National university of life and environmental sciencesof ukraine
Chair of molecular geneticsand biosafety
Termpaper
VectorsFor The Molecular cloning
done by:
third year student
group №2
department of ecology
and biotechnology
Pereguda Olga
Scientific advisor
Professor StarodubN. F.
Kyiv 2010/>
Abstract
Term paper on“Vectors for the moleculars cloning” consist of two sections: conclusions list andof the references.
The object ofresearch:different vectors for the moleculars cloning.
The tasks of termpaper:
1) Learned the vectors for themoleculars cloning
2) Consider and study vectorsof molecular cloning, and functions, properties etc.
The results presentedin the form of conclusions at the end of term paper.
Contents
Key words
Abstract
Conditional shortenings
Introducting
Literature review
1. Plasmid vectors
2. Cosmids
3. Phagemids
4. Bacteriophagevectors
4.1. Filamentousphage
4.2.Double-strandedphage
5. Scope of PresentReview
6. Life cycle and geneticsof Lambda
6.1. Development of Lambda
7. Phage Lambda as a vector
7.1. Size Limitation for Packaging
7.2. Transfection of RecombinantMolecules
7.3. Biological Containment
8. Phage vectors
8.1.Replacement Vectors
8.2. Insertion Vectors
8.3.Storage of Lambda Stocks
Conclusion
Literature
Key words
Cosmids — an extrachromosomal circularDNA molecule that combines features of plasmids and phage; cloning limit — 35-50 kb.
DNA –a longchain polymer ofdeoxyribonucleotides.DNA constitutesthe genetic material ofmost knownorganismsand organelles,and usuallyis inthe formof a double helix,although some viralgenomes consistof a singlestrandof DNA, andothersof a single- or a double-strandedRNA.
Enzyme –a biological catalyst,usuallya protein,that canspeed up a chemicalreactionby loweringit’s energyof activationwithoutbeing used up inthe reaction.Helicase –a type of enzyme thatbreaks hydrogenbonds between complementarybase pairsof DNA,therebycausingthe doublestrandto spit into separate singlestrands.
Molecular cloning– is processof creating an identical copy of DNA fragments. Phage — derivatives of bacteriophagelambda; linear DNA molecules, whose region can be replaced with foreign DNA withoutdisrupting its life cycle. Plasmid — an extrachromosomal circular DNA moleculethat autonomously replicates inside the bacterial cell.
Promoter — a specificDNA sequence that serves as a binding site for RNA polymerase near each gene.
Replicon – a block of DNA betweentwo adjacent replication origins.
Vector – is an agent that can carryout a DNA fragment into a host cell.
Conditional shortenings
BAC– Bacterial Artificial Chromosome
cos– cohesice end site
DNA — deoxyribonucleicacid
Kb– Kilobases
Kbp– Kilobase pair
nt- necleotides
PCR– Polymarase chain reaction
pUc,pBluscript – phagemid vectors
RNA –ribonucleic acid
Sp6,T7 — promoters
Introduction
Cloning- is the processof creating an identical copy of something. In Biology, it collectively refersto processes used to create copies of DNA fragments (Molecular Cloning), cells(Cell Cloning), or organisms. The term also encompases situations, whereby organismsreproduce asexually, but in common parlance refers to intentionally created copiesof organisms.
In1972, Paul Berg and colleagues made the first “artificial” recombinant DNA molecule.The molecular analysis of DNA has been made possible by the cloning of DNA. Thetwo molecules that are required for cloning are the DNA to be cloned and a cloningvector.
Cloning vector — a DNA molecule that carriesforeign DNA into a host cell, replicates inside a bacterial (or yeast) cell andproduces many copies of itself and the foreign DNA. Types of Cloning Vectors arePlasmid, Phage, Cosmids.
Molecular cloningrefers to the process of making multiple molecules. Cloning is commonly used toamplify DNA fragments containing whole genes, but it can also be used to amplifyany DNA sequence such as promoters, non-coding sequences and randomly fragmentedDNA. It is used in a wide array of biological experiments and practical applicationsranging from genetic fingerprinting to large scale protein production. Occasionally,the term cloning is misleadingly used to refer to the identification of the chromosomallocation of a gene associated with a particular phenotype of interest, such as inpositional cloning. In practice, localization of the gene to a chromosome or genomicregion does not necessarily enable one to isolate or amplify the relevant genomicsequence. To amplify any DNA sequence in a living organism, that sequence mustbe linked to an origin of replication, which is a sequence of DNA capable of directingthe propagation of itself and any linked sequence. However, a number of other featuresare needed and a variety of specialised cloning vectors (small piece of DNA intowhich a foreign DNA fragment can be inserted) exist that allow protein expression,tagging, single stranded RNA and DNA production and a host of other manipulations.
Cloning of anyDNA fragment essentially involves four steps
· fragmentation — breaking apart a strand ofDNA
· ligation — gluing together pieces of DNAin a desired sequence
· transfection — inserting the newly formedpieces of DNA into cells
· screening/selection — selecting out the cellsthat were successfully transfected with the new DNA
Recombinant DNA techniques have allowed the isolation and propagation ofspecific DNA fragments which can be easily sequenced and/or used as highly specificprobes. In vitro site-directed modifications of these fragments and their reintroductioninto the genome result in a modified genetic makeup of an organism. In addition,it is now possible to induce overproduction of commercially important proteinsby genetically tailored microorganisms[8].
Several cloning strategies have been developed to meet various specific requirements.Cloning protocols have been designed for a variety of host systems. However, Escherichiacoli still remains the most popular host of choice since its genetics, physiology,and molecular biology have been studied in great detail and a wealth of informationis readily available. Many cloning vectors have also been constructed for use withE. coli as a host. Although this review focuses on the basic and applied aspectsof bacteriophage lambda ectors, an overview of other vectors is included for comparison.
In general, cloning vectors can be broadly classified as plasmid and phagevectors.
So, the aim ofthis work is: to consider and study vectors of molecular cloning, and functions,properties etc.
Literaturereview
Plasmids are usefulfor a wide range of molecular genetic, genomic and proteomic approaches. In recentyears, plasmid clone production has increased dramatically in response to the availabilityof genome information and new technologies.[9]
In 1952, Joshua Lederberg coined the term plasmid to describe anybacterial genetic element that exists in an extrachromosomal state for at leastpart of its replication cycle. As this description included bacterial viruses,the definition of what constitutes a plasmid was subsequently refined to describeexclusively or predominantly extrachromosomal genetic elements that replicate autonomously.[1]
Most plasmids possess a circular geometry, there are now many examplesin a variety of bacteria of plasmids that are linear. As linear plasmids requirespecialized mechanisms to replicate their ends, which circular plasmids and chromosomesdo not, linear plasmids tend to exist in bacteria that also have linear chromosomes[1]
Plasmids, like chromosomes, are replicated during the bacterial cell cycleso that the new cells can each be provided with at least one plasmid copy at celldivision [1]
Frederick Twort (1915) and Felix d’Herelle (1917) were the first to recognizeviruses which infect bacteria, which d'Herelle called bacteriophages (eaters ofbacteria).[7]
Lambda (λ) bacteriophages are viruses that specifically infect bacteria.The genome of λ-phage is a double-stranded DNA molecule approx 50 kb in length.In bacterial cells, λ-phage employs one of two pathways of replication:lytic or lysogenic. [2]
In lytic growth, approx 100 new virions are synthesized and packaged beforelysing the host cell, releasing the progeny phage to infect new hosts. In lysogeny,the phage genome undergoes recombination into the host chromosome, where it isreplicated and inherited along with the host DNA. [2]
Cosmids- an extrachromosomal circular DNA molecule that combines features of plasmids andphage. [8]
Cosmids are conventional vectors that contain a small region of bacteriophageλDNA containing the cohesiveend site (cos). This contains all of the cis-acting elementsfor packaging of viral DNAinto λ particles [4]
1. PlasmidVectors
In 1952, Joshua Lederberg coined the term plasmid to describe anybacterial genetic element that exists in an extrachromosomal state for at leastpart of its replication cycle. As this description included bacterial viruses,the definition of what constitutes a plasmid was subsequently refined to describeexclusively or predominantly extrachromosomal genetic elements that replicate autonomously.
Figure 1. Joshua Lederberg
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Plasmid vectors are convenient for cloning of small DNA fragments for restrictionmapping and for studying regulatory regions. However, these vectors have a relativelysmall insert capacity. Therefore, a large number of clones are required for screeningof a single-copy DNA fragment of higher eukaryotes. Second, the handling and storageof these clones is time-consuming and difficult. The repeated subcultures of recombinantsmay result in deletions in the inserts.
The plasmid vectors can be of three main types:
· generalpurpose cloning vectors,
· expression vectors,
· promoter probe or terminator probe vectors.
Figure 2. Cloning into a plasmid
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General-purpose cloning vectors
Cloning of foreign DNA fragments in general-purpose cloning vectors [11]selectively inactivates one of the markers (insertional inactivation) or derepressesa silent marker (positive selection) so as to differentiate the recombinants fromthe native phenotype of the vector.
Expression vectors
In expression vectors, DNA to be cloned and expressed is inserted downstreamof a strong promoter present in the vector. The expression of the foreign gene isregulated by the vector promoter irrespective of the recognition of its own regulatorysequence.
Promoter probe and terminator probe vectors
Promoter probe and terminator probe vectors are useful for the isolation ofregulatory sequences such as promoters or terminators and for studying their recognitionby a specific host. They possess a structural gene devoid of the promoter or theterminator sequence [8].
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Figure 3. Replication of rolling-circle plasmids
2. Cosmids
A cosmid, firstdescribed by Collins and Hohn in 1978, is a type of hybrid plasmid (often used asa cloning vector) that contains cos sequences, DNA sequences originally from theLambda phage. Cosmids can be used to build genomic libraries.
Cosmids are ableto contain 37 to 52 kb of DNA, while normal plasmids are able to carry only1–20 kb. They can replicate as plasmids if they have a suitable origin of replication:for example SV40 ori in mammalian cells, ColE1 ori for double-stranded DNA replicationor f1 ori for single-stranded DNA replication in prokaryotes. They frequently alsocontain a gene for selection such as antibiotic resistance, so that the transfectedcells can be identified by plating on a medium containing the antibiotic. Thosecells which did not take up the cosmid would be unable to grow.
Unlike plasmids,they can also be packaged in phage capsids, which allows the foreign genes to betransferred into or between cells by transduction. Plasmids become unstable aftera certain amount of DNA has been inserted into them, because their increased sizeis more conducive to recombination. To circumvent this, phage transduction is usedinstead. This is made possible by the cohesive ends, also known as cos sites. Inthis way, they are similar to using the lambda phage as a vector, but only thatall the lambda genes have been deleted with the exception of the cos sequence.
Cos sequences are~200 base pairs long and essential for packaging. They contain a cosN site whereDNA is nicked at each strand, 12bp apart, by terminase. This causes linearizationof the circular cosmid with two «cohesive» or «sticky ends»of 12bp. (The DNA must be linear to fit into a phage head.) The cosB site holdsthe terminase while it is nicking and separating the strands. The cosQ site ofnext cosmid (as rolling circle replication often results in linear concatemers)is held by the terminase after the previous cosmid has been packaged, to preventdegradation by cellular DNases.
Figure 4. Cloning by using Cosmid method
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Cosmid featuresand uses
Cosmids are predominantlyplasmids with a bacterial oriV, an antibiotic selection marker and a cloning site,but they carry one, or more recently two cos sites derived from bacteriophage lambda.Depending on the particular aim of the experiment broad host range cosmids, shuttlecosmids or 'mammalian' cosmids (linked to SV40 oriV and mammalian selection markers)are available. The loading capacity of cosmids varies depending on the size ofthe vector itself but usually lies around 40–45 kb. The cloning procedure involvesthe generation of two vector arms which are then joined to the foreign DNA. Selectionagainst wildtype cosmid DNA is simply done via size exclusion. Cosmids, however,always form colonies and not plaques. Also the clone density is much lower witharound 105 — 106 CFU per µg of ligated DNA.
After the constructionof recombinant lambda or cosmid libraries the total DNA is transferred into an appropriateE.coli host via a technique called in vitro packaging. The necessary packaging extractsare derived from E.coli cI857 lysogens (red- gam- Sam and Dam (head assembly) andEam (tail assembly) respectively). These extracts will recognize and package therecombinant molecules in vitro, generating either mature phage particles (lambda-basedvectors) or recombinant plasmids contained in phage shells (cosmids). These differencesare reflected in the different infection frequencies seen in favour of lambda-replacementvectors. This compensates for their slightly lower loading capacity. Phage libraryare also stored and screened easier than cosmid (colonies!) libraries.
Target DNA: thegenomic DNA to be cloned has to be cut into the appropriate size range of restrictionfragments. This is usually done by partial restriction followed by either sizefractionation or dephosphorylation (using calf-intestine phosphatase) to avoid chromosomescrambling, i.e. the ligation of physically unlinked fragments.
3. Phagemids
Phagemids combine desirable properties of both plasmids and filamentous phages.They carry
· the ColEl origin of replication,
· a selectable marker such as antibiotic resistance,
· the major intergenic region of a filamentousphage .
The segments of foreign DNA cloned in these vectors can be propagated as plasmids.When cells harboring these plasmids are infected with a suitable helper bacteriophage,the mode of replication of the plasmid changes under the influence of the gene IIproduct of the incoming virus.
Interaction of the intergenic region of the plasmid with the gene II proteininitiates the rolling-circle replication to generate copies of one strand of theplasmid DNA, which are packaged into progeny bacteriophage particles. The single-strandedDNA purified from these particles is used as a template to determine the nucleotidesequence of one strand of the foreign DNA segment, for site-directed mutagenesisor as a strand-specific probe. Phagemids provide high yields of double-strandedDNA and render unnecessary the time-consuming process of subcloning DNA fragmentsfrom plasmids to filamentous bacteriophages.
4. BacteriophageVectors
Both single-stranded (filamentous) and double-stranded E.coli phages havebeen exploited as cloning vectors.
Frederick Twort (1915) and Felix d’Herelle (1917) were the first to recognizeviruses which infect bacteria, which d'Herelle called bacteriophages (eaters ofbacteria). [7]
Figure 5. Frederick Twort and Felix d’Herelle
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4.1 Filamentous phages
Filamentous phages are not lytic. They coexist with the infected cells forseveral generations and are convenient for cloning genes which produce toxic products.Among the filamentous phages, fd, fl, and M13 have been well characterized andtheir genomes have been sequenced [4]. Their gene functions and molecular mode ofpropagation are very similar. They infect cells via F pili, and the first maturephage appears within 15 min [6].
Phage M13 is widely used in nucleotide sequencing and site-directed mutagenesissince its genome can exist either in a single-stranded form inside a phage coator as a doublestranded replicative form within the infected cell. During replication,only the plus strand of the replicative form is selectively packaged by the phageproteins [1]. The replicative form is a covalently closed circular molecule andhence can be used as a plasmid vector and transformed into the host by the usualtransformation procedures. The vectors derived from M13, have the same polylinkeras that of pUC18 and pUC19, respectively [2]. The DNA fragments having noncomplementaryends can be directionally cloned in this pair of vectors, and the two strands ofDNA can be sequenced independently.
4.2.Double-stranded phage vectors
Of the double-stranded phages, bacteriophage lambda-derived vectors are themost popular tools for several reasons:
· acceptance by the phage of large foreignDNA fragments, thereby increasing the chances of screening a single clone carryinga DNA sequence corresponding to a complete gene;
· development and availability of refinedtechniques aimed at minimizing the problems of background due to nonrecombinants;
· the possibility of screening several thousandclones at a time from a single petri plate; and, finally,
· the ease with which the phage library canbe stored as a clear lysate at 4°C for months without significant loss in plaque-formingactivity [7].
Recently, a bacteriophage P1 cloning system has been developed which permitscloning of DNA fragments as large as 100 kbp with an efficiency that is intermediatebetween cosmids and yeast artificial chromosomes .
5. Scopeof Present Review
The extensive knowledge of the basic biology of lambda has permitted modificationsof its genome to suit the given experimental conditions. In the present reviewwe describe how the utility of lambda as a cloning vector rests essentially in itsintrinsic molecular organization. The following sections give an account of variousproblems encountered in constructing lambda vectors and the strategies that havebeen adopted to overcome them. A few commonly used vectors are described in detail,taking into account their special values and limitations. The different methodsfor screening and storage of genomic and cDNA libraries in lambda vectors are alsodiscussed.
6. Lifecycle and genetics of Lambda
An understanding of the basic biology of lambda, its mode of propagation,and the genetic and molecular mechanisms that control its life cycle is neededbefore its applications for genetic manipulations are discussed. This section dealswith the basic biology of lambda.
The lambda virus particle contains a linear DNA of 48,502 bp with a single-stranded5' extension of 12 bases at both ends; these extensions are complementary to eachother.
These ends are called cohesive ends or cos. During infection, the right5' extension (cosR), followed by the entire genome, enters the host cell. Boththe cos ends are ligated by E. coli DNA ligase, forming a covalently closed circularDNA which is acted upon by the host DNA gyrase, resulting in a supercoiled structure.
6.1 Development of Lambda
Two Alternative Modes. After infecting the host, the lambda genome may startits replication; this results in the formation of multiple copies of the genome.The protein components necessary for the assembly of mature phage particles aresynthesized by the coordinated expression of phage genes. Phage DNA is packagedinside a coat, and the mature phages are released into the environment after celllysis. This mode of propagation is called the lytic cycle.
Alternatively, the phage genome may enter a dormant stage (prophage) by integratingitself into a bacterial genome by site-specific recombination; during this stageit is propagated along with the host in the subsequent progeny. This stage is termedlysogeny. Changes in environmental and physiological conditions may activate theprophage stage and trigger lytic events.
7. PhageLambda as a vector
Figure 6. Bacteriophage
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The large genome size and complex genetic organization of lambda had posedinitial problems with its use as a vector. The problems, however, were surmountedthrough the sustained efforts of researchers, and lambda has been developed intoan efficient vector.
The broad objectives in constructing various phage vectors are
— thepresence of cloning sites only in the dispensable fragments,
— thecapacity to accommodate foreign DNA fragments of various sizes,
— thepresence of multiple cloning sites,
— anindication of incorporation of DNA fragments by a change in the plaque type,
— theability to control transcription of a cloned fragment from promoters on the vector,
— thepossibility of growing vectors and clones to high yield,
— easyand ready recovery of cloned DNA,
— introductionof features contributing to better biological containment.
There are several difficulties in the use of lambda as a vector.
Some of the problems and the general strategies adopted to overcome themare discussed in this section. Manipulation of Restriction Sites The major obstacleto the use of phage lambda as a cloning vector was essentially the presence ofmultiple recognition sites for a number of restriction enzymes in its genome.
Initially, all attempts were directed toward minimizing the number of EcoRIsites. Murray and Murray in 1974 were able to construct derivatives of lambda withonly one or two EcoRI sites. Similarly, Rambach and Toillais constructed lambdaderivatives with EcoRI sites only in the nonessential region of the genome by repeatedtransfer on restrictive and nonrestrictive hosts. After several cycles of digestion,packaging, and growth, phage derivatives with desirable restriction sites and fullretention of infectivity were obtained. All but one HindIII sites were removedby recombination of known deletion mutants or substitutions. Recently, oligonucleotideswith specific sequences have been synthesized and introduced into the bacteriophagelambda genome. This has provided a variety of cloning sites in the genome [5].
7.1 Size Limitation for Packaging
The second problem was the requirement of a minimum and maximum genome length(38 and 53 kbp, respectively) for the efficient packaging and for the productionof viable phage particles. The viability of the bacteriophage decreases when itsgenome length is greater than 105% or less than 78% of that of wild-type lambda.Genetic studies of specialized transducing bacteriophages showed, however, thatthe central one-third of the genome, i.e., the region between the J and Ngenes,is not essential for lytic growth. The presence of a nonessential middle fragmentof the phage genome was also revealed during construction of viable deletion mutants.These mutants lack most of the two central EcoRI B fragments which are not essentialfor lytic growth. However, too much DNA cannot be deleted because there is a minimum38-kbp requirement essential for efficient packaging. The de novo insertion ofDNA (even if heterogeneous) is essential for the formation of viable phages. Thisconstitutes a positive selection for recombinant phages carrying insertions. Thisapproach was successfully exploited in constructing recombinant phages carryingE. coli and Drosophila melanogaster DNA [8].
7.2 Transfection of Recombinant Molecules
The problem of transfection of recombinant molecules constructed in vitrowas overcome by the successful in vitro assembly of viable and infectious phageparticles. Two types of in vitro packaging systems have been developed so far, i.e.,two-strain packaging and single-strain packaging.
Two-strain packaging.
The basis of the two-strain in vitrop ackaging system is the complementationof two amber mutations. Two lambda lysogens, each carrying a single amber mutationin a distinctly different gene, are induced and grown separately so that they cansynthesize the necessary proteins. Neither of the lysogens alone is capable of packagingthe phage DNA. The role of various phage products in DNA packaging has been studiedin detail[3]. The E protein is the major component of the bacteriophage head, andin its absence all the viral capsid components accumulate. The D protein is involvedin the coupled process of insertion of bacteriophage DNA into the prehead precursorand the subsequent maturation of the head. The A protein is required for the cleavageof the concatenated precursor DNA at the cos sites. Two phage lysogens carryingA and E or D and E mutations in the phage genome are induced separately, and cellextracts are prepared. Neither of the extracts can produce infectious phage particles.However, when the extracts are mixed, mature phage particles are produced by complementation.
The major drawback of the two-strain system is the competition of native phageDNA with recombinant molecules. In both the cell extracts, native phage DNA is alsopresent and can be packaged with an efficiency equal to that of the chimeric DNA.This reduces the proportion of recombinants obtained in a library. The problem ofregeneration of endogenous phages obtained in the library was partially overcomeby the use of b2-deleted prophages, which poorly excise out of the host chromosomeor by UV irradiation of packaging extracts.
Single-strain packaging.
Rosenberg have successfully developed a single-strain packaging system byintroducing deletion in the cos region of prophage, rendering the prophage DNA unpackagablebecause cos is the packaging origin. Induction of the lysogen results in the intracellularaccumulation of all protein components needed for packaging.
However, packaging of phage DNA is prevented by the lack of cos sites onthe prophage DNA. On the other hand, exogenous DNA with cos sites is packaged efficientlyto produce an infectious bacteriophage particle. The single-strain system is superiorto two-strain system in having a lower background of parental phages. In addition,it uses E. coli C, which lacks the EcoK restriction system, as the host for thelysogen.
7.3 Biological Containment
The biological containment of recombinant phages is an important aspectfrom the point of view of ethics and eventual biohazards. It is desirable that cloningvectors and recombinants have poor survival in the natural environment and requirespecial laboratory conditions for their replication and survival. According toBlattner, the lytic phages offer a natural advantage in this respect since the phageand the sensitive bacteria coexist only briefly. A newly inserted segment may notbe compatible with E. coli metabolism for extended periods. To make the phage vectorsmore safe, three amber mutations were introduced in its genome. The new vectorXgt WES XC is safer because an amber suppressor host strain is a very rare occurrencein the natural environment. Many vectors carry one of the amber mutations on thegenome so that they can be propagated only on an appropriate suppressor host.
8. Phagevectors
Many phage vectors have been constructed in the recent past, each with itsown special features. There is no universal lambda vector which can fulfill allthe desired objectives of the cloning experiments.
Thechoice of a vector depends mainly on
— thesize of a DNA fragment to be inserted,
— therestriction enzymes to be used,
— thenecessity for expression of the cloned fragment,
— themethod of screening to be used to select the desired clones.
Bacteriophage lambda vectors can be broadly classified into two types:
1. replacement vectors ,
2. insertion vectors.
Figure 7. Lambda Phage genome
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8.1 Replacement Vectors
Taking advantage of the maximum and minimum genome size essential for efficientpackaging and the presence of the nonessential central fragment, it is possibleto remove the stuffer fragment and replace it with a foreign DNA fragment in thedesired size range. This forms the basis of lambdaderived replacement vectors.
Cloning of a foreign DNA in these vectors involves
· preparation of left and right arms by physicalelimination of the nonessential region,
· ligation of the foreign DNA fragment betweenthe arms,
· in vitro packaging and infection.
The replacement vectors contain a pair of restriction sites to excise thecentral stuffer fragments, which can be replaced by a desired DNA sequence withcompatible ends. The presence of identical sites within the stuffer fragment butnot in the arms facilitates the separation of the arms and the stuffer on densitygradient centrifugation. In many vectors, sets of such sites are provided on attachedpolylinkers so that an insert can be easily excised. Two purified arms cannot bepackaged despite their being ligated to each other, because they fall short ofthe minimum length required for packaging. This provides positive selection ofrecombinants. The replacement vectors are convenient for cloning of large (in somecases up to 24 kbp) DNA fragments and are useful in the construction of genomiclibraries of higher eukaryotes. Charon and EMBL are among the popular replacementvectors.
8.2 Insertion Vectors
Because the maximum packagable size of lambda genome is 53 kb, small DNAfragments can be introduced without removal of the nonessential (stuffer) fragment.These vectors are therefore called insertion vectors. Cloning of foreign DNA inthese vectors exploits the insertional inactivation of the biological function,which differentiates recombinants from nonrecombinants. Insertion vectors are particularlyuseful in cloning of small DNA fragments such as cDNA.
AgtlO and Agtll are examples of this type of vector. In recent years a multitudeof lambda vectors have been constructed. Many innovative approaches have been usedto introduce desired properties into the vectors. The following section deals withthe strategies adopted for the construction of some of the commonly used vectorsand their salient features, utilities, and limitations.
8.3 Storage of Lambda Stocks
Most of the lambda strains are stable for several years when stored at 4°Cin SM buffer containing 0.3% freshly distilled chloroform (94). The master stocksof bacteriophage lambda are kept in 0.7% (vol/vol) dimethyl sulfoxide at -70°Cfor long-term storage. Klinman and Cohen have developed a method for storage ofa phage library at -70°C by using top agar containing 30% glycerol.
Conclusion
In my work I determinedinvestigations in Molecular cloning, familiarized with vectors for molecular cloning,summarized the received information and made consequences of scientists researches,defined the main tasks of molecular cloning, and made such conclusions:
1. sequencesthat permit the propagation of itself in bacteria (or in yeast for YACs) .
2. a cloningsite to insert foreign DNA; the most versatile vectors contain a site that canbe cut by many restriction enzymes .
3. a methodof selecting for bacteria (or yeast for YACs) containing a vector with foreignDNA; uually accomplished by selectable markers for drug resistance .
Cloning vector — a DNA molecule that carriesforeign DNA into a host cell, replicates inside a bacterial (or yeast) cell andproduces many copies of itself and the foreign DNA .
General Steps ofCloning with Any Vector :
1. preparethe vector and DNA to be cloned by digestion with restriction enzymes to generatecomplementary ends ;
2. ligatethe foreign DNA into the vector with the enzyme DNA ligase;
3. introducethe DNA into bacterial cells (or yeast cells for YACs) by transformation ;
4. selectcells containing foreign DNA by screening for selectable markers (usually drugresistance);
/>Literature
1. Finbar Hayes The Function and Organizationof Plasmids// E. coli Plasmid Vectors Methods and Applications.- 2007.- vol.235– pp. 1-18.
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3. Tim S. Poulsen and Hans E. Johnsen BACEnd Sequencing // Bacterial Artificial Chromosomes Volume 1: Library Construction,Physical Mapping, and Sequencing.- 2007. – vol.255 — pp.157-162.
4. Andrew Preston Choosing a Cloning Vector//E. coli Plasmid Vectors Methods and Applications.- 2007.- vol.235 – pp.19-22.
5. Sambrook, J., Fritsch, E. F., and Maniatis,T. (eds.) (1989) Bacteriophage λvectors, in Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp.2.3–2.125.
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7. www.Microbiologybytes.com
8. www.wikigenes.org
9. http:// plasmid.hms.harvard.edu
10. www. Bookrags.com/YAC