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DNA gel electrophoresis is a process used to separate proteins and nucleic acids in molecular 

 biology. The gel is usually composed of a crossed-linked polymer and acrylamide which aid in

separating and analyzing different parts of a DNA molecule. DNA gel electrophoresis iscommonly used in forensics to determine the specific sequence of DNA to help find the leading

suspect. It is also commonly used in genetics and the fields of molecular biology and

 biochemistry.

 Nucleic acids and proteins carry either a positive or negative charge. When placed in an electric

chamber in DNA gel electrophoresis, each electrical charge will move toward either end of thechamber. The negative electrical charged molecules will move to the positive end of the

chamber, while the positive electrical charged molecules will move to the negative end. The gel

separates each of these molecules so they evenly spread throughout the reading. The gel is

composed of long fibers which prevent the molecules from moving to aid in analyzing theindividual molecules.

Southern blotting is a technique used in DNA gel electrophoresis to determine the presence or 

absence of a specific nucleotide sequence in a DNA molecule. Northern blotting is a differenttechnique used to identify gene expression in the presence of  RNA, or ribonucleic acid. Eastern

 blotting is used to determine the presence of lipids, proteins and carbohydrates in a DNAsequence. To simply detect proteins in a DNA sequence, the technique of western blotting is

used in the electrophoresis chamber through gel isolation.

Zymography is also an approach to detect certain biological functions through DNA gel

electrophoresis. It is mainly used to study extracellular matrix-degrading enzymes, which

catalyze the destruction of the matrix outside the cell, as well as the basement membrane of a

cell. The use of polyacrylamide gel aids in separating the enzymes, which are proteins, by usinga protein substrate such as gelatin or casein. This technique is used to measure the amount of 

degradation activity occurring outside the cell.

DNA gel electrophoresis is used quite often in forensics. It is used to separate blood proteins and

DNA found at a crime scene to determine correlations with the available suspects. Gel

electrophoresis is also used to determine a specific inheritance within genetics, as certain DNAand proteins are associated with different races. It is also used to solve paternity cases to

determine the relative relationship between individuals. Digital image of 3 plasmid restriction

digests run on a 1% w/v agarose gel, 3 volt/cm, stained with ethidium bromide. The DNA sizemarker is a commercial 1 kbp ladder. The position of the wells and direction of DNA migration

is noted.

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Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and

 proteins) and their fragments, based on their size and charge.

It is used in clinical chemistry to separate proteins by charge and/or size (IEF agarose, essentially

size independent) and in  biochemistry and molecular biology to separate a mixed population of 

DNA and RNA fragments by length, to estimate the size of  DNA and RNA fragments or toseparate proteins  by charge.[1]

  Nucleic acid molecules are separated by applying an electric field 

to move the negatively charged molecules through an agarose matrix. Shorter molecules move

faster and migrate farther than longer ones because shorter molecules migrate more easilythrough the pores of the gel. This phenomenon is called sieving.

[2] Proteins are separated by

charge in agarose because the pores of the gel are too large to sieve proteins. Gel electrophoresis

can also be used for separation of nanoparticles.

Gel electrophoresis uses a gel as an anticonvective medium and/or sieving medium during

electrophoresis, the movement of a charged particle in an electrical field. Gels suppress the

thermal convection caused by application of the electric field, and can also act as a sieving

medium, retarding the passage of molecules; gels can also simply serve to maintain the finishedseparation, so that a post electrophoresis stain can be applied.[3]

 DNA Gel electrophoresis is

usually performed for analytical purposes, often after amplification of DNA via PCR , but may beused as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, 

PCR , cloning, DNA sequencing, or  Southern blotting for further characterization.

Separation

In simple terms: Electrophoresis is a process which enables the sorting of molecules based on

size. Using an electric field, molecules (such as DNA) can be made to move through a gel madeof  agar  or   polyacrylamide. The molecules being sorted are dispensed into a well in the gel

material. The gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric current is applied, the larger molecules move more slowly through the

gel while the smaller molecules move faster. The different sized molecules form distinct bandson the gel.

[citation needed ] 

The term "gel" in this instance refers to the matrix used to contain, then separate the target

molecules. In most cases, the gel is a crosslinked polymer  whose composition and porosity is

chosen based on the specific weight and composition of the target to be analyzed. Whenseparating  proteins or small nucleic acids (DNA, RNA, or  oligonucleotides) the gel is usually

composed of different concentrations of  acrylamide and a cross-linker , producing different sized

mesh networks of   polyacrylamide. When separating larger nucleic acids (greater than a few

hundred bases), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet porous matrix. Acrylamide, in contrast to  polyacrylamide, is a neurotoxin and must be handledusing appropriate safety precautions to avoid poisoning. Agarose is composed of long

unbranched chains of uncharged carbohydrate without cross links resulting in a gel with large

 pores allowing for the separation of macromolecules and macromolecular complexes.[citation needed ]

 

"Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules

through the gel matrix. By placing the molecules in wells in the gel and applying an electric

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field, the molecules will move through the matrix at different rates, determined largely by their 

mass when the charge to mass ratio (Z) of all species is uniform, toward the (negatively charged)

cathode if positively charged or toward the (positively charged) anode if negatively charged.[4]

 

If several samples have been loaded into adjacent wells in the gel, they will run parallel in

individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per 

component. Incomplete separation of the components can lead to overlapping bands, or to

indistinguishable smears representing multiple unresolved components.[citation needed ]

Bands indifferent lanes that end up at the same distance from the top contain molecules that passed

through the gel with the same speed, which usually means they are approximately the same size.

There are molecular weight size markers available that contain a mixture of molecules of known

sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bandsobserved can be compared to those of the unknown in order to determine their size. The distance

a band travels is approximately inversely proportional to the logarithm of the size of the

molecule.[citation needed ]

 

There are limits to electrophoretic techniques. Since passing current through a gel causes

heating, gels may melt during electrophoresis. Electrophoresis is performed in buffer solutions toreduce pH changes due to the electric field, which is important because the charge of DNA and

RNA depends on pH, but running for too long can exhaust the buffering capacity of the solution.

Further, different preparations of genetic material may not migrate consistently with each other,

for morphological or other reasons.

Types of gel

Agarose

Agarose gels are easily cast and handled compared to other matrices, because the gel setting is a

 physical rather than chemical change. Samples are also easily recovered. After the experiment isfinished, the resulting gel can be stored in a plastic bag in a refrigerator.

Agarose gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pair  to several megabases (millions of bases) using specialized apparatus. The distance

 between DNA bands of a given length is determined by the percent agarose in the gel. The

disadvantage of higher concentrations is the long run times (sometimes days). Instead high

 percentage agarose gels should be run with a  pulsed field electrophoresis (PFE), or  fieldinversion electrophoresis. 

"Most agarose gels are made with between 0.7% (good separation or resolution of large 5 – 10kbDNA fragments) and 2% (good resolution for small 0.2 – 1kb fragments) agarose dissolved in

electrophoresis buffer. Up to 3% can be used for separating very tiny fragments but a vertical

 polyacrylamide gel is more appropriate in this case. Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set evenly. 1%

gels are common for many applications."[5]

 

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Agarose gels do not have a uniform pore size, but are optimal for electrophoresis of proteins that

are larger than 200 kDa.[6]

 

Polyacrylamide

Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins ranging in size from 5to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Pore size is

controlled by controlling the concentrations of acrylamide and bis-acrylamide powder used in

creating a gel. Care must be used when creating this type of gel, as acrylamide is a potent

neurotoxin in its liquid and powdered form.

Traditional DNA sequencing techniques such as Maxam-Gilbert or  Sanger  methods used

 polyacrylamide gels to separate DNA fragments differing by a single base-pair in length so thesequence could be read. Most modern DNA separation methods now use agarose gels, except for 

 particularly small DNA fragments. It is currently most often used in the field of  immunology and

 protein analysis, often used to separate different proteins or  isoforms of the same protein into

separate bands. These can be transferred onto a nitrocellulose or  PVDF membrane to be probedwith antibodies and corresponding markers, such as in a western blot. 

Typically resolving gels are made in 6%, 8%, 10%, 12% or 15%. Stacking gel (5%) is poured on

top of the resolving gel and a gel comb (which forms the wells and defines the lanes where

 proteins, sample buffer and ladders will be placed) is inserted. The percentage chosen dependson the size of the protein that one wishes to identify or probe in the sample. The smaller the

known weight, the higher the percentage that should be used. Changes on the buffer system of 

the gel can help to further resolve proteins of very small sizes.[7]

 

Starch

Partially hydrolysed potato starch makes for another non-toxic medium for proteinelectrophoresis. The gels are slightly more opaque than acrylamide or agarose. Non-denatured

 proteins can be separated according to charge and size. They are visualised using Napthal Black 

or Amido Black staining. Typical starch gel concentrations are 5% to 10%.[8][9][10]

 

Gel conditions

Denaturing

A denaturing gel is a type of electrophoresis in which the native structure of  macromolecules that

are run within the gel is not maintained. For instance, gels used in SDS-PAGE (sodium dodecyl

sulfate  polyacrylamide gel electrophoresis) will unfold and denature the native structure of a

 protein. In contrast to native gel electrophoresis, quaternary structure cannot be investigatedusing this method.

Native

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Specific enzyme-linked staining: Glucose-6-Phosphate Dehydrogenase isoenzymes in

 Plasmodium falciparum infected Red blood cells[11]

 

 Native gel electrophoresis is an electrophoretic separation method typically used in  proteomics and metallomics.[12]

 However, native SDS-PAGE, is also used to scan genes (DNA) for unknown

mutations as in Single-strand_conformation_polymorphism. 

 Native PAGE separations are run in non-denaturing conditions. Detergents are used only to the

extent that they are necessary to lyse lipid membranes in the cell. Complexes remain — for the

most part — associated and folded as they would be in the cell. One downside, however, is thatcomplexes may not separate cleanly or predictably, since they cannot move through the

 polyacrylamide gel as quickly as individual, denatured proteins.

Unlike denaturing methods, such as SDS-PAGE, native gel electrophoresis does not use a

charged denaturing agent. The molecules being separated (usually  proteins or  nucleic acids) therefore differ not only in molecular mass and intrinsic charge, but also the cross-sectional area,

and thus experience different electrophoretic forces dependent on the shape of the overallstructure. Since the proteins remain in the native state they may be visualised not only by general

 protein staining reagents but also by specific enzyme-linked staining.

Buffers

Buffers in gel electrophoresis are used to provide ions that carry a current and to maintain the pH

at a relatively constant value. There are a number of buffers used for electrophoresis. The mostcommon being, for nucleic acids Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA (TBE). Many

other buffers have been proposed, e.g. lithium borate, which is almost never used, based onPubmed citations (LB), iso electric histidine, pK matched goods buffers, etc.; in most cases the purported rationale is lower current (less heat) and or matched ion mobilities, which leads to

longer buffer life. Borate is problematic; Borate can polymerize, and/or interact with cis diols

such as those found in RNA. TAE has the lowest buffering capacity but provides the bestresolution for larger DNA. This means a lower voltage and more time, but a better product. LB is

relatively new and is ineffective in resolving fragments larger than 5 kbp; However, with its low

conductivity, a much higher voltage could be used (up to 35 V/cm), which means a shorter analysis time for routine electrophoresis. As low as one base pair size difference could be

resolved in 3% agarose gel with an extremely low conductivity medium (1 mM Lithium

 borate).[13]

 

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Visualization

DNA gel electrophoresis 

After the electrophoresis is complete, the molecules in the gel can be stained to make them

visible. DNA may be visualized using ethidium bromide which, when intercalated into DNA,fluoresce under  ultraviolet light, while protein may be visualised using silver stain or  Coomassie

Brilliant Blue dye. Other methods may also be used to visualize the separation of the mixture's

components on the gel. If the molecules to be separated contain radioactivity, for example in

DNA sequencing gel, an autoradiogram can be recorded of the gel. Photographs can be taken of gels, often using Gel Doc. 

The most common dye used to make DNA or RNA bands visible for agarose gel electrophoresisis ethidium bromide, usually abbreviated as EtBr. It fluoresces under UV light when intercalated

into the major groove of DNA (or RNA). By running DNA through an EtBr-treated gel and

visualizing it with UV light, any band containing more than ~20 ng DNA becomes distinctly

visible. EtBr is a known mutagen, and safer alternatives are available, such as GelRed, which binds to the minor groove.

SYBR Green I is another dsDNA stain, produced by Invitrogen. It is more expensive, but 25

times more sensitive, and possibly safer than EtBr, though there is no data addressing its

mutagenicity or toxicity in humans.[14]

 

SYBR Safe is a variant of SYBR Green that has been shown to have low enough levels of 

mutagenicity and toxicity to be deemed nonhazardous waste under U.S. Federal regulations.[15]

 It

has similar sensitivity levels to EtBr ,[15]

  but, like SYBR Green, is significantly more expensive.In countries where safe disposal of hazardous waste is mandatory, the costs of EtBr disposal can

easily outstrip the initial price difference, however.

Since EtBr stained DNA is not visible in natural light, scientists mix DNA with negatively

charged loading buffers before adding the mixture to the gel. Loading buffers are useful because

they are visible in natural light (as opposed to UV light for EtBr stained DNA), and they co-sediment with DNA (meaning they move at the same speed as DNA of a certain length). Xylene

cyanol and Bromophenol blue are common dyes found in loading buffers; they run about the

same speed as DNA fragments that are 5000 bp and 300 bp in length respectively, but the precise position varies with percentage of the gel. Other less frequently used progress markers are Cresol

Red and Orange G which run at about 125 bp and 50 bp, respectively.

Visualization can also be achieved by transferring DNA to a nitrocellulose membrane followed by exposure to a hybridization probe. This process is termed Southern blotting. 

Analysis

After electrophoresis the gel is illuminated with an ultraviolet lamp (usually by placing it on a

light box, while using protective gear to limit exposure to ultraviolet radiation). The illuminator apparatus mostly also contains imaging apparatus that takes an image of the gel, after 

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illumination with UV radiation. The ethidium bromide fluoresces reddish-orange in the presence

of DNA, since it has intercalated with the DNA. The DNA band can also be cut out of the gel,

and can then be dissolved to retrieve the purified DNA. The gel can then be photographedusually with a digital or polaroid camera. Although the stained nucleic acid fluoresces reddish-

orange, images are usually shown in black and white (see figures).

Even short exposure of nucleic acids to UV light causes significant damage to the sample. UV

damage to the sample will reduce the efficiency of subsequent manipulation of the sample, such

as ligation and cloning. If the DNA is to be used after separation on the agarose gel, it is best toavoid exposure to UV light by using a blue light excitation source such as the XcitaBlue UV to

 blue light conversion screen from Bio-Rad or Dark Reader from Clare Chemicals. A blue

excitable stain is required, such as one of the SYBR Green or GelGreen stains. Blue light is also

 better for visualization since it is safer than UV (eye-protection is not such a critical requirement)and passes through transparent plastic and glass. This means that the staining will be brighter 

even if the excitation light goes through glass or plastic gel platforms.

Downstream processing

After separation, an additional separation method may then be used, such as isoelectric focusing 

or  SDS-PAGE. The gel will then be physically cut, and the protein complexes extracted fromeach portion separately. Each extract may then be analysed, such as by  peptide mass

fingerprinting or  de novo sequencing after  in-gel digestion. This can provide a great deal of 

information about the identities of the proteins in a complex.

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Genetic engineering, also called genetic modification, is the direct manipulation of an

organism's genome using  biotechnology. New DNA may be inserted in the host genome by first

isolating and copying the genetic material of interest using molecular cloning methods togenerate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the

host organism. Genes may be removed, or "knocked out", using a nuclease. Gene targeting is a

different technique that uses homologous recombination to change an endogenous gene, and can be used to delete a gene, remove exons, add a gene, or introduce  point mutations. 

An organism that is generated through genetic engineering is considered to be a geneticallymodified organism (GMO). The first GMOs were bacteria in 1973; GM mice were generated in

1974. Insulin- producing bacteria were commercialized in 1982 and genetically modified food

has been sold since 1994.

Genetic engineering techniques have been applied in numerous fields including research,

agriculture, industrial biotechnology, and medicine. Enzymes used in laundry detergent and

medicines such as insulin and human growth hormone are now manufactured in GM cells,

experimental GM cell lines and GM animals such as mice or  zebrafish are being used for research purposes, and genetically modified crops have been commercialized.

Definition

Comparison of conventional plant breeding with transgenic and cisgenic genetic modification.

Genetic engineering alters the genetic makeup of an organism using techniques that removeheritable material or that introduce DNA prepared outside the organism either directly into the

host or into a cell that is then fused or  hybridized with the host.[1]

 This involves usingrecombinant nucleic acid (DNA or  RNA) techniques to form new combinations of heritable

genetic material followed by the incorporation of that material either indirectly through a vector  

system or directly through micro-injection, macro-injection and micro-encapsulation techniques.

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Genetic engineering does not normally include traditional animal and  plant breeding, in vitro

fertilisation, induction of   polyploidy, mutagenesis and cell fusion techniques that do not use

recombinant nucleic acids or a genetically modified organism in the process.[1]

 However theEuropean Commission has also defined genetic engineering broadly as including selective

 breeding and other means of artificial selection.[2]

 Cloning and stem cell research, although not

considered genetic engineering,

[3]

 are closely related and genetic engineering can be used withinthem.[4]

 Synthetic biology is an emerging discipline that takes genetic engineering a step further  by introducing artificially synthesized genetic material from raw materials into an organism.

[5] 

If genetic material from another species is added to the host, the resulting organism is called

transgenic. If genetic material from the same species or a species that can naturally breed with

the host is used the resulting organism is called cisgenic.[6]

 Genetic engineering can also be used

to remove genetic material from the target organism, creating a gene knockout organism.[7]

 InEurope genetic modification is synonymous with genetic engineering while within the United

States of America it can also refer to conventional breeding methods.[8][9]

 The Canadian

regulatory system is based on whether a product has novel features regardless of method of 

origin. In other words, a product is regulated as genetically modified if it carries some trait not previously found in the species whether it was generated using traditional breeding methods

(e.g., selective breeding, cell fusion, mutation breeding) or genetic engineering.[10][11][12]

 Withinthe scientific community, the term genetic engineering is not commonly used; more specificterms such as transgenic are preferred.

Genetically modified organisms

Main article: Genetically modified organism 

Plants, animals or micro organisms that have changed through genetic engineering are termed

genetically modified organisms or GMOs.[13] Bacteria were the first organisms to be geneticallymodified. Plasmid DNA containing new genes can be inserted into the bacterial cell and the

 bacteria will then express those genes. These genes can code for medicines or enzymes that process food and other  substrates.

[14][15] Plants have been modified for insect protection,

herbicide resistance, virus resistance, enhanced nutrition, tolerance to environmental pressures

and the production of edible vaccines.[16]

 Most commercialised GMO's are insect resistant and/or herbicide tolerant crop plants.

[17] Genetically modified animals have been used for research,

model animals and the production of agricultural or pharmaceutical products. They include

animals with genes knocked out, increased susceptibility to disease, hormones for extra growthand the ability to express proteins in their milk .

[18] 

History

Main article: History of genetic engineering 

Humans have altered the genomes of species for thousands of years through artificial selection and more recently mutagenesis. Genetic engineering as the direct manipulation of DNA by

humans outside breeding and mutations has only existed since the 1970s. The term "genetic

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engineering" was first coined by Jack Williamson in his science fiction novel  Dragon's Island ,

 published in 1951,[19]

 one year before DNA's role in heredity was confirmed by Alfred Hershey 

and Martha Chase,[20]

 and two years before James Watson and Francis Crick  showed that theDNA molecule has a double-helix structure.

In 1974 Rudolf Jaenisch created the first GM animal. 

In 1972 Paul Berg created the first recombinant DNA molecules by combining DNA from themonkey virus SV40 with that of the lambda virus.

[21] In 1973 Herbert Boyer  and Stanley Cohen 

created the first transgenic organism  by inserting antibiotic resistance genes into the  plasmid of an  E. coli  bacterium.

[22][23] A year later  Rudolf Jaenisch created a transgenic mouse  by

introducing foreign DNA into its embryo, making it the world’s first transgenic animal.[24]

 These

achievements led to concerns in the scientific community about potential risks from geneticengineering, which were first discussed in depth at the Asilomar Conference in 1975. One of the

main recommendations from this meeting was that government oversight of recombinant DNA

research should be established until the technology was deemed safe.[25][26]

 

In 1976 Genentech, the first genetic engineering company was founded by Herbert Boyer and

Robert Swanson and a year later and the company produced a human protein (somatostatin) in E.coli. Genentech announced the production of genetically engineered human insulin in 1978.[27]

 In 1980, the U.S. Supreme Court in the Diamond v. Chakrabarty case ruled that genetically

altered life could be patented.[28]

 The insulin produced by bacteria, branded humulin, was

approved for release by the Food and Drug Administration in 1982.[29]

 

In the 1970s graduate student Stephen Lindow of the University of Wisconsin – Madison with

D.C. Arny and C. Upper found a bacterium he identified as P. syringae that played a role in icenucleation and in 1977, he discovered a mutant ice-minus strain. Later, he successfully created a

recombinant ice-minus strain.[30]

 In 1983, a biotech company, Advanced Genetic Sciences (AGS)

applied for U.S. government authorization to perform field tests with the ice-minus strain of  P.

 syringae to protect crops from frost, but environmental groups and protestors delayed the fieldtests for four years with legal challenges.[31]

 In 1987, the ice-minus strain of  P. syringae became

the first genetically modified organism (GMO) to be released into the environment[32]

 when a

strawberry field and a potato field in California were sprayed with it.[33]

 Both test fields wereattacked by activist groups the night before the tests occurred: "The world's first trial site

attracted the world's first field trasher".[32]

 

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The first field trials of  genetically engineered plants occurred in France and the USA in 1986,

tobacco plants were engineered to be resistant to herbicides.[34]

 The People’s Republic of China

was the first country to commercialize transgenic plants, introducing a virus-resistant tobacco in1992.

[35] In 1994 Calgene attained approval to commercially release the Flavr Savr  tomato, a

tomato engineered to have a longer shelf life.[36]

 In 1994, the European Union approved tobacco

engineered to be resistant to the herbicide  bromoxynil, making it the first genetically engineeredcrop commercialized in Europe.[37]

 In 1995, Bt Potato was approved safe by the EnvironmentalProtection Agency, after having been approved by the FDA, making it the first pesticide

 producing crop to be approved in the USA.[38]

 In 2009 11 transgenic crops were grown

commercially in 25 countries, the largest of which by area grown were the USA, Brazil,Argentina, India, Canada, China, Paraguay and South Africa.

[39] 

In the late 1980s and early 1990s, guidance on assessing the safety of genetically engineered plants and food emerged from organizations including the FAO and WHO.

[40][41][42][43] 

In 2010, scientists at the J. Craig Venter Institute, announced that they had created the first

synthetic bacterial genome, and added it to a cell containing no DNA. The resulting bacterium,named Synthia, was the world's first synthetic life form.[44][45]

 

Process

Main article: Techniques of genetic engineering 

The first step is to choose and isolate the gene that will be inserted into the genetically modified

organism. Presently, most genes transferred into plants provide protection against insects or 

tolerance to herbicides.[46]

 In animals the majority of genes used are growth hormone genes.[47]

 The gene can be isolated using restriction enzymes to cut DNA into fragments and gel

electrophoresis to separate them out according to length.[48] Polymerase chain reaction (PCR) canalso be used to amplify up a gene segment, which can then be isolated through gelelectrophoresis.

[49] If the chosen gene or the donor organism's genome has been well studied it

may be present in a genetic library. If the DNA sequence is known, but no copies of the gene are

available, it can be artificially synthesized.[50]

 

The gene to be inserted into the genetically modified organism must be combined with other 

genetic elements in order for it to work properly. The gene can also be modified at this stage for  better expression or effectiveness. As well as the gene to be inserted most constructs contain a

 promoter  and terminator  region as well as a selectable marker  gene. The promoter region

initiates transcription of the gene and can be used to control the location and level of gene

expression, while the terminator region ends transcription. The selectable marker, which in mostcases confers antibiotic resistance to the organism it is expressed in, is needed to determine

which cells are transformed with the new gene. The constructs are made using recombinant DNA 

techniques, such as restriction digests, ligations and molecular cloning.[51]

 The manipulation of the DNA generally occurs within a  plasmid. 

The most common form of genetic engineering involves inserting new genetic material randomlywithin the host genome. Other techniques allow new genetic material to be inserted at a specific

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location in the host genome or generate mutations at desired genomic loci capable of  knocking

out endogenous genes. The technique of  gene targeting uses homologous recombination to target

desired changes to a specific endogenous gene. This tends to occur at a relatively low frequencyin plants and animals and generally requires the use of  selectable markers. The frequency of gene

targeting can be greatly enhanced with the use of engineered nucleases such as zinc finger 

nucleases,

[52]

 

[53]

 engineered homing endonucleases,

[54]

 

[55]

 or nucleases created from TALeffectors.[56]

 [57]

 In addition to enhancing gene targeting, engineered nucleases can also be used tointroduce mutations at endogenous genes that generate a gene knockout

[58] .

[59] 

Transformation

Main article: Transformation (genetics) 

 A. tumefaciens attaching itself to a carrot cell

About 1% of bacteria are naturally able to take up foreign DNA  but it can also be induced inother bacteria.

[60] Stressing the bacteria for example, with a heat shock  or an electric shock, can

make the cell membrane permeable to DNA that may then incorporate into their genome or exist

as extrachromosomal DNA. DNA is generally inserted into animal cells using microinjection, where it can be injected through the cells nuclear envelope directly into the nucleus or through

the use of  viral vectors. In plants the DNA is generally inserted using  Agrobacterium-mediated

recombination or   biolistics.[61]

 

In Agrobacterium-mediated recombination the  plasmid construct contains T-DNA, DNA which

is responsible for insertion of the DNA into the host plants genome. This plasmid is transformed

into Agrobacterium that contains no plasmids and then plant cells are infected. The Agrobacterium will then naturally insert the genetic material into the plant cells.[62] In biolistics

transformation particles of gold or tungsten are coated with DNA and then shot into young plant

cells or plant embryos. Some genetic material will enter the cells and transform them. Thismethod can be used on plants that are not susceptible to Agrobacterium infection and also allows

transformation of plant plastids. Another transformation method for plant and animal cells is

electroporation. Electroporation involves subjecting the plant or animal cell to an electric shock,which can make the cell membrane permeable to plasmid DNA. In some cases the electroporated

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cells will incorporate the DNA into their genome. Due to the damage caused to the cells and

DNA the transformation efficiency of biolistics and electroporation is lower than agrobacterial

mediated transformation and microinjection.[63]

 

As often only a single cell is transformed with genetic material the organism must be regenerated

from that single cell. As bacteria consist of a single cell and reproduce clonally regeneration isnot necessary. In plants this is accomplished through the use of  tissue culture. Each plant species

has different requirements for successful regeneration through tissue culture. If successful an

adult plant is produced that contains the transgene in every cell. In animals it is necessary toensure that the inserted DNA is present in the embryonic stem cells. Selectable markers are used

to easily differentiate transformed from untransformed cells. These markers are usually present

in the transgenic organism, although a number of strategies have been developed that can remove

the selectable marker from the mature transgenic plant.[64]

 When the offspring is produced theycan be screened for the presence of the gene. All offspring from the first generation will be

heterozygous for the inserted gene and must be mated together to produce a homozygous animal.

Further testing uses PCR , Southern hybridization, and DNA sequencing is conducted to confirmthat an organism contains the new gene. These tests can also confirm the chromosomal location

and copy number of the inserted gene. The presence of the gene does not guarantee it will beexpressed at appropriate levels in the target tissue so methods that look for and measure the gene

 products (RNA and protein) are also used. These include northern hybridization, quantitative

RT-PCR , Western blot, immunofluorescence, ELISA and  phenotypic analysis. For stable

transformation the gene should be passed to the offspring in a Mendelian inheritance  pattern, sothe organism's offspring are also studied.

Applications

Genetic engineering has applications in medicine, research, industry and agriculture and can beused on a wide range of plants, animals and micro organism.

Medicine

In medicine genetic engineering has been used to mass-produce insulin, human growthhormones, follistim (for treating infertility), human albumin, monoclonal antibodies, 

antihemophilic factors, vaccines and many other drugs.[65]

 Vaccination generally involves

injecting weak live, killed or inactivated forms of  viruses or their toxins into the person being

immunized.[66]

 Genetically engineered viruses are being developed that can still confer immunity, but lack the infectious sequences.

[67] Mouse hybridomas, cells fused together to create

monoclonal antibodies, have been humanised through genetic engineering to create humanmonoclonal antibodies.

[68] 

Genetic engineering is used to create animal models of human diseases. Genetically modified

mice are the most common genetically engineered animal model.[69]

 They have been used tostudy and model cancer (the oncomouse), obesity, heart disease, diabetes, arthritis, substance

abuse, anxiety, aging and Parkinson disease.[70]

 Potential cures can be tested against these mouse

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models. Also genetically modified pigs have been bred with the aim of increasing the success of 

 pig to human organ transplantation.[71]

 

Gene therapy is the genetic engineering of humans  by replacing defective human genes with

functional copies. This can occur in somatic tissue or  germline tissue. If the gene is inserted into

the germline tissue it can be passed down to that person's descendants .

[72]

 Gene therapy has beenused to treat patients suffering from immune deficiencies (notably Severe combined

immunodeficiency) and trials have been carried out on other  genetic disorders.[73]

 The success of 

gene therapy so far has been limited and a patient (Jesse Gelsinger ) has died during a clinicaltrial testing a new treatment.

[74] There are also ethical concerns should the technology be used

not just for treatment, but for enhancement, modification or alteration of a human beings'

appearance, adaptability, intelligence, character or behavior .[75]

 The distinction between cure and

enhancement can also be difficult to establish.[76]

 Transhumanists consider the enhancement of humans desirable.

Human cells in which some proteins are fused with green fluorescent protein to allow them to bevisualised

Genetic engineering is an important tool for  natural scientists. Genes and other genetic

information from a wide range of organisms are transformed into bacteria for storage and

modification, creating genetically modified bacteria in the process. Bacteria are cheap, easy togrow, clonal, multiply quickly, relatively easy to transform and can be stored at -80 °C almost

indefinitely. Once a gene is isolated it can be stored inside the bacteria providing an unlimited

supply for research.

Organisms are genetically engineered to discover the functions of certain genes. This could be

the effect on the phenotype of the organism, where the gene is expressed or what other genes itinteracts with. These experiments generally involve loss of function, gain of function, trackingand expression.

  Loss of function experiments, such as in a gene knockout experiment, in which an

organism is engineered to lack the activity of one or more genes. A knockout experiment

involves the creation and manipulation of a DNA construct in vitro, which, in a simpleknockout, consists of a copy of the desired gene, which has been altered such that it is

non-functional. Embryonic stem cells incorporate the altered gene, which replaces the

already present functional copy. These stem cells are injected into  blastocysts, which are

implanted into surrogate mothers. This allows the experimenter to analyze the defectscaused by this mutation and thereby determine the role of particular genes. It is used

especially frequently in developmental biology. Another method, useful in organisms

such as Drosophila (fruit fly), is to induce mutations in a large population and then screen

the progeny for the desired mutation. A similar process can be used in both plants and prokaryotes. 

  Gain of function experiments, the logical counterpart of knockouts. These are

sometimes performed in conjunction with knockout experiments to more finely establishthe function of the desired gene. The process is much the same as that in knockout

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engineering, except that the construct is designed to increase the function of the gene,

usually by providing extra copies of the gene or inducing synthesis of the protein more

frequently.

  Tracking experiments, which seek to gain information about the localization and

interaction of the desired protein. One way to do this is to replace the wild-type gene with

a 'fusion' gene, which is a juxtaposition of the wild-type gene with a reporting elementsuch as green fluorescent protein (GFP) that will allow easy visualization of the productsof the genetic modification. While this is a useful technique, the manipulation can destroy

the function of the gene, creating secondary effects and possibly calling into question the

results of the experiment. More sophisticated techniques are now in development that cantrack protein products without mitigating their function, such as the addition of small

sequences that will serve as binding motifs to monoclonal antibodies.

  Expression studies aim to discover where and when specific proteins are produced. In

these experiments, the DNA sequence before the DNA that codes for a protein, known asa gene's  promoter , is reintroduced into an organism with the protein coding region

replaced by a reporter gene such as GFP or an enzyme that catalyzes the production of a

dye. Thus the time and place where a particular protein is produced can be observed.Expression studies can be taken a step further by altering the promoter to find which

 pieces are crucial for the proper expression of the gene and are actually bound by

transcription factor proteins; this process is known as  promoter bashing. 

Industrial

Using genetic engineering techniques one can transform microorganisms such as bacteria or yeast, or insect mammalian cells with a gene coding for a useful protein, such as an enzyme, so

that the transformed organism will overexpress the desired protein. One can manufacture mass

quantities of the protein by growing the transformed organism in  bioreactor  equipment using

techniques of  industrial fermentation, and then  purifying the protein.

[77]

 Some genes do not work well in bacteria, so yeast, insect cells, or mammalians cells, each a eukaryote, can also be

used.[78]

 These techniques are used to produce medicines such as insulin, human growth

hormone, and vaccines, supplements such as tryptophan, aid in the production of food (chymosin in cheese making) and fuels.

[79] Other applications involving genetically engineered bacteria

 being investigated involve making the bacteria perform tasks outside their natural cycle, such as

cleaning up oil spills, carbon and other toxic waste[80]

 and detecting arsenic in drinking water .[81]

 

Experimental, lab scale industrial applications

In materials science, a genetically modified virus has been used in an academic lab as a scaffold

for assembling a more environmentally friendly lithium-ion battery.

[82][83]

 

Bacteria have been engineered to function as sensors by expressing a fluorescent protein under 

certain environmental conditions.[84]

 

Agriculture

Main article:Genetically modified crops 

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 Bt-toxins present in  peanut leaves (bottom image) protect it from extensive damage caused by

European corn borer  larvae (top image).[85]

 

One of the best-known and controversial applications of genetic engineering is the creation and

use of  genetically modified crops or  genetically modified organisms, such as geneticallymodified fish, which are used to produce genetically modified food and materials with diverse

uses. There are four main goals in generating genetically modified crops.[86]

 

One goal, and the first to be realized commercially, is to provide protection from environmental

threats, such as cold (in the case of  Ice-minus bacteria), or pathogens, such as insects or viruses,

and/or resistance to herbicides. There are also fungal and virus resistant crops developed or indevelopment.

[87][88] They have been developed to make the insect and weed management of crops

easier and can indirectly increase crop yield.[89]

 

Another goal in generating GMOs, is to modify the quality of the produce, for instance,

increasing the nutritional value or providing more industrially useful qualities or quantities of the produce.[90]

 The Amflora  potato, for example, produces a more industrially useful blend of 

starches. Cows have been engineered to produce more protein in their milk to facilitate cheese production.

[91] Soybeans and canola have been genetically modified to produce more healthy

oils.[92][93]

 

Another goal consists of driving the GMO to produce materials that it does not normally make.

One example is "pharming", which uses crops as  bioreactors to produce vaccines, drug

intermediates, or drug themselves; the useful product is purified from the harvest and then usedin the standard pharmaceutical production process.

[94] Cows and goats have been engineered to

express drugs and other proteins in their milk, and in 2009 the FDA approved a drug produced in

goat milk .

[95][96]

 

Another goal in generating GMOs, is to directly improve yield by accelerating growth, or 

making the organism more hardy (for plants, by improving salt, cold or drought tolerance).[90]

 Some agriculturally important animals have been genetically modified with growth hormones to

increase their size.[97]

 

The genetic engineering of agricultural crops can increase the growth rates and resistance to

different diseases caused by  pathogens and  parasites.[98]

 This is beneficial as it can greatly

increase the production of food sources with the usage of fewer resources that would be required

to host the world's growing populations. These modified crops would also reduce the usage of chemicals, such as fertilizers and  pesticides, and therefore decrease the severity and frequency of 

the damages produced by these chemical  pollution.[98][99]

 

Ethical and safety concerns have been raised around the use of genetically modified food.[100]

 A

major safety concern relates to the human health implications of eating genetically modified

food, in particular whether toxic or allergic reactions could occur .[101]

 Gene flow into relatednon-transgenic crops, off target effects on  beneficial organisms and the impact on  biodiversity 

are important environmental issues.[102]

 Ethical concerns involve religious issues, corporate 

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control of the food supply, intellectual property rights and the level of labeling needed on

genetically modified products.

BioArt and Entertainment

Genetic engineering is also being used to create BioArt.[103]

 Some bacteria have been geneticallyengineered to create black and white photographs

[104] 

Genetic engineering has also been used to create novelty items such as lavender-coloredcarnations,

[105]  blue roses,

[106] and glowing fish.

[107][108] 

Regulation

Main article: Regulation of genetic engineering 

The regulation of genetic engineering concerns the approaches taken by governments to assess

and manage the risks associated with the development and release of genetically modified crops.There are differences in the regulation of GM crops between countries, with some of the most

marked differences occurring between the USA and Europe. Regulation varies in a given countrydepending on the intended use of the products of the genetic engineering. For example, a crop

not intended for food use is generally not reviewed by authorities responsible for food safety.

Controversy

Main articles: Genetically modified food controversies and Human genetic engineering 

Critics have objected to use of genetic engineering per se on several grounds, including ethicalconcerns, ecological concerns, and economic concerns raised by the fact GM techniques and GM

organisms are subject to intellectual property law. GMOs also are involved in controversies over GM food with respect to whether food produced from GM crops is safe, whether it should be

labeled, and whether GM crops are needed to address the world's food needs. See the genetically

modified food controversies article for discussion of issues about GM crops and GM food. Thesecontroversies have led to litigation, international trade disputes, and protests, and to restrictive

regulation of commercial products in most countries.