Term Paper of Genetic Engineering

8/8/2019 Term Paper of Genetic Engineering

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CONTENTS

Introduction Electrophoresis and its principle ElectrophoresisEquipment Rate of Migration Types of Support Media Types ofelectrophoresis Conclusion Bibliography


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INTRODUCTION

Electrophoresis is the main technique for molecular se paration in today's cell biology. It is apowerful technique, which iseasy and inexpensive. In spite of the many physical arrangmentsfor the apparatus, and regardless of the medium through which molecules are allowed to migrate,all electrophoretic separations depend upon the charge distribution of the molecules beingseparated.

Electrophoresis can be one dimensional (i.e. one plane ofseparation) or two dimensional. Onedimensionalelectrophoresis is used for most routine protein and nucleic acid separations.Twodimensional se paration of proteins is used for finger printing , and when properly constructed

can be extremely accurate in resolving all of the proteins present within a cell (greater than1,500).

The support medium for electrophoresis can be formed into a gel within a tube or it can belayered into flat sheets.The tubes are used for easy one dimensional separations , while thesheets have a larger surface area and are better for twodimensional separations dimensionalseparations.

Typicalslab electrophoresis unit.

When the detergent SDS (sodium dodecyl sulfate) is used with proteins, all of the proteinsbecome negatively charged by their attachment to the SDS anions. When separated on apolyacrylamide gel, the procedure is abbreviated as SDS--PAGE (for Sodium Dodecyl SulfatePolyAcrylamide GelElectrophoresis).The technique has become a standard means for molecularweight determination.


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Polyacrylamide gels are formed from the polymerization of two compounds, acrylamide and N,N-methylene- bis-acrylamide (Bis, for short). Bis is a cross-linking agent for the gels. The

polymerization is initiated by the addition of ammonium persulfate along with either -dimethyl

amino-propionitrile (DMAP) or N,N,N ,N ,- tetramethylethylenediamine (TEMED). The gelsare neutral, hydrophillic, three-dimensional networks of long hydrocarbons crosslinked bymethylene groups.

Theseparation of molecules within a gel is determined by the relativesize of the pores formedwithin the gel. The pore size of a gel is determined by two factors, the total amount ofacrylamide present (designated as %T) and the amount of cross-linker (%C). As the total amountof acrylamide increases, the pore size decreases. With cross- linking,5%C gives the smallestporesize. Any increase or decrease in %C increases the poresize. Gels are designated as percentsolutions and will have two necessary parameters.The total acrylamide is given as a % (w/v) ofthe acrylamide plus the bis-acrylamide.Thus, a 7 1/2 %T would indicate that there is a total of

7.5 gms of acrylamide and bis per 100 ml of gel. A gel designated as 7.5%T:5%C

would have atotal of 7.5% (w/v) acrylamide + bis, and the bis would be5% of the total (with pure acrylamidecomposing the remaining 2.5%).

Proteins with molecular weights ranging from 10,000 to 1,000,000 may be separated with 71/2% acrylamide gels, while proteins with higher molecular weights require lower acrylamidegel concentrations.Conversely, gels up to 30% have been used to separatesmall polypeptides.The higher the gel concentration, thesmaller the poresize of the gel and the better it will be ableto separate smaller molecules.The percent gel to use depends on the molecular weight of theprotein to beseparated. Use5% gels for proteins ranging from 60,000 to 200,000 daltons, 10%gels for a range of 16,000 to 70,000 daltons and 15% gels for a range of 12,000 to 45,000

daltons.

Cationic vs anionic systems

In electrophoresis, proteins areseparated on the basis of charge, and the charge of a protein canbeeither + or -- , depending upon the pH of the buffer. In normal operation, a column of gel ispartitioned into threesections, known as the Separating orRunning Gel, the Stacking Gel and theSample Gel.The sample gel may be eliminated and the sample introduced via a dense non-convective medium such assucrose.Electrodes are attached to theends of the column and an

electric current passed through the partitioned gels. If theelectrodes are arranged in such a waythat the upper bath is -- (cathode), while thelower bath is + (anode), and -- anions are allowed toflow toward the anode, thesystem is known as an anionic system. Flow in the opposite direction,with + cations flowing to the cathode is a cationic system.


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Tube vs Slab Systems

Electrophoretic separations of proteins

Two basic approaches have been used in the design ofelectrophoresis protocols. One, columnelectrophoresis, uses tubular gels formed in glass tubes, while the other,slab gelelectrophoresis,uses flat gels formed between two plates of glass. Tube gels have an advantage in that themovement of molecules through the gels is less prone to lateral movement and thus there is aslightly improved resolution of the bands, particularly for proteins. It is also moreeconomical,since it is relativelyeasy to construct homemadesystems from materials on hand. However,slabgels have the advantage of allowing for two dimensional analysis, and of running multiple

samplessimultaneously in thesame gel.

Slab gels are designed with multiplelanesset up such that samples run in parallel.Thesize andnumber of the lanes can be varied and,since thesamples run in thesame medium, there is lesslikelihood of sample variation due to minor changes in the gel structure. Slab gels areunquestionably the the technique of choice for any blot analyses and for autoradiographicanalysis. Consequently, for laboratories performing routine nucleic acid analyses, and thoseemploying antigenic controls, slab gels have become standard.The availability of reasonablypriced commercialslab gel units has increased the use ofslab gelsystems, and the use of tubegels is becoming rare.

The theory and operation of slab gel electrophoresis is identical to tube gel electrophoresis.Which system is used depends more on the experience of the investigator than on any otherfactor, and the availability ofequipment

Continuous vs discontinuous gel system

The original use of gels asseparating media involved using a single gel with a uniform pHthroughout. Molecules wereseparated on the basis of their mobility through a single gel matrix..It has been replaced with discontinous, multiple gelsystems. In multiple gelsystems, aseparating gel is augmented with a stacking gel and an optionalsample gel.These gels can havedifferent concentrations of thesamesupport media, or may be completely different agents.The

key difference is how the moleculesseparate when theyenter theseparating gel.The proteins inthesample gel will concentrate into a small zone in thestacking gel beforeentering theseparating gel.The zone within thestacking gel can range in thickness from a few microns to afull millimeter. As the proteins arestacked in concentrated bands, they continue to migrate intotheseparating gel in concentrated narrow bands.The bands then areseparated from each otheron a discontinuous (i.e. disc ) pH gel.


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Once the protein bands enter the se parating gel, se paration of the bands is enhanced by ionspassing through the gel column in pairs.Each ioin in the pair has thesame charge polarity as theprotein (usually negative), but differ in charge magnitude. One ion will have a much greatercharge magnitude than the proteins, while the other has a lesser charge magnitude than theproteins.The ion having a greater charge will move faster and is thus theleading ion, while the

ion with thelesser charge will be the trailing ion. When an anionic system isemployed, theCland glycinate (glycine as its acid derivative) ions are derived from the reservoir buffer (Tris-

Glycine).Theleading ion is usuallyClglycinate is the trailing ion.

A schematic of this anionic system isshown in Figure.

Chloride ions enter the se parating gel first and rapidly move down the gel, followed by theproteins and then the glycinate ions.The glycinate ions overtake the proteins and ultimatelyestablish a uniform linear voltage gradient within the gel. The proteins then sort themselves

within this gradient according to their charge and size.

What is Electrophoresis ?

Electrophoresis is an analytical method frequently used in molecular biology and medicine. It isapplied for the se paration and characterization of proteins, nucleic acids and subcellular-sizedparticles like viruses and small organelles. Its principle is that the charged particles of a samplemigrate in an applied electrical field. If conducted in solution,samples areseparated according to

their surface net charge density. The most frequent applications, however, use gels(polyacrylamide, agarose) as a support medium.The presence ofsuch a matrix adds a sievingeffect so that particles can be characterized by both charge and size. Protein electrophoresis isoften performed in the presence of a charged detergent likesodium dodecylsulfate (SDS) whichusuallyequalizes thesurface charge and, therefore, allows for the determination of proteinsizeson a single gel. Additives are not necessary for nucleic acids which have a similarsurface chargeirrespective of theirsize.


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Principle of Electrophoresis

Charge of the particle:

The Charged particles under the influence of a liquid media placed in an electric field will

migrate to the electrode of the opposite charge. Positive ions (cations) will migrate to thecathode, the negativeelectrode. Negative ions (anions) will migrate to the anode, the positiveelectrode.

Size of the molecule:

Smaller thesize of the molecule,faster is the migration and greater thesize,lesser is the migrationof molecules

Electrophoresis Equipment

In addition to thespecimen sample,support medium and buffer forelectrophoresis, apowersupply, positive and negativeelectrodes, chamber, and identification or detectionmethod are needed.

The powersupply is a source of constant voltage or current that providesenergy to theelectrodes.This drives the movement of the ions in the medium and results in themovement and separation of the molecules orsolutes in thespecimen.Control of currentor voltage comes with the powersource in order to make adjustments.

The chamber is divided into two sections or has two reservoirs for the buffer and oneelectrode is placed in each.Thesupport medium islaid over the chamber in such a waythat it connects the two reservoirs. A lid or cover is placed over the chamber duringelectrophoresis. during electrophoresis.


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Rate of Migration

The net charge of a molecule is the most important factor affecting the mobility of that molecule.The greater the net charge, the greater the mobility or the morequickly the molecule migrates.The net charge of a particular compound depends upon the buffer and the resultant pH set by that

buffer.

Thesize and shape of a molecule also influence the rate of migration in that the larger thesize,theslower the molecule will move in electrophoresis.

The viscosity and the poresize in thesupport media or gels used forelectrophoresis influence therate of migration. Increased viscosityslows the migration and increasing poresizespeeds up themigration.

Increased heat increases the rate of migration. Increasing thestrength of theelectrical field byincreasing voltage and increasing the temperature used for theelectrophoresis both increase the

mobility and rate of migration. When increasing these factors that affect mobility, caution isnecessary.Each willlead to an increase in temperature that can possibly denature thesample andalter the characteristics of thesupport medium.

The ionic strength of the buffer and its effect on mobility are more complicated. The ionicstrength of the buffer affects the thickness of the ionic cloud, the rate of migration, and thesharpness of theseparated solutes. In electrophoresis, a cloud of ions forms over the medium andis composed of buffer ions, sample ions and other nonbuffer ions. Increasing the buffer ionicstrength increases the buffer ions in the cloud and slows the movement ofsolutes and createssharper bands. However, this also increases heat production.

Buffers and pH

The isoelectric point of most proteins is between pH 4.0 and 7.5. In pH 8-9, proteins will take ona negative charge and migrate to the anode. Most protein electrophoresis is performed at pH8.6.Buffers most commonly used are barbital or tris-boric acid-EDTA buffers.They fix the pH at8.6,leading to sharper bands and good separations

Role ofBuffers:-The two important purposes of the buffer are to create the pH and to conductthe current.The buffer ions will carry the current during electrophoresis.

The pH set by the buffer determines the net charge on thesolutes.The pH ionizes thesesolutes

and the resulting net charge determines which electrode the solutes migrate toward.Besidessetting the pH, the buffer also maintains the pH throughout theelectrophoresis of thesample.


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Types of Support Media

Forelectrophoretic separation ofsolutes, thesample ofsolutes is placed on a gel or membrane incontact with buffer for separation. Common gels are cellulose acetate, agarose, andpolyacrylamide gels.These gels are formed into sheets,slabs, or inserted into columns or tubes.

The gel can be positioned horizontally or vertically.

Cellulose is chemically reacted with acetic anyhdride to form a cellulose acetate gel.Becausecellulose requires soaking before sample application and a clearing ste p for detection ofseparated solutes or bands, agarose gel is more often used than cellulose acetate gel for clinicalelectrophoresis.

Agarose Gels

Agarose gels are chemically purified forms of agar, a polysaccharideextracted from seaweed.The gel pores allow for se paration of proteins based on their individual charge and mass.

Agarose gel will naturally clear after drying theseparated proteins.

Common clinical uses of agarose gelelectrophoresis (AGE) areseparations of plasma proteins,hemoglobin variants, lipoproteins, and isoenzymes.The gels come prepackaged with a plastictemplate to lay over gel forsample application orslotsetched in the gel for thesesamples.

Agarose Gels

Agaroseseparation of cDNA

Polyacrylamide Gels

Polyacrylamide electrophoresis (PAGE) is performed on a gel formed by polymerizing andcross-linking acrylamides.These gels arestronger than agarose gels and also thermostable and

transparent.The matrix created by cross-linking the polymer chains is more regular and the poresizes are more uniform in an individual gel.The pore size can be changed by changing theconcentrations of the acrylamides used.

In addition to se parating fragments by charge and mass, PAGE also separates solutes bymolecularsize. When using PAGE, the gel allows more fractions ofsmallersize to be detected

than the traditional agarose gel methods.


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Types of Electrophoresis

Gel Electrophoresis

Agarose gel electrophoresis is a method used in biochemistry and molecular biology to

separateDNA, orRNA molecules by size however proteins can also be separated on agarosegels. Se paration of the molecules is achieved by moving negatively charged nucleic acidmolecules through an agarose matrix in an electric field. Shorter molecules move faster andmigrate further than longer ones.

Agarose gels allow:

1. Se paration of restriction enzyme digested DNA including genomic DNA, prior toSouthern Blot transfer. It is also often used forseparating RNA prior to Northern transfer.

2. Analysis of PCR products after polymerase chain reaction to assess for target DNAamplification.

3.

Allows for theestimation of thesize of DNA molecules using a DNA marker or ladderwhich contains DNA fragments of various known sizes.4. Allows the rough estimation of DNA quantity and quality.5. Quantity is assessed using lambda DNA ladder which containsspecific amounts of DNA

in different bands.6. Quality of DNA is assessed by observing the absence of streaking or fragments (or

contaminating DNA bands).7. Other techniques rely on agarose gelelectrophoresis for DNA separation including DNA

fingerprinting.

The advantages are that the gel iseasily poured, does not denature thesamples.Thesamples canalso be recovered.

The disadvantages are that gels can melt during electrophoresis, the buffer can becomeexhausted, and different forms of genetic material may run in unpredictable forms.


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Two-dimensional gel electrophoresis

Two-dimensional gelelectrophoresis (2-D electrophoresis) is a powerful and widely used method

for the analysis of complex protein mixtures extracted from cells, tissues, or other biological

samples.This techniqueseparate proteins in two steps, according to two independent properties:

the first-dimension is isoelectric focusing (IEF), which separates proteins according to their

isoelectric points (pI); the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-

PAGE), which separates proteins according to their molecular weights (MW). In this way,

complex mixtures consisted of thousands of different proteins can be resolved and the relative

amount ofeach protein can be determined.The procedure involves placing the sample in gel with a pH gradient, and applying a potential

difference across it. In the electrical field, the protein migrates a long the pH gradient, until it

carries no overall charge.Thislocation of the protein in the gel constitutes the apparent pI of the

protein.


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There are two alternatives methods to create the pH gradient - carrier ampholites and immobilized

pH gradient (IPG) gels.

The IEF is the most critical ste p of the 2-D electrophoresis process. The proteins must

besolubilize without charged detergents, usually in high concentrated urea solution, reducing

agents and chaotrophs.To obtain high quality data it is essential to achieve low ionic strength

conditions before the IEF itself. Since different types ofsamples differ in their ion content, it is

necessary to adjust the IEF buffer and theelectrical profile to each type ofsample.

The se paration in the second dimension by molecular size is performed in slab SDS- PAGE.

Twelve parallel gels can beseparated in a fixed temperature to minimize theseparation variations

between individual gels.


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CapillaryElectrophoresis:-

Capillaryelectrophoresis (CE) is relatively new separation technique compared to the traditional

techniquessuch as high pressureliquid chromatography (HPLC) or gas chromatography (GC). It

provides very attractive features which make it both competitive and a good alternative. One ofthe major advantages ofCE over otherseparation technique is the ability to separate both charged

and non-charged molecules.In CE, se paration of analyte ions is performed in an electrolyte

solution (background electrolyte) present in a narrow fused-silica capillary. The ends of the

capillary are immersed into vials (inlet and outlet) filled with electrolyte solution, which also

contain electrodes connected to a high voltage supply (see Figure). The sample solution is

introduced in the capillary as a small plug by applying pressure (hydrodynamic injection) or

voltage (electrokinetic injection). With the application of high voltage (5 30 kV) across the

capillary, zones of analyte are formed due to different electrophoretic mobilities of ionic species

and migrate toward the outlet side of the capillary. In fact different ions can beseparated when

their charge/size ratio differs.Before reaching theend of the capillary, theseparated analyte bands

are detected directly through the capillary wall. Some of its main application fields include: i)

food analysis, ii) pharmaceutical analysis, iii) bioanalysis, iv) environmental pollutants analysis


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CONCLUSION:-

Electrophoresis is the technique of se paration of charged molecules under the influence of anelectrical field so that they migrate in the direction ofelectrode bearing the opposite charge, viz,cationic (positively charged) molecules move toward cathode (-ve electrode) and anionic

(negatively charged) molecules travel towards anode (+ve electrode).The molecules to be separated are maintained in aqueous phase. The speed of migration(electrophoretic mobility) of a molecule depends on its charge and molecular mass.

Charge of a molecule is influenced by the following:(1) the type, concentration and pH of buffer,(2) the temperature,(3) strength of theelectrical field, and(4) the nature of thesupport material (matrix) used forelectrophoresis.

In electrophoresis, chemicals such as blood proteins, DNA or inorganic ions can be separated

according to differences in their mass and/or charge.Thesolid medium used in electrophoresis isusually an agarose or polyacrylamide gel

Electrophoretic se paration has uses in forensic science because it can be used to isolate andcompare DNA, blood proteins and inorganic substances such as gunshot residues from crimescenes with suspects, victims orstandard reference material.

Electrophoresis is most frequently used in forensic science to produce DNA fingerprints. DNAevidence from a crime scene can be compared to DNA samples from different suspects, forinstance, and suspects can either be included orexcluded from suspicion using the results ofsuchtests

In gel electrophoresis, DNA strands from crime scenes, victims or suspects are applied to anagarose gel that is subjected to an electric potential. The more traditional RFLP (restrictionfragment length polymorphism) profiling procedure is now being replaced by the PCR(polymerase chain reaction) method, which often involves the use of shorter DNA segmentsknown as STRs (single tandem repeats).This method is faster and requiresless DNA.

Capillary electrophoresis in which a fused silica capillary is used instead of a gel slab, is nowbeing used more frequently in DNA electrophoresis. Although applying the same principles ofse paration as the more traditional gel slab electrophoresis, it is more rapid and has a higherresolution.

Future Directions in Electrophoresis

Research is currently being undertaken in the US to develop portable microchip DNA profilingdevices that can be used in the field. In this method, STRanalysis of a small DNA sample can beachieved on thesurface of microchips in much less time than traditional techniques. Pulsed fieldelectrophoresis is another innovation being investigated here, the direction of theelectric fieldis alternated, allowing for the separation of DNA molecules up to several million base pairs in


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length.

These, and other advances in electrophoretic technology, will ensure faster and more effectiveanalysis of crimesceneevidence in theyears ahead.

BIBLIOGRAPHY

y Dr. William H. Heidcamp,Biology Department, Gustavus AdolphusCollege,St. Peter,http://homepages.gac.edu/~cellab/chpts/chpt4/intro4.html

y http://www.his.com/~djt/elphoexplain.htmly http://www.medialabinc.net/electrophoresis-keyword.aspxy http://www.molecularstation.com/molecular-biology-techniques/gel-electrophoresis/y http://www.tau.ac.il/lifesci/units/proteomics/2dimgel.htmly http://www.scitopics.com/Capillary_Electrophoresis.htmy Tissue,B.M, 2010, "Electrophoresis",TheChemistry Hypermedia Project, chem vt.edu,

accessed 1/2/2010, http://www.suite101.com/content/the-use-of-electrophoresis-in-

forensic-science-


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Term Paper of Genetic Engineering