are non-hazardous they can be included

in the buffer solution in which the agarose or polyacrylamide is dissolved when the gel

is prepared. Some of these dyes require UV irradiation in order to make the bands

visible, but others are visualized by illumination at other wavelengths, for example

under blue light. This removes a second hazard as UV radiation can cause severe burns.

The most sensitive dyes are able to detect bands that contain less than 1 ng DNA.


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Estimation the Molecular sizes of DNA fragments:

The molecular size of an unknown piece of DNA can be estimated by comparison of

the distance that it travels with that of the molecular weight standards.

Gel electrophoresis separates different-sized DNA molecules, with the smallest

molecules travelling the greatest distance toward the positive electrode. If several DNA

fragments of varying sizes are present (the result of a successful restriction digest, for

example), then a series of bands appears in the gel. But how can the sizes of these

fragments be determined?

The most accurate method is to make use of the mathematical relationship that links

migration rate to molecular mass. The relevant formula is:

where D is the distance moved, M is the molecular mass, and a and b are constants

that depend on the electrophoresis conditions.

Because extreme accuracy in estimating DNA fragment sizes is not always

necessary, a much simpler – but less precise – method is more generally used. A standard

restriction digest, comprising fragments of known size, is usually included in each

electrophoresis gel that is run. Restriction digests of λ DNA are often used in this way

as size markers. For example, HindIII cleaves λ DNA into eight fragments that range

in size from 125 bp for the smallest to over 23 kb for the largest. As the sizes of the

fragments in this digest are known, the fragment sizes in the experimental digest can be

estimated by comparing the positions of the bands in the two tracks. Special mixtures of

DNA fragments called DNA ladders, whose sizes are multiples of 100 bp or of 1 kb,

can also be used as size markers. Although not precise, size estimation by comparison

with DNA markers can be performed with as little as 5%error, which is satisfactory for

most purposes.

Estimation of the sizes of DNA fragments in an agarose gel:

(a) A rough estimate of fragment size can be obtained by eye (Log paper).

(b) A more accurate measurement of fragment size is gained by using the mobilities

of the HindIII–λ fragments to construct a calibration curve; the sizes of the unknown

fragments can then be determined from the distances they have migrated.


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Gel electrophoresis of DNA. Fragments of different sizes were mixed and placed in a

well. Electrophoresis was in the downward direction. The DNA has been made visible

by the addition of a dye (ethidium bromide) that binds only to DNA and that fluoresces

when the gel is illuminated with short-wavelength ultraviolet light.

biotechnology-theory lec. 2 3rd student-medical analysis

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