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[email protected] Cloning Vectors

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Cloning vector

A cloning vectoris a small piece ofDNA,taken from avirus,aplasmid,or thecellof a higher organism,

that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted

forcloningpurposes. The vector therefore contains features that allow for the convenient insertion orremoval of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA

with arestriction enzymethat creates the same overhang, thenligatingthe fragments together. After a

DNA fragment has been cloned into a cloning vector, it may be furthersub clonedinto another vector

designed for more specific use.

There are many types of cloning vectors, but the most commonly-used ones are genetically

engineeredplasmids.Cloning is generally first performed usingEscherichia coli,and cloning vectors in E.

coli include plasmids,bacteriophages (such asphage ),Cosmids, and bacterial artificial

chromosomes(BACs). Some DNA however cannot be stably maintained in E. coli, for example very large

DNA fragment, and other organisms such as yeast may be used. Cloning vectors in yeast includeyeast

artificial chromosomes(YACs).

Features of a cloning vector

All commonly-used cloning vectors inmolecular biologyhave key features necessary for their function

such as a suitable cloning site and selectable marker. Others may have additional features specific to

their use. For reason of ease and convenience, cloning is often performed using E. coli, the cloning

vectors used therefore often have elements necessary for their propagation and maintenance in E.

coli such as a functionalorigin of replication(ori). TheColE1origin of replication is found in many

plasmids. Some vectors also include elements that allow them to be maintained in another organism in

addition to E. coli, and these vectors are calledshuttle vector.

Cloning site

All cloning vectors have features that allow a gene to be conveniently inserted into the vector or removed

from it. This may be amultiple cloning site(MCS) which contains many uniquerestriction sites. The

restriction sites in the MCS are first cleaved by restriction enzymes, and aPCR-amplified target gene,

also digested with the same enzymes, is then ligated into the vectors usingDNA ligase.The target DNA

sequence can be inserted into the vector in a specific direction if so desired. The restriction sites may be

further used forsub-cloninginto another vector if necessary.

Other cloning vectors may usetopo isomeraseinstead of ligase and cloning may be done more rapidly

without the need for restriction digest of the vector or insert. In thisTOPO cloningmethod a linearized

vector is activated by attaching topo isomerase I to its ends, and this "TOPO-activated" vector may then

accept a PCR product by ligating both the 5' ends of the PCR product, releasing the topo isomerase and
http://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Virushttp://en.wikipedia.org/wiki/Virushttp://en.wikipedia.org/wiki/Virushttp://en.wikipedia.org/wiki/Plasmidhttp://en.wikipedia.org/wiki/Plasmidhttp://en.wikipedia.org/wiki/Plasmidhttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Molecular_cloninghttp://en.wikipedia.org/wiki/Molecular_cloninghttp://en.wikipedia.org/wiki/Molecular_cloninghttp://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/DNA_ligasehttp://en.wikipedia.org/wiki/DNA_ligasehttp://en.wikipedia.org/wiki/DNA_ligasehttp://en.wikipedia.org/wiki/Subclonedhttp://en.wikipedia.org/wiki/Subclonedhttp://en.wikipedia.org/wiki/Subclonedhttp://en.wikipedia.org/wiki/Plasmidhttp://en.wikipedia.org/wiki/Plasmidhttp://en.wikipedia.org/wiki/Plasmidhttp://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Bacteriophagehttp://en.wikipedia.org/wiki/Bacteriophagehttp://en.wikipedia.org/wiki/Bacteriophagehttp://en.wikipedia.org/wiki/Lambda_phagehttp://en.wikipedia.org/wiki/Lambda_phagehttp://en.wikipedia.org/wiki/Lambda_phagehttp://en.wikipedia.org/wiki/Cosmidshttp://en.wikipedia.org/wiki/Cosmidshttp://en.wikipedia.org/wiki/Cosmidshttp://en.wikipedia.org/wiki/Bacterial_artificial_chromosomehttp://en.wikipedia.org/wiki/Bacterial_artificial_chromosomehttp://en.wikipedia.org/wiki/Bacterial_artificial_chromosomehttp://en.wikipedia.org/wiki/Yeast_artificial_chromosomehttp://en.wikipedia.org/wiki/Yeast_artificial_chromosomehttp://en.wikipedia.org/wiki/Yeast_artificial_chromosomehttp://en.wikipedia.org/wiki/Yeast_artificial_chromosomehttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/E._colihttp://en.wikipedia.org/wiki/E._colihttp://en.wikipedia.org/wiki/E._colihttp://en.wikipedia.org/wiki/Origin_of_replicationhttp://en.wikipedia.org/wiki/Origin_of_replicationhttp://en.wikipedia.org/wiki/Origin_of_replicationhttp://en.wikipedia.org/wiki/ColE1http://en.wikipedia.org/wiki/ColE1http://en.wikipedia.org/wiki/ColE1http://en.wikipedia.org/wiki/Shuttle_vectorhttp://en.wikipedia.org/wiki/Shuttle_vectorhttp://en.wikipedia.org/wiki/Shuttle_vectorhttp://en.wikipedia.org/wiki/Multiple_cloning_sitehttp://en.wikipedia.org/wiki/Multiple_cloning_sitehttp://en.wikipedia.org/wiki/Multiple_cloning_sitehttp://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/PCRhttp://en.wikipedia.org/wiki/PCRhttp://en.wikipedia.org/wiki/PCRhttp://en.wikipedia.org/wiki/DNA_ligasehttp://en.wikipedia.org/wiki/DNA_ligasehttp://en.wikipedia.org/wiki/DNA_ligasehttp://en.wikipedia.org/wiki/Sub-cloninghttp://en.wikipedia.org/wiki/Sub-cloninghttp://en.wikipedia.org/wiki/Sub-cloninghttp://en.wikipedia.org/wiki/Topoisomerasehttp://en.wikipedia.org/wiki/Topoisomerasehttp://en.wikipedia.org/wiki/Topoisomerasehttp://en.wikipedia.org/wiki/TOPO_cloninghttp://en.wikipedia.org/wiki/TOPO_cloninghttp://en.wikipedia.org/wiki/TOPO_cloninghttp://en.wikipedia.org/wiki/TOPO_cloninghttp://en.wikipedia.org/wiki/Topoisomerasehttp://en.wikipedia.org/wiki/Sub-cloninghttp://en.wikipedia.org/wiki/DNA_ligasehttp://en.wikipedia.org/wiki/PCRhttp://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/Multiple_cloning_sitehttp://en.wikipedia.org/wiki/Shuttle_vectorhttp://en.wikipedia.org/wiki/ColE1http://en.wikipedia.org/wiki/Origin_of_replicationhttp://en.wikipedia.org/wiki/E._colihttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Yeast_artificial_chromosomehttp://en.wikipedia.org/wiki/Yeast_artificial_chromosomehttp://en.wikipedia.org/wiki/Bacterial_artificial_chromosomehttp://en.wikipedia.org/wiki/Bacterial_artificial_chromosomehttp://en.wikipedia.org/wiki/Cosmidshttp://en.wikipedia.org/wiki/Lambda_phagehttp://en.wikipedia.org/wiki/Bacteriophagehttp://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Plasmidhttp://en.wikipedia.org/wiki/Subclonedhttp://en.wikipedia.org/wiki/DNA_ligasehttp://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/Molecular_cloninghttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Plasmidhttp://en.wikipedia.org/wiki/Virushttp://en.wikipedia.org/wiki/DNA


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forming a circular vector in the process. Another method of cloning without the use of ligase is byDNA

recombination, for example as used in theGateway cloning system, The gene, once cloned into the

cloning vector (called entry clone in this method), may be conveniently introduced into a variety of

expression vectors by recombination.

Selectable marker

Aselectable markeris carried by the vector to allow the selection of

positivelytransformedcells.Antibioticresistance is often used as marker, an example is thebeta-

lactamasegene which confers resistance to thepenicillingroup ofbeta-lactam antibioticslikeampicillin.

Some vectors contain two selectable markers, for example the plasmid pACYC177 has both ampicillin

andkanamycinresistance gene. Shuttle vector which is designed to be maintained in two different

organisms may also require two selectable markers, although some selectable markers such as

resistance tozeocinandhygromycin Bare effective in different cell types.Auxotrophicselection markers

that allow an auxotrophic organism to grow inminimal growth mediummay also be used; examples of

these areLEU2andURA3which are used with their corresponding auxotrophic strains of yeast.

Reporter gene

In order to facilitate the screening of successful clone, some cloning vectors contain features that allow

successful clone to be identified. Such features present in cloning vectors may be the lacZ fragmentfor

complementation inblue-white selection, and/ormarker geneorreporter genesin frame with and

flanking theMCSto facilitate the production offusion proteins.Examples of fusion partners that may be

used for screening are thegreen fluorescent protein(GFP) andluciferase.

Elements for expression

A cloning vector need not contain suitable elements for theexpressionof a cloned target gene, many

however do, and may then work as anexpression vector.The targetDNAmay be inserted into a site that

is under the control of a particularpromoternecessary for the expression of the target gene in the chosen

host. Where the promoter is present, the expression of the gene is preferably tightly controlled

andinducibleso that proteins are only produced when required. Some commonly used promoters areT7

promoters,lacpromoters. The presence of a promoter is necessary when screening techniques such

asblue-white selectionare used.

Cloning vectors without promoter andribosomal binding site(RBS) for the cloned DNA sequence are

sometimes used, for example when cloning genes whose products are toxic toE. colicells. Promoter and

RBS for the cloned DNA sequence are also unnecessary when first making a genomicorcDNA libraryof

clones since the cloned genes are normally sub cloned into a more appropriate expression vector if their

expression is required.
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Types of cloning vectors

A large number of cloning vectors are available, and choosing the vector may depend a number of

factors, such as the size of the insert, copy number and cloning method. Large insert may not be stably

maintained in a general cloning vector, especially for those with a high copy number, therefore cloninglarge fragments may require more specialized cloning vector.

The pUC plasmid has a high copy number, contains a multiple cloning site, a gene for ampicillin antibiotic

selection, and can be used for blue-white screen.

Plasmid

Plasmids are the standard cloning vectors and the most commonly used. Most general plasmids may be

used to clone DNA insert of up to 15 kb in size. One of the earliest commonly used cloning vectors is

thepBR322plasmid. Other cloning vectors include thepUCseries of plasmids, and a large number of

different cloning plasmid vectors are available. Many plasmids have high copy number, for

examplepUC19which has a copy number of 500-700 copies per cell, and high copy number is useful as it

produces greater yield of recombinant plasmid for subsequent manipulation. However low-copy-number

plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned

gene is toxic to the cells.

Some plasmids contain anM13 bacteriophageorigin of replication and may be used to generate single-

stranded DNA. These are calledphagemid,and examples are thepBluescriptseries of cloning vectors.

Bacteriophage

The bacteriophage used for cloning are thephage andM13 phage. There is an upper limit on the

amount of DNA that can be packed into a phage (a maximum of 53 kb), therefore to allow foreign DNA to

be inserted into phage DNA, phage cloning vectors need to have some non-essential genes deleted, for
http://en.wikipedia.org/wiki/PBR322http://en.wikipedia.org/wiki/PBR322http://en.wikipedia.org/wiki/PBR322http://en.wikipedia.org/wiki/PUC19http://en.wikipedia.org/wiki/PUC19http://en.wikipedia.org/wiki/PUC19http://en.wikipedia.org/wiki/PUC19http://en.wikipedia.org/wiki/PUC19http://en.wikipedia.org/wiki/PUC19http://en.wikipedia.org/wiki/M13_bacteriophagehttp://en.wikipedia.org/wiki/M13_bacteriophagehttp://en.wikipedia.org/wiki/M13_bacteriophagehttp://en.wikipedia.org/wiki/Phagemidhttp://en.wikipedia.org/wiki/Phagemidhttp://en.wikipedia.org/wiki/Phagemidhttp://en.wikipedia.org/wiki/PBluescripthttp://en.wikipedia.org/wiki/PBluescripthttp://en.wikipedia.org/wiki/PBluescripthttp://en.wikipedia.org/wiki/Bacteriophage_lambdahttp://en.wikipedia.org/wiki/Bacteriophage_lambdahttp://en.wikipedia.org/wiki/Bacteriophage_lambdahttp://en.wikipedia.org/wiki/M13_phagehttp://en.wikipedia.org/wiki/M13_phagehttp://en.wikipedia.org/wiki/M13_phagehttp://en.wikipedia.org/wiki/File:PUC19.svghttp://en.wikipedia.org/wiki/M13_phagehttp://en.wikipedia.org/wiki/Bacteriophage_lambdahttp://en.wikipedia.org/wiki/PBluescripthttp://en.wikipedia.org/wiki/Phagemidhttp://en.wikipedia.org/wiki/M13_bacteriophagehttp://en.wikipedia.org/wiki/PUC19http://en.wikipedia.org/wiki/PUC19http://en.wikipedia.org/wiki/PBR322


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example the genes forlysogenyin phage .There are two kinds of phage vectors - insertion vector and

replacement vector. Insertion vectors contain a unique cleavage site whereby foreign DNA with size of 5

11 kb may be inserted. In replacement vectors, the cleavage sites flank a region containing genes not

essential for the lytic cycle, and this region may be deleted and replaced by the DNA insert in the cloning

process, and a larger sized DNA of 824 kb may be inserted.

There is also a lower size limit for DNA that can be packed into a phage, and vectors that are too small

cannot be properly packaged into the phage. This property can be used for selection - vector without

insert may be too small, therefore only vectors with insert may be selected for propagation.

Cosmid

Cosmidsare plasmids that incorporate a segment of bacteriophage DNA that has the cohesive end site

(cos) which contains elements required for packaging DNA into particles. It is normally used to clone

large DNA fragments between 28 to 45 Kb.

Bacterial artificial chromosome

Insert size of up to 350 kb can be cloned inbacterial artificial chromosome(BAC). BACs are maintained

in E. coli with a copy number of only 1 per cell. BACS are based onF plasmid, another artificial

chromosome called thePACis based on theP1 phage.

Yeast artificial chromosome

Insert of up to 3,000 kb may be carried byyeast artificial chromosome.

Human artificial chromosome

Human artificial chromosome may be potentially useful as a gene transfer vectors for gene delivery into

human cells, and a tool for expression studies and determining human chromosome function. It can carry

very large DNA fragment (there is no upper limit on size for practical purposes), therefore it does not have

the problem of limited cloning capacity of other vectors, and it also avoids possible insertional

mutagenesis caused by integration into host chromosomes by viral vector.

Screening: example of the blue/white screen

Many general purpose vectors such aspUC19usually include a system for detecting the presence of a

cloned DNA fragment, based on the loss of an easily scored phenotype. The most widely used is the

gene coding for E. coli-galactosidase,whose activity can easily be detected by the ability of the enzyme

it encodes to hydrolyze the soluble, colour less substrateX-gal(5-bromo-4-chloro-3-indolyl-beta-d-

galactoside) into an insoluble, blue product (5,5'-dibromo-4,4'-dichloro indigo). Cloning a fragment of DNA

within the vector-based lacZ sequence of the -galactosidase prevents the production of an active

enzyme. If X-gal is included in the selective agar plates, transformant colonies are generally blue in the
http://en.wikipedia.org/wiki/Lysogenyhttp://en.wikipedia.org/wiki/Lysogenyhttp://en.wikipedia.org/wiki/Lysogenyhttp://en.wikipedia.org/wiki/Cosmidshttp://en.wikipedia.org/wiki/Cosmidshttp://en.wikipedia.org/wiki/Bacterial_artificial_chromosomehttp://en.wikipedia.org/wiki/Bacterial_artificial_chromosomehttp://en.wikipedia.org/wiki/Bacterial_artificial_chromosomehttp://en.wikipedia.org/wiki/Fertility_factorhttp://en.wikipedia.org/wiki/Fertility_factorhttp://en.wikipedia.org/wiki/Fertility_factorhttp://en.wikipedia.org/wiki/P1-derived_artificial_chromosomehttp://en.wikipedia.org/wiki/P1-derived_artificial_chromosomehttp://en.wikipedia.org/wiki/P1-derived_artificial_chromosomehttp://en.wikipedia.org/wiki/P1_phagehttp://en.wikipedia.org/wiki/P1_phagehttp://en.wikipedia.org/wiki/P1_phagehttp://en.wikipedia.org/wiki/Yeast_artificial_chromosomehttp://en.wikipedia.org/wiki/Yeast_artificial_chromosomehttp://en.wikipedia.org/wiki/Yeast_artificial_chromosomehttp://en.wikipedia.org/wiki/PUC19http://en.wikipedia.org/wiki/PUC19http://en.wikipedia.org/wiki/PUC19http://en.wikipedia.org/wiki/Beta-galactosidasehttp://en.wikipedia.org/wiki/Beta-galactosidasehttp://en.wikipedia.org/wiki/Beta-galactosidasehttp://en.wikipedia.org/wiki/Beta-galactosidasehttp://en.wikipedia.org/wiki/X-galhttp://en.wikipedia.org/wiki/X-galhttp://en.wikipedia.org/wiki/X-galhttp://en.wikipedia.org/wiki/X-galhttp://en.wikipedia.org/wiki/Beta-galactosidasehttp://en.wikipedia.org/wiki/PUC19http://en.wikipedia.org/wiki/Yeast_artificial_chromosomehttp://en.wikipedia.org/wiki/P1_phagehttp://en.wikipedia.org/wiki/P1-derived_artificial_chromosomehttp://en.wikipedia.org/wiki/Fertility_factorhttp://en.wikipedia.org/wiki/Bacterial_artificial_chromosomehttp://en.wikipedia.org/wiki/Cosmidshttp://en.wikipedia.org/wiki/Lysogeny


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case of a vector with no inserted DNA and white in the case of a vector containing a fragment of cloned

DNA.

SV40

SV40 is an abbreviation for Simian vacuolating virus 40or Simian virus 40, apolyomavirusthat is

found in bothmonkeysand humans. Like other polyomaviruses, SV40 is aDNA virusthat has the

potential to causetumors,but most often persists as a latent infection.

SV40 became a highly controversial subject after it was revealed that millions were exposed to the virus

after receiving a contaminated polio vaccine.

History

The virus was first identified byBernice E. Eddy(based on a work of her andSarah

StewartaboutPolyoma viruses) in 1960 in cultures ofrhesus monkeykidneycellsthat were being used

to producepolio vaccineIt was named for the effect it produced on infectedgreen monkeycells, which

developed an unusual number ofvacuoles. This observation was repeated and confirmed by Hilleman

and Sweet who were employed by Merck in their vaccine division. The complete viral genomewas

sequenced byWalter Fiersand his team at theUniversity of Ghent(Belgium) in 1978.[ The virus is

dormant and is asymptomatic in Rhesus monkeys. The virus has been found in

manymacaquepopulations in the wild, where it rarely causes disease. However, in monkeys that

areimmunodeficientdue to, for example, infection withSimian immunodeficiency virusSV40 acts

much like the humanJCandBKpolyomaviruses, producingkidneydisease and sometimes

ademyelinating diseasesimilar toPML.In other species, particularlyhamsters,SV40 causes a variety of

tumors, generallysarcomas. In rats, the oncogenicSV40 Large T-antigenwas used to establish a brain

tumor model forPNETsandmedulloblastomas.

The molecular mechanisms by which the virus reproduces and alters cell function were previously

unknown, and research into SV40 vastly increased biologists' understanding ofgene expressionand the

regulation of cell growth.

Virology

SV40 consists of an unenveloped icosahedral virion with a closed circular dsDNA genome of 5kb. The

virion adheres to cell surface receptors of MHC class 1 by the virion glycoprotein VP1. Penetration into

the cell is through a caveolin vesicle. Inside the cell nucleus, the cellular RNA polymerase II acts to
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rg/wiki/Polio_vaccinehttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Kidneyhttp://en.wikipedia.org/wiki/Rhesus_Macaquehttp://en.wikipedia.org/wiki/Polyomahttp://en.wikipedia.org/wiki/Sarah_Stewart_(cancer_researcher)http://en.wikipedia.org/wiki/Sarah_Stewart_(cancer_researcher)http://en.wikipedia.org/wiki/Bernice_Eddyhttp://en.wikipedia.org/wiki/Tumorhttp://en.wikipedia.org/wiki/DNA_virushttp://en.wikipedia.org/wiki/Humanhttp://en.wikipedia.org/wiki/Monkeyhttp://en.wikipedia.org/wiki/Polyomavirus


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promote early gene expression. This results in an mRNA that is spliced into two segments. The small and

large T antigens result from this. The large T antigen has two functions: 5% will go to the plasma

membrane of the cell and 95% will return to the nucleus. Once in the nucleus the large T antigen binds

three viral DNA sites, I, II, and III. Binding of sites I and II autoregulates early RNA synthesis. Binding to

site II takes place in each cell cycle. Binding site I initiates DNA replication at theorigin of replication.

Early transcription gives two spliced RNAs that are both 19s. Late transcription gives both a longer 16s,

which synthesizes the major viral capsid protein VP1; and the smaller 19s, which gives VP2, and VP3

throughleaky scanning. All of the proteins, besides the 5% of large T, return to the nucleus because

assembly of the viral particle happens in the nucleus. Eventual release of the viral particles is cytolytic

and results in cell death.

Multiplicity Reactivation

SV40 is capable of multiplicity reactivation (MR). MR is the process by which two, or more, virus genomes

containing otherwise lethal damage interact within an infected cell to form a viable virus genome.

Yamamato and Shimojo observed MR when SV40 virions were irradiated with UV light and allowed to

undergo multiple infection of host cells. Hall studied MR when SV 40 virions were exposed to the DNA

crosslinking agent 4, 5, 8-trimethylpsoralen.Under conditions where only a single virus particle entered

each host cell, approximately one DNA cross-link was lethal to the virus, and could not be repaired. In

contrast, when multiple viral genomes infected a host cell, psoralen induced DNA cross-links were

repaired; that is, MR occurred. Hall suggested that the virions with cross-linked DNA were repaired by

recombinational repair. Michod et al. reviewed numerous examples of MR in different viruses, and

suggested that MR is a common form of sexual interaction that provides the advantage of

recombinational repair of genome damages.

Transcription

The early promoter for SV40 contains three elements. TheTATA boxis located approximately 20 base-

pairs upstream from the transcriptional start site. The 21 base-pair repeats contain six GC boxes and are

the site that determines the direction of transcription. Also, the 72 base-pair repeats are transcriptional

enhancers. When the SP1 protein interacts with the 21 bp repeats it binds either the first or the last three

GC boxes. Binding of the first three initiates early expression and binding of the last three initiates late

expression. The function of the 72 bp repeats is to enhance the amount of stable RNA and increase the

rate of synthesis. This is done by binding (dimerization) with the AP1 (activator protein 1) to give a

primary transcript that is 3' polyadenylated and 5' capped.

Theorized role in human disease
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The hypothesis that SV40 might cause cancer in humans has been a particularly controversial area of

research. Several different methods have been used to detect SV40 in a variety of human cancers,

although how reliable these detection methods are, and whether SV40 has any role in causing these

tumors, remains unclear. As a result of these uncertainties, academic opinion remains divided, with some

arguing that this hypothesis is not supported by the data, and others arguing that some cancers may

involve SV40. However, theUnited StatesNational Cancer Instituteannounced in 2004 that although

SV40 does cause cancer in someanimal models,"substantial epidemiological evidence has accumulated

to indicate that SV40 likely does not cause cancer in humans" This announcement is based on two recent

studies.

p53 Damage and carcinogenicity

SV40 is believed to suppress the transcriptional properties of the tumor-suppressingp53in humans

through theSV40 Large T-antigenandSV40 Small T-antigen.p53 is responsible for initiating regulated

cell death ("apoptosis"), or cell cycle arrest when a cell is damaged. A mutated p53 gene may contribute

to uncontrolled cellular proliferation, leading to atumor.

SV40 may act as aco-carcinogenwithcrocidoliteasbestos to cause bothPeritonealandPleural

Mesothelioma

When SV40 infects nonpermissive cells, such as 3T3 mouse cells, the dsDNA of SV40 becomes

covalently integrated. In nonpermissive cells only the early gene expression occurs and this leads to

transformation, or oncogenesis. The nonpermissive host needs the Large T-antigen and the Small t-

antigen in order to function. The Small T-antigen interacts with and integrates with the cellularphosphatase pp2A. This causes the cell to lose the ability to initiate transcription.

Polio vaccine contamination

Soon after its discovery, SV40 was identified in the oral form of thepolio vaccineproduced between 1955

and 1961 produced by American Home Products (dba Lederle). This is believed to be due to two sources:

1) SV40 contamination of the original seed strain (coded SOM); 2) contamination of the substrate -

primary kidney cells from infected monkeys used to grow the vaccine virus during production. Both

theSabinvaccine (oral, live virus) and theSalkvaccine (injectable, killed virus) were affected; the

technique used to inactivate the polio virus in the Salk vaccine, by means offormaldehyde, did not

reliably kill SV40.

It was difficult to detect small quantities of virus until the advent ofPCR;since then, stored samples of

vaccine made after 1962 have tested negative for SV40, but no samples prior to 1962 could initially be

found. Then, in 1997,Herbert RatnerofOak Park, Illinois, gave some vials of 1954 Salk vaccine to
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researcher Michele Carbone. Ratner, the Health Commissioner of Oak Park at the time the Salk vaccine

was introduced, had kept these vials of vaccine in a refrigerator for over forty years.

Upon testing this vaccine, Carbone discovered that it contained not only the SV40 strain already known to

have been in the Salk vaccine (containing two 72-bp enhancers) but also the same slow-growing SV40

strain currently being found in some malignant tumors and lymphomas (containing one 72-bp

enhancers). It is unknown how widespread the virus was among humans before the 1950s, though one

study found that 12% of a sample of German medical students in 1952 had SV40antibodies.

Althoughhorizontal transmissionbetween people has been proposed,[ it is not clear if this actually

happens and if it does, how frequently it occurs.

An analysis presented at the Vaccine Cell Substrate Conference in 2004[24]

suggested that vaccines used

in the formerSoviet bloccountries, China, Japan, and Africa, could have been contaminated up to 1980,

meaning that hundreds of millions more could have been exposed to the virus unknowingly.

Baculo viral vectors:

Baculoviruses a diverse group of insect viruses. Autograpaha californica is a multinuclear polyhedral virus(ACMNPV), and Bombax mori polyhedron viruses (BMNPV) are just two such examples.Genome size of these viruses is about 128 to 200 kbp (ACMNPV =128 KBP) AND IN CIRCULARMODE. Insect cell lines used for the expression of viral genomes are Spodofora fugiperda (Sf-9 celllines). On infection viruses multiply within epithelial cells of gut.Viral genes are expressed in temporal fashion, such as early, late and very late.

Early gene products initiate its DNA replication and also activate the expression of late genes. Thisresults in the multiplication of the genome as well as viruses, which in turn are budded off as envelopedviruses (Env), which further infect new set of cells.Expression of very late genes is about 18 to 20 hrs after infection, which code for polyhydrin proteins. Atthis stage the host cell nuclear membrane proliferates and primary viruses are occluded among thepolyhydrin matrix proteins (29KD).In order to use them for expression foreign genes first clone polyhydrin gene along with flanking regionsinto a plasmid for recombination purposes.
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~~~--Col.E1oriI-V-DNA-I----polyhydrin----I-V-DNA-I---Amp^+--~~~

~~~ = End of a circular plasmid.V-DNA = Viral DNA as flanking regions.

Then delete the polyhydrin gene and in its place put a desired gene under a proper polyhydrinpromoter. Then co-infect insect lines with plasmid DNA and the viral DNA. In insect cells homologousrecombination results in the incorporation of the desired gene. The viral DNA replicates to 800-1000copies per cell and later generates primary virions, which can further infect.Mouse IgG against Pseudomonas aeuriginosa lipoprotein was expressed in Baculo viralrecombinants. In this case Transfer vector was constructed in such a way both light and heavy chaingenes are cloned separately under specific polyhydrin promoters with flanking DNA from the virus. Whenwild type viral DNA and the transfer DNA are co infected into insect cell, homologous recombinationresults in the integration of both IgG genes under promoters which are expressed in insect cells. In orderto scale up the production one can directly infect larvae instead if cells.For example recombinant Trichplasi larvae containing human Adenosine deaminase gene, found theexpressed protein level was found to be 3-5% of the cellular proteins. For continuous production the

desired gene has to be to be constructed under the early gene promoter for they dont require any viralgene products for expression. It is also possible to integrate the gene construct into host cells andexpress continuously to get more of the recombinant protein. Advantage of using Baculo viral viruses isone can clone a DNA fragme4nt of the size of 15-20 kbp long. Direct injecting modified virus can achieveinfection and growth. Proteins expressed in insect are found to have all characteristic post-translationalmodification, but in some case glycosylation is same as that of mammalian system.


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pAC UW31 transfer Vector:

Derived from Baculo viral genome.

~~-M13 oriAmp^+->---ColE1 ori----ACNPV 2099SV40P-X


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In order to develop a live recombinant virus first one has to transfer viral capsid genes into specific hoistcells, where the genes are inserted in expression mode but regulatable. Such cells can be maintainedand expanded; such cells are called helper cell lines.Such cells are transfected with above-mentioned viral construct. Then the cells are stimulated to producecapsid proteins. As the vector DNA has sequences for packaging, the capsid proteins bind to suchsequences and complete packaging leads to full viral particle production. If such cell line also havespecific envelop protein genes, then the envelope proteins that incorporate into the viral envelop, whichcan targeted to specific type of tissue.When the tissue is infected with the recombinant viruses, the released DNA goes into the nucleus whereit gets integrated into the chromosomal DNA using LTR sequences and rest of the DNA gets degrades.Application of this method is very important for one can deliver the viruses into specific tissues, withoutcausing any adverse effects. Any constructs with LTR sequences can also used directly transfer theconstruct into the cells by direct transfer by any one of the Transfection methods. The transfected DNAgets integrated into the host genome using LTR sequences. It is greatly facilitated if the cells aremitotically active. This method has been employed in gene therapy.

-U3-R-U5-I~~-P---X-I-I-geneTtrI---PKan+ --TtrU3-R-U5-

U3RU5 = LTR sequences,~~ = Packaging sequences

II = Introns.Ttr = transcriptional terminator.


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Expression vector

An expression vector, otherwise known as an expression construct, is usually aplasmidor virus

designed forprotein expressionin cells. Thevectoris used to introduce a specificgeneinto a target cell,

and can commandeer the cell's mechanism forprotein synthesisto produce theproteinencodedby the

gene. The plasmid is engineered to contain regulatory sequences that act

asenhancerandpromoterregions and lead to efficient transcription of the gene carried on the expression

vector. The goal of a well-designed expression vector is the production of significant amount of

stablemessenger RNA,and therefore proteins. Expression vectors are basic tools forbiotechnologyand

the production of proteins, for exampleinsulinwhich is important for medical treatments ofdiabetes.

Elements of expression vectors
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An expression vector has features that anyvectormay have, such as anorigin of replication,aselectable

marker,and a suitable site for the insertion of a gene such as themultiple cloning site.The cloned gene

may be transferred from a specialized cloning vectorsto an expression vector, although it is possible to

clone directly into an expression vector. The cloning process is normally performed inEscherichia coli,

and vectors used for protein expression in organisms other than E.coli may have, in addition to a suitable

origin of replication for its propagation in E. coli, elements that allow them to be maintained in another

organism, and these vectors are calledshuttle vector.

Elements for expression

An expression vectors must have elements necessary for protein expression. These include a strong

promoter, the correct translation initiation sequence such as aribosomal binding siteandstart codon,a

strongtermination codon, and atranscription termination sequence. There are differences in the

machinery for protein synthesis between prokaryotes and eukaryotes, therefore the expression vectors

must have the elements for expression that is appropriate for the chosen host. For example, prokaryotes

expression vectors would have aShine-Dalgarno sequenceat its translation initiation site for the binding

of ribosomes, while eukaryotes expression vectors would contain theKozak consensus sequence.

Thepromoterinitiates thetranscriptionand is therefore the point of control for the expression of the

cloned gene. The promoters used in expression vector are normallyinducible, meaning that protein

synthesis is only initiated when required by the introduction of aninducersuch asIPTG. Protein

expression however may also be constitutive (i.e. protein is constantly expressed) in some expression

vectors. Low level of constitutive protein synthesis may occur even in expression vectors with tightly-

controlled promoters.

Protein tags

After the expression of the gene product, it is usually necessary to purify the expressed protein. However,

separating the protein of interest from the great majority of proteins of the host cell can be a protracted

process. To make this purification process easier, apurification tagmay be added to the cloned gene.

This tag could behistidine (His) tag,other marker peptides, or afusion partnerssuch asglutathione S-

transferaseormaltose-binding protein. Other fusion proteins such asgreen fluorescent proteinmay act

as areporter gene.

Others

The expression vector istranformedortransfectedinto the host cell for protein synthesis. Some

expression vectors may have element for transformation or the insertion of DNA into the host

chromosome, for example thevir genesfor plant transformation, andintegrasesites for chromosomal

insertion.
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ikipedia.org/wiki/Kozak_consensus_sequencehttp://en.wikipedia.org/wiki/Shine-Dalgarno_sequencehttp://en.wikipedia.org/wiki/Terminator_(genetics)http://en.wikipedia.org/wiki/Termination_codonhttp://en.wikipedia.org/wiki/Start_codonhttp://en.wikipedia.org/wiki/Ribosomal_binding_sitehttp://en.wikipedia.org/wiki/Shuttle_vectorhttp://en.wikipedia.org/wiki/Escherichia_coli_(molecular_biology)http://en.wikipedia.org/wiki/Cloning_vectorshttp://en.wikipedia.org/wiki/Multiple_cloning_sitehttp://en.wikipedia.org/wiki/Selectable_markerhttp://en.wikipedia.org/wiki/Selectable_markerhttp://en.wikipedia.org/wiki/Origin_of_replicationhttp://en.wikipedia.org/wiki/Vector_(molecular_biology)


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Some vectors may include targeting sequence that may target the expressed protein to a specific location

such as theperiplasmic spaceof bacteria.

Expression systems

Different organism may be used to express a target protein, the expression vector used therefore will

have elements specific for use in the particular organism. The most commonly-used organism forprotein

expressionis the bacteriumEscherichia coli.However not all proteins can be successfully expressed in E.

coli, and other systems may therefore be used.

Bacterial

An example of a bacterial expression vector is the pGEX-3x plasmid

The expression host of choice for the expression of many proteins is Escherichia coli as the production of

heterologous protein in E. coliis relatively simple and convenient, as well as being rapid and cheap. A

large number of E. coli expression plasmids are also available suitable for a wide variety of needs. Other

bacteria used for protein expression includeBacillus subtilis.

Most proteins are expressed in the cytoplasm of E. coli, but where necessarily, for example when the

protein can only fold correctly in an oxidizing environment due to the presence ofdisulphide bonds, the

protein may be targeted to theperiplasmic spaceby the use of an N-terminalsignal sequence. Other

more sophisticated systems are being developed; such systems may allow for the expression of proteins

previously thought impossible in E. coli, such asglycosylatedproteins.

The promoters used for these vector are usually based on the promoter of the lac operonor

theT7promoter, and they are normally regulated by theoperator.These promoters may also be hybrids

of different promoters, for example, the tac promoter is a hybrid oftrpand lac promoters. Note that most

commonly-used lac or lac-derived promoters are based on the lacUV5 mutant which is insensitive to

catabolite repression.This mutant allows for expression of protein under the control of the lac promoter
http://en.wikipedia.org/wiki/Periplasmic_spacehttp://en.wikipedia.org/wiki/Periplasmic_spacehttp://en.wikipedia.org/wiki/Periplasmic_spacehttp://en.wikipedia.org/wiki/Protein_expression_(biotechnology)http://en.wikipedia.org/wiki/Protein_expression_(biotechnology)http://en.wikipedia.org/wiki/Protein_expression_(biotechnology)http://en.wikipedia.org/wiki/Protein_expression_(biotechnology)http://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Bacillus_subtilishttp://en.wikipedia.org/wiki/Bacillus_subtilishttp://en.wikipedia.org/wiki/Bacillus_subtilishttp://en.wikipedia.org/wiki/Disulphide_bondshttp://en.wikipedia.org/wiki/Disulphide_bondshttp://en.wikipedia.org/wiki/Disulphide_bondshttp://en.wikipedia.org/wiki/Periplasmic_spacehttp://en.wikipedia.org/wiki/Periplasmic_spacehttp://en.wikipedia.org/wiki/Periplasmic_spacehttp://en.wikipedia.org/wiki/Signal_peptidehttp://en.wikipedia.org/wiki/Signal_peptidehttp://en.wikipedia.org/wiki/Signal_peptidehttp://en.wikipedia.org/wiki/Glycosylatedhttp://en.wikipedia.org/wiki/Glycosylatedhttp://en.wikipedia.org/wiki/Glycosylatedhttp://en.wikipedia.org/wiki/Lac_operonhttp://en.wikipedia.org/wiki/Lac_operonhttp://en.wikipedia.org/wiki/Lac_operonhttp://en.wikipedia.org/wiki/T7_phagehttp://en.wikipedia.org/wiki/T7_phagehttp://en.wikipedia.org/wiki/T7_phagehttp://en.wikipedia.org/wiki/Operator_(biology)http://en.wikipedia.org/wiki/Operator_(biology)http://en.wikipedia.org/wiki/Operator_(biology)http://en.wikipedia.org/wiki/Trp_operonhttp://en.wikipedia.org/wiki/Trp_operonhttp://en.wikipedia.org/wiki/Catabolite_repressionhttp://en.wikipedia.org/wiki/Catabolite_repressionhttp://en.wikipedia.org/wiki/File:PGEX-3X_cloning_vector.pnghttp://en.wikipedia.org/wiki/File:PGEX-3X_cloning_vector.pnghttp://en.wikipedia.org/wiki/File:PGEX-3X_cloning_vector.pnghttp://en.wikipedia.org/wiki/File:PGEX-3X_cloning_vector.pnghttp://en.wikipedia.org/wiki/Catabolite_repressionhttp://en.wikipedia.org/wiki/Trp_operonhttp://en.wikipedia.org/wiki/Operator_(biology)http://en.wikipedia.org/wiki/T7_phagehttp://en.wikipedia.org/wiki/Lac_operonhttp://en.wikipedia.org/wiki/Glycosylatedhttp://en.wikipedia.org/wiki/Signal_peptidehttp://en.wikipedia.org/wiki/Periplasmic_spacehttp://en.wikipedia.org/wiki/Disulphide_bondshttp://en.wikipedia.org/wiki/Bacillus_subtilishttp://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Protein_expression_(biotechnology)http://en.wikipedia.org/wiki/Protein_expression_(biotechnology)http://en.wikipedia.org/wiki/Periplasmic_space


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when thegrowth medium contains glucose since glucose would inhibits protein expression if wild-

type lac promoter is used.

Examples of E. coli expression vectors are the pGEX series of vectors where glutathione-S-transferase is

used as a fusion partner, and the pET series of vectors which uses a T7 promoter.

It is possible to simultaneously express two or more different proteins in E. coli using different plasmids.

However, when 2 or more plasmids used, each plasmid needs to use a different antibiotic selection as

well as a different origin of replication, otherwise the plasmids may not be stably maintained. Another

approach would be to use a single bicistronic vector or design the coding sequences in tandem as a bi- or

poly-cistronic construct.

Yeast

A yeast commonly used for protein expression isPichia pastoris.Examples of yeast expression vector

in Pichia are the pPIC series of vectors, and these vectors use the AOX1promoter which is inducible

withmethanol.The plasmids may contain elements for insertion of foreign DNA into the yeast genome

and signal sequence for the secretion of expressed protein. Proteins with disulphide bonds and

glycosylation can be efficiently produced in yeast. Another yeast used for protein expression

isKluyveromyces lactisand the protein is expressed driven by a variant of the strong lactaseLAC4

promoter.

Saccharomyces cerevisiaeis also commonly used for protein expression, for example inyeast two-hybrid

systemfor the study of protein-protein interaction. The vectors used in yeast two-hybrid system contain

fusion partners for two cloned genes that allow the transcription of a reporter gene when there is

interaction between the two proteins expressed from the cloned genes.

Baculovirus

Baculovirus,a rod-shaped virus which infect insect cells, is used as the expression vector in this system.

Insect cell lines derived fromLepidopterans(moths and butterflies), such asSpodoptera frugiperda,are

used as host. Theshuttle vectoris called bacmid, and protein expression is under the control of a strong

promoter pPolh. It is normally used for production ofglycoproteins,although the glycosylations may be

different from those found in vertebrates. It is safer to use than mammalian virus as it has a limited host

range and does not infect vertebrates.

Plant

Many plant expression vectors are based on theTi plasmidofAgrobacterium tumefaciens.[17]

In these

expression vectors, DNA to be inserted into plant is cloned into the T-DNA,a stretch of DNA flanked by a
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25-bp direct repeat sequence at either end, and which can integrate into the plant genome. The T-DNA

also contains the selectable marker. TheAgrobacterium provides a mechanism fortranformation,

integration of into the plant genome, and the promoters for its vir genes may also be used for the cloned

genes.

Some plant viruses may be used as vectors since Agrobacterium does not work for all plants. Examples

of plant virus used are thetobacco mosaic virus(TMV),potato virus X,andcowpea mosaic virus.[18]

The

protein may be expressed as a fusion to the coat protein of the virus and is displayed on the surface of

assembled viral particles, or as an unfused protein that accumulates within the plant. Expression in plant

using plant vectors is often constitutive, and a commonly-used constitutive promoter in plant expression

vectors is thecauliflower mosaic virus(CaMV) 35S promoter.

Mammalian

Cultured mammalian cell lines such as theChinese hamster ovary (CHO),HEKandCOS celllines are

used to produce protein. Vectors aretransfectedinto the cells and the DNA may be integrated into the

genome byhomologous recombinationin the case of stable transfection, or the cells may be transiently

transfected. Examples of mammalian expression vectors include theadenoviralvectors, the pSV and the

pCMV series of vectors. The promoters forcytomegalovirus(CMV) andSV40are commonly used in

mammalian expression vectors to drive protein expression.

Cell-free systems

E. colicell lysatecontaining the cellular components required for transcription and translation are used in

this in vitro method of protein expression. The advantage of such system is that protein may be producedmuch faster than those produced in vivo, but it is more expensive. Vectors used for E. coli expression can

be used in this system although specifically-design vectors for this system are also available. Eukaryotic

cell extracts may also be used in other cell-free systems.

Applications

Laboratory use

Expression vector in an expression host is now the usual method used in laboratories to produce proteins

for research. Most proteins are produced in E. coli, but for glycosylated proteins and those with disulphide

bonds, yeast, baculovirus and mammalian systems may be used.

Production of peptide and protein pharmaceuticals

Most proteinpharmaceuticalsare now produced through recombinant DNA technology using expression

vectors. These peptide and protein pharmaceuticals may be hormones, vaccines, antibiotics, antibodies,

and enzymes. The first human recombinant protein used for disease management was insulin and it was
http://en.wikipedia.org/wiki/Transformation_(genetics)http://en.wikipedia.org/wiki/Transformation_(genetics)http://en.wikipedia.org/wiki/Transformation_(genetics)http://en.wikipedia.org/wiki/Tobacco_mosaic_virushttp://en.wikipedia.org/wiki/Tobacco_mosaic_virushttp://en.wikipedia.org/wiki/Tobacco_mosaic_virushttp://en.wikipedia.org/wiki/Potato_virus_Xhttp://en.wikipedia.org/wiki/Potato_virus_Xhttp://en.wikipedia.org/wiki/Potato_virus_Xhttp://en.wikipedia.org/wiki/Cowpea_mosaic_virushttp://en.wikipedia.org/wiki/Cowpea_mosaic_virushttp://en.wikipedia.org/wiki/Expression_vector#cite_note-18http://en.wikipedia.org/wiki/Expression_vector#cite_note-18http://en.wikipedia.org/wiki/Expression_vector#cite_note-18http://en.wikipedia.org/wiki/Cauliflower_mosaic_virushttp://en.wikipedia.org/wiki/Cauliflower_mosaic_virushttp://en.wikipedia.org/wiki/Cauliflower_mosaic_virushttp://en.wikipedia.org/wiki/Chinese_hamster_ovary_cellhttp://en.wikipedia.org/wiki/Chinese_hamster_ovary_cellhttp://en.wikipedia.org/wiki/Chinese_hamster_ovary_cellhttp://en.wikipedia.org/wiki/HEK_cellhttp://en.wikipedia.org/wiki/HEK_cellhttp://en.wikipedia.org/wiki/HEK_cellhttp://en.wikipedia.org/wiki/COS_cellhttp://en.wikipedia.org/wiki/COS_cellhttp://en.wikipedia.org/wiki/COS_cellhttp://en.wikipedia.org/wiki/Transfectedhttp://en.wikipedia.org/wiki/Transfectedhttp://en.wikipedia.org/wiki/Transfectedhttp://en.wikipedia.org/wiki/Homologous_recombinationhttp://en.wikipedia.org/wiki/Homologous_recombinationhttp://en.wikipedia.org/wiki/Homologous_recombinationhttp://en.wikipedia.org/wiki/Adenoviralhttp://en.wikipedia.org/wiki/Adenoviralhttp://en.wikipedia.org/wiki/Adenoviralhttp://en.wikipedia.org/wiki/Cytomegalovirushttp://en.wikipedia.org/wiki/Cytomegalovirushttp://en.wikipedia.org/wiki/Cytomegalovirushttp://en.wikipedia.org/wiki/SV40http://en.wikipedia.org/wiki/SV40http://en.wikipedia.org/wiki/SV40http://en.wikipedia.org/wiki/Cell_lysatehttp://en.wikipedia.org/wiki/Cell_lysatehttp://en.wikipedia.org/wiki/Cell_lysatehttp://en.wikipedia.org/wiki/Pharmaceuticalshttp://en.wikipedia.org/wiki/Pharmaceuticalshttp://en.wikipedia.org/wiki/Pharmaceuticalshttp://en.wikipedia.org/wiki/Pharmaceuticalshttp://en.wikipedia.org/wiki/Cell_lysatehttp://en.wikipedia.org/wiki/SV40http://en.wikipedia.org/wiki/Cytomegalovirushttp://en.wikipedia.org/wiki/Adenoviralhttp://en.wikipedia.org/wiki/Homologous_recombinationhttp://en.wikipedia.org/wiki/Transfectedhttp://en.wikipedia.org/wiki/COS_cellhttp://en.wikipedia.org/wiki/HEK_cellhttp://en.wikipedia.org/wiki/Chinese_hamster_ovary_cellhttp://en.wikipedia.org/wiki/Cauliflower_mosaic_virushttp://en.wikipedia.org/wiki/Expression_vector#cite_note-18http://en.wikipedia.org/wiki/Cowpea_mosaic_virushttp://en.wikipedia.org/wiki/Potato_virus_Xhttp://en.wikipedia.org/wiki/Tobacco_mosaic_virushttp://en.wikipedia.org/wiki/Transformation_(genetics)


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introduced in 1982. Biotechnology allows these peptide and protein pharmaceuticals, some of which were

previously rare or difficult to obtain, to be produced in large amount. It also reduces the risks of

contaminants such as host viruses, toxins andprions. For example,growth hormoneextracted

frompituitary glandsharvested from human cadavers had causedCreutzfeldtJakob diseasein patients

receiving treatment fordwarfismdue to prion contamination, and viral contaminants in clottingfactor

VIIIisolated from human blood had resulted in the transmission of viral diseases such

ashepatitisandAIDS,and such risk is reduced or removed completely when these proteins are produced

in non-human cell-lines.

Transgenic plants

In recent years, expression vectors have been used to introduce specific genes inorganisms,for example

inagricultureit is used to producetransgenic plants. Expression vectors have been used to introduce

avitamin Aprecursor,beta-carotene,into rice plants. This product is calledgolden rice.This process has

also been used to introduce a gene into plants that produces an insecticide,calledBacillus thuringiensis

toxinorBt toxinwhich reduces the need for farmers to apply insecticides since it is produced by the

modified organism. In addition expression vectors are used to extend the ripeness of tomatoes by altering

the plant so that it produces less of the chemical that causes the tomatoes to rot.There has been

controversy over using expression vectors to modify crops due to the fact that there might be unknown

health risks, possibilities of companies patenting certaingenetically modified foodcrops, and ethical

concerns. Nevertheless, this technique is still being used and heavily researched.

Gene therapy

Gene therapyis a promising treatment for a number of diseases where a "normal" gene carried by the

vector is inserted into the genome, to replace an "abnormal" gene or supplement the expression of

particular gene. Viral vectors are generally used but other nonviral methods of delivery are being

developed. The treatment is still a risky option due to the viral vector used which can cause ill-effects, for

example giving rise toinsertional mutationthat can result in cancer. However, there have been promising

results.

Paired-end tag ( PET)Paired-end tags (PET)(sometimes "Paired-End diTags", or simply "ditags") are the short sequences at

the 5 and 3 ends of aDNAfragment which are unique enough that they (theoretically) exist together only

once in agenome,therefore making the sequence of the DNA in between them available upon search (if

full-genome sequence data is available) or upon further sequencing (since tag sites are unique enough to

serve asprimerannealing sites). Paired-end tags (PET) exist in PET libraries with the intervening DNA
http://en.wikipedia.org/wiki/Prionshttp://en.wikipedia.org/wiki/Prionshttp://en.wikipedia.org/wiki/Prionshttp://en.wikipedia.org/wiki/Growth_hormonehttp://en.wikipedia.org/wiki/Growth_hormonehttp://en.wikipedia.org/wiki/Growth_hormonehttp://en.wikipedia.org/wiki/Pituitary_glandhttp://en.wikipedia.org/wiki/Pituitary_glandhttp://en.wikipedia.org/wiki/Pituitary_glandhttp://en.wikipedia.org/wiki/Creutzfeldt%E2%80%93Jakob_diseasehttp://en.wikipedia.org/wiki/Creutzfeldt%E2%80%93Jakob_diseasehttp://en.wikipedia.org/wiki/Creutzfeldt%E2%80%93Jakob_diseasehttp://en.wikipedia.org/wiki/Creutzfeldt%E2%80%93Jakob_diseasehttp://en.wikipedia.org/wiki/Creutzfeldt%E2%80%93Jakob_diseasehttp://en.wikipedia.org/wiki/Dwarfismhttp://en.wikipedia.org/wiki/Dwarfismhttp://en.wikipedia.org/wiki/Dwarfismhttp://en.wikipedia.org/wiki/Factor_VIIIhttp://en.wikipedia.org/wiki/Factor_VIIIhttp://en.wikipedia.org/wiki/Factor_VIIIhttp://en.wikipedia.org/wiki/Factor_VIIIhttp://en.wikipedia.org/wiki/Hepatitishttp://en.wikipedia.org/wiki/Hepatitishttp://en.wikipedia.org/wiki/Hepatitishttp://en.wikipedia.org/wiki/AIDShttp://en.wikipedia.org/wiki/AIDShttp://en.wikipedia.org/wiki/AIDShttp://en.wikipedia.org/wiki/Organismhttp://en.wikipedia.org/wiki/Organismhttp://en.wikipedia.org/wiki/Organismhttp://en.wikipedia.org/wiki/Agriculturehttp://en.wikipedia.org/wiki/Agriculturehttp://en.wikipedia.org/wiki/Agriculturehttp://en.wikipedia.org/wiki/Transgenic_plantshttp://en.wikipedia.org/wiki/Transgenic_plantshttp://en.wikipedia.org/wiki/Transgenic_plantshttp://en.wikipedia.org/wiki/Vitamin_Ahttp://en.wikipedia.org/wiki/Vitamin_Ahttp://en.wikipedia.org/wiki/Vitamin_Ahttp://en.wikipedia.org/wiki/Beta-carotenehttp://en.wikipedia.org/wiki/Beta-carotenehttp://en.wikipedia.org/wiki/Beta-carotenehttp://en.wikipedia.org/wiki/Golden_ricehttp://en.wikipedia.org/wiki/Golden_ricehttp://en.wikipedia.org/wiki/Golden_ricehttp://en.wikipedia.org/wiki/Insecticidehttp://en.wikipedia.org/wiki/Insecticidehttp://en.wikipedia.org/wiki/Insecticidehttp://en.wikipedia.org/wiki/Bacillus_thuringiensishttp://en.wikipedia.org/wiki/Bacillus_thuringiensishttp://en.wikipedia.org/wiki/Bacillus_thuringiensishttp://en.wikipedia.org/wiki/Bacillus_thuringiensishttp://en.wikipedia.org/wiki/Bacillus_thuringiensishttp://en.wikipedia.org/wiki/Bacillus_thuringiensishttp://en.wikipedia.org/wiki/Bacillus_thuringiensishttp://en.wikipedia.org/wiki/Genetically_modified_foodhttp://en.wikipedia.org/wiki/Genetically_modified_foodhttp://en.wikipedia.org/wiki/Genetically_modified_foodhttp://en.wikipedia.org/wiki/Gene_therapyhttp://en.wikipedia.org/wiki/Gene_therapyhttp://en.wikipedia.org/wiki/Insertional_mutationhttp://en.wikipedia.org/wiki/Insertional_mutationhttp://en.wikipedia.org/wiki/Insertional_mutationhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Primer_(molecular_biology)http://en.wikipedia.org/wiki/Primer_(molecular_biology)http://en.wikipedia.org/wiki/Primer_(molecular_biology)http://en.wikipedia.org/wiki/Primer_(molecular_biology)http://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Insertional_mutationhttp://en.wikipedia.org/wiki/Gene_therapyhttp://en.wikipedia.org/wiki/Genetically_modified_foodhttp://en.wikipedia.org/wiki/Bacillus_thuringiensishttp://en.wikipedia.org/wiki/Bacillus_thuringiensishttp://en.wikipedia.org/wiki/Bacillus_thuringiensishttp://en.wikipedia.org/wiki/Insecticidehttp://en.wikipedia.org/wiki/Golden_ricehttp://en.wikipedia.org/wiki/Beta-carotenehttp://en.wikipedia.org/wiki/Vitamin_Ahttp://en.wikipedia.org/wiki/Transgenic_plantshttp://en.wikipedia.org/wiki/Agriculturehttp://en.wikipedia.org/wiki/Organismhttp://en.wikipedia.org/wiki/AIDShttp://en.wikipedia.org/wiki/Hepatitishttp://en.wikipedia.org/wiki/Factor_VIIIhttp://en.wikipedia.org/wiki/Factor_VIIIhttp://en.wikipedia.org/wiki/Dwarfismhttp://en.wikipedia.org/wiki/Creutzfeldt%E2%80%93Jakob_diseasehttp://en.wikipedia.org/wiki/Pituitary_glandhttp://en.wikipedia.org/wiki/Growth_hormonehttp://en.wikipedia.org/wiki/Prions


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absent, that is, a PET "represents" a larger fragment of genomic orcDNAby consisting of a short 5' linker

sequence, a short 5' sequence tag, a short 3' sequence tag, and a short 3' linker sequence. It was shown

conceptually that 13 bp is sufficient to map tags uniquely.

However, longer sequences are more practical for mapping reads uniquely.

Theendonucleases(discussed below) used to produce PETs give longer tags (18/20 bp and 25/27 bp)

but sequences of 50100 base pairs would be optimal for both mapping and cost efficiency.[1]After

extracting the PETs from many DNA fragments, they are linked (concatenated) together for efficient

sequencing. On average, 2030 tags could be sequenced with theSangermethod, which has a longer

read length. Since the tag sequences are short, individual PETs are well suited fornext-generation

sequencingthat has short read lengths and higher throughput. The main advantages of PET sequencing

are its reduced cost by sequencing only short fragments, detection of structural variants in the genome,

and increased specificity when aligning back to the genome compared to single tags, which involves only

one end of the DNA fragment.

Constructing the PET library

Workflow of Cloning and Cloning-free based PET library construction.
http://en.wikipedia.org/wiki/CDNAhttp://en.wikipedia.org/wiki/CDNAhttp://en.wikipedia.org/wiki/CDNAhttp://en.wikipedia.org/wiki/Restriction_enzymeshttp://en.wikipedia.org/wiki/Restriction_enzymeshttp://en.wikipedia.org/wiki/Restriction_enzymeshttp://en.wikipedia.org/wiki/Paired-end_tag#cite_note-fullwood-1http://en.wikipedia.org/wiki/Paired-end_tag#cite_note-fullwood-1http://en.wikipedia.org/wiki/DNA_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencing#High-throughput_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencing#High-throughput_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencing#High-throughput_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencing#High-throughput_sequencinghttp://en.wikipedia.org/wiki/File:PETworkflow.pnghttp://en.wikipedia.org/wiki/File:PETworkflow.pnghttp://en.wikipedia.org/wiki/File:PETworkflow.pnghttp://en.wikipedia.org/wiki/File:PETworkflow.pnghttp://en.wikipedia.org/wiki/DNA_sequencing#High-throughput_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencing#High-throughput_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencinghttp://en.wikipedia.org/wiki/Paired-end_tag#cite_note-fullwood-1http://en.wikipedia.org/wiki/Restriction_enzymeshttp://en.wikipedia.org/wiki/CDNA


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PET libraries are typically prepared in two general methods: cloning based and cloning-free based.

Cloning based

Fragmented genomic DNA or complementary DNA (cDNA) of interest is cloned into plasmid vectors.The

cloning sites are flanked with adaptor sequences that contain restriction sites for endonucleases(discussed below). Inserts are ligated to the plasmid vectors and individual vectors are

thentransformedinto E. coli making the PET library. PET sequences are obtained by purifying plasmid

an

micro r dna technology

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