Vectors for moleculars cloning

National university of life and environmental sciences
of ukraine

Chair of molecular genetics
and biosafety

Term
paper

Vectors
For The Molecular cloning

done by:

third year student

group №2

department of ecology

and biotechnology

Pereguda Olga

Scientific advisor

Professor Starodub
N. F.

Kyiv 2010

Abstract

Term paper on
“Vectors for the moleculars cloning” consist of two sections: conclusions list and
of the references.

The object of
research:different vectors for the moleculars cloning.

The tasks of term
paper:

1)  Learned the vectors for the
moleculars cloning

2)  Consider and study vectors
of molecular cloning, and functions, properties etc.

The results presented
in 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. Bacteriophage
vectors

4.1. Filamentous
phage

4.2.Double-stranded
phage

5. Scope of Present
Review

6. Life cycle and genetics
of Lambda

6.1. Development of Lambda

7. Phage Lambda as a vector

7.1. Size Limitation for Packaging

7.2. Transfection of Recombinant
Molecules

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 circular
DNA molecule that combines features of plasmids and phage; cloning limit -
35-50 kb.   

DNA –
a long
chain polymer of
deoxyribonucleotides.
DNA constitutes
the genetic material of
most known
organisms
and organelles,
and usually
is in
the form
of a double helix,
although some viral
genomes consist
of a single
strand
of DNA, and
others
of a single- or a double-stranded
RNA. 

Enzyme –
a biological catalyst,
usually
a protein,
that can
speed up a chemical
reaction
by lowering
it’s energy
of activation
without
being used up in
the reaction.
Helicase –
a type of enzyme that
breaks hydrogen
bonds between complementary
base pairs
of DNA,
thereby
causing
the double
strand
to spit into separate single
strands.

Molecular cloning
– is process
of creating an identical copy of DNA fragments. Phage - derivatives of bacteriophage
lambda; linear DNA molecules, whose region can be replaced with foreign DNA without
disrupting its life cycle. Plasmid - an extrachromosomal circular DNA molecule
that autonomously replicates inside the bacterial cell.

Promoter
- a specific
DNA sequence that serves as a binding site for RNA polymerase near each gene.

Replicon – a block of DNA between
two adjacent replication origins.

Vector – is an agent that can carry
out a DNA fragment into a host cell.

Conditional shortenings

BAC – Bacterial Artificial Chromosome

cos
– cohesice end site

DNA - deoxyribonucleic acid

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 process
of creating an identical copy of something. In Biology, it collectively refers
to processes used to create copies of DNA fragments (Molecular Cloning), cells
(Cell Cloning), or organisms. The term also encompases situations, whereby organisms
reproduce asexually, but in common parlance refers to intentionally created copies
of organisms.

In
1972, 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. The
two molecules that are required for cloning are the DNA to be cloned and a cloning
vector.

Cloning vector - a DNA molecule that carries
foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell and
produces many copies of itself and the foreign DNA. Types of Cloning Vectors are
Plasmid, Phage, Cosmids.

Molecular cloning
refers to the process of making multiple molecules. Cloning is commonly used to
amplify DNA fragments containing whole genes, but it can also be used to amplify
any DNA sequence such as promoters, non-coding sequences and randomly fragmented
DNA. It is used in a wide array of biological experiments and practical applications
ranging from genetic fingerprinting to large scale protein production. Occasionally,
the term cloning is misleadingly used to refer to the identification of the chromosomal
location of a gene associated with a particular phenotype of interest, such as in
positional cloning. In practice, localization of the gene to a chromosome or genomic
region does not necessarily enable one to isolate or amplify the relevant genomic
sequence. To amplify any DNA sequence in a living organism, that sequence must
be linked to an origin of replication, which is a sequence of DNA capable of directing
the propagation of itself and any linked sequence. However, a number of other features
are needed and a variety of specialised cloning vectors (small piece of DNA into
which 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 any
DNA fragment essentially involves four steps

· 
fragmentation - breaking apart a strand of
DNA

· 
ligation - gluing together pieces of DNA
in a desired sequence

· 
transfection - inserting the newly formed
pieces of DNA into cells

· 
screening/selection - selecting out the cells
that were successfully transfected with the new DNA

Recombinant DNA techniques have allowed the isolation and propagation of
specific DNA fragments which can be easily sequenced and/or used as highly specific
probes. In vitro site-directed modifications of these fragments and their reintroduction
into the genome result in a modified genetic makeup of an organism. In addition,
it is now possible to induce overproduction of commercially important proteins
by 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, Escherichia
coli 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 information
is readily available. Many cloning vectors have also been constructed for use with
E. coli as a host. Although this review focuses on the basic and applied aspects
of bacteriophage lambda ectors, an overview of other vectors is included for comparison.

In general, cloning vectors can be broadly classified as plasmid and phage
vectors.

So, the aim of
this work is: to consider and study vectors of molecular cloning, and functions,
properties etc.

Literature
review

Plasmids are useful
for a wide range of molecular genetic, genomic and proteomic approaches. In recent
years, plasmid clone production has increased dramatically in response to the availability
of genome information and new technologies.[9]

In 1952, Joshua Lederberg coined the term plasmid to describe any
bacterial genetic element that exists in an extrachromosomal state for at least
part of its replication cycle. As this description included bacterial viruses,
the definition of what constitutes a plasmid was subsequently refined to describe
exclusively or predominantly extrachromosomal genetic elements that replicate autonomously.
[1]

Most plasmids possess a circular geometry, there are now many examples
in a variety of bacteria of plasmids that are linear. As linear plasmids require
specialized mechanisms to replicate their ends, which circular plasmids and chromosomes
do not, linear plasmids tend to exist in bacteria that also have linear chromosomes
[1]

Plasmids, like chromosomes, are replicated during the bacterial cell cycle
so that the new cells can each be provided with at least one plasmid copy at cell
division [1]

Frederick Twort (1915) and Felix d’Herelle (1917) were the first to recognize
viruses which infect bacteria, which d'Herelle called bacteriophages (eaters of
bacteria).
[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 before
lysing the host cell, releasing the progeny phage to infect new hosts. In lysogeny,
the phage genome undergoes recombination into the host chromosome, where it is
replicated and inherited along with the host DNA. [2]

Cosmids
- an extrachromosomal circular DNA molecule that combines features of plasmids and
phage. [8]

Cosmids are conventional vectors that contain a small region of bacteriophage
λ DNA containing the cohesive
end site (cos). This contains all of the cis-acting elements for packaging of viral DNA
into λ particles [4]

1. 
Plasmid
Vectors

In 1952, Joshua Lederberg coined the term plasmid to describe any
bacterial genetic element that exists in an extrachromosomal state for at least
part of its replication cycle. As this description included bacterial viruses,
the definition of what constitutes a plasmid was subsequently refined to describe
exclusively or predominantly extrachromosomal genetic elements that replicate autonomously.

Figure 1. Joshua Lederberg

Plasmid vectors are convenient for cloning of small DNA fragments for restriction
mapping and for studying regulatory regions. However, these vectors have a relatively
small insert capacity. Therefore, a large number of clones are required for screening
of a single-copy DNA fragment of higher eukaryotes. Second, the handling and storage
of these clones is time-consuming and difficult. The repeated subcultures of recombinants
may 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

General-purpose cloning vectors

Cloning of foreign DNA fragments in general-purpose cloning vectors [11]
selectively inactivates one of the markers (insertional inactivation) or derepresses
a silent marker (positive selection) so as to differentiate the recombinants from
the native phenotype of the vector.

Expression vectors

In expression vectors, DNA to be cloned and expressed is inserted downstream
of a strong promoter present in the vector. The expression of the foreign gene is
regulated by the vector promoter irrespective of the recognition of its own regulatory
sequence.

Promoter probe and terminator probe vectors

Promoter probe and terminator probe vectors are useful for the isolation of
regulatory sequences such as promoters or terminators and for studying their recognition
by a specific host. They possess a structural gene devoid of the promoter or the
terminator sequence [8].

Figure 3. Replication of rolling-circle plasmids

2. 
Cosmids

A cosmid, first
described by Collins and Hohn in 1978, is a type of hybrid plasmid (often used as
a cloning vector) that contains cos sequences, DNA sequences originally from the
Lambda phage. Cosmids can be used to build genomic libraries.

Cosmids are able
to contain 37 to 52 kb of DNA, while normal plasmids are able to carry only
1–20 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 replication
or f1 ori for single-stranded DNA replication in prokaryotes. They frequently also
contain a gene for selection such as antibiotic resistance, so that the transfected
cells can be identified by plating on a medium containing the antibiotic. Those
cells 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 be
transferred into or between cells by transduction. Plasmids become unstable after
a certain amount of DNA has been inserted into them, because their increased size
is more conducive to recombination. To circumvent this, phage transduction is used
instead. This is made possible by the cohesive ends, also known as cos sites. In
this way, they are similar to using the lambda phage as a vector, but only that
all 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 where
DNA is nicked at each strand, 12bp apart, by terminase. This causes linearization
of 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 holds
the terminase while it is nicking and separating the strands. The cosQ site of
next cosmid (as rolling circle replication often results in linear concatemers)
is held by the terminase after the previous cosmid has been packaged, to prevent
degradation by cellular DNases.

Figure 4. Cloning by using Cosmid method

Cosmid features
and uses

Cosmids are predominantly
plasmids 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, shuttle
cosmids or 'mammalian' cosmids (linked to SV40 oriV and mammalian selection markers)
are available. The loading capacity of cosmids varies depending on the size of
the vector itself but usually lies around 40–45 kb. The cloning procedure involves
the generation of two vector arms which are then joined to the foreign DNA. Selection
against wildtype cosmid DNA is simply done via size exclusion. Cosmids, however,
always form colonies and not plaques. Also the clone density is much lower with
around 105 - 106 CFU per µg of ligated DNA.

After the construction
of recombinant lambda or cosmid libraries the total DNA is transferred into an appropriate
E.coli host via a technique called in vitro packaging. The necessary packaging extracts
are derived from E.coli cI857 lysogens (red- gam- Sam and Dam (head assembly) and
Eam (tail assembly) respectively). These extracts will recognize and package the
recombinant molecules in vitro, generating either mature phage particles (lambda-based
vectors) or recombinant plasmids contained in phage shells (cosmids). These differences
are reflected in the different infection frequencies seen in favour of lambda-replacement
vectors. This compensates for their slightly lower loading capacity. Phage library
are also stored and screened easier than cosmid (colonies!) libraries.

Target DNA: the
genomic DNA to be cloned has to be cut into the appropriate size range of restriction
fragments. This is usually done by partial restriction followed by either size
fractionation or dephosphorylation (using calf-intestine phosphatase) to avoid chromosome
scrambling, 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 filamentous
phage .

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 II
product of the incoming virus.

Interaction of the intergenic region of the plasmid with the gene II protein
initiates the rolling-circle replication to generate copies of one strand of the
plasmid DNA, which are packaged into progeny bacteriophage particles. The single-stranded
DNA purified from these particles is used as a template to determine the nucleotide
sequence of one strand of the foreign DNA segment, for site-directed mutagenesis
or as a strand-specific probe. Phagemids provide high yields of double-stranded
DNA and render unnecessary the time-consuming process of subcloning DNA fragments
from plasmids to filamentous bacteriophages.

4. 
Bacteriophage
Vectors

Both single-stranded (filamentous) and double-stranded E.coli phages have
been exploited as cloning vectors.

Frederick Twort (1915) and Felix d’Herelle (1917) were the first to recognize
viruses which infect bacteria, which d'Herelle called bacteriophages (eaters of
bacteria). [7]

Figure 5. Frederick Twort and Felix d’Herelle

4.1 Filamentous phages

Filamentous phages are not lytic. They coexist with the infected cells for
several generations and are convenient for cloning genes which produce toxic products.
Among the filamentous phages, fd, fl, and M13 have been well characterized and
their genomes have been sequenced [4]. Their gene functions and molecular mode of
propagation are very similar. They infect cells via F pili, and the first mature
phage appears within 15 min [6].

Phage M13 is widely used in nucleotide sequencing and site-directed mutagenesis
since its genome can exist either in a single-stranded form inside a phage coat
or as a doublestranded replicative form within the infected cell. During replication,
only the plus strand of the replicative form is selectively packaged by the phage
proteins [1]. The replicative form is a covalently closed circular molecule and
hence can be used as a plasmid vector and transformed into the host by the usual
transformation procedures. The vectors derived from M13, have the same polylinker
as that of pUC18 and pUC19, respectively [2]. The DNA fragments having noncomplementary
ends can be directionally cloned in this pair of vectors, and the two strands of
DNA can be sequenced independently.

4.2.Double-stranded phage vectors

Of the double-stranded phages, bacteriophage lambda-derived vectors are the
most popular tools for several reasons:

· 
acceptance by the phage of large foreign
DNA fragments, thereby increasing the chances of screening a single clone carrying
a DNA sequence corresponding to a complete gene;

· 
development and availability of refined
techniques aimed at minimizing the problems of background due to nonrecombinants;

· 
the possibility of screening several thousand
clones at a time from a single petri plate; and, finally,

· 
the ease with which the phage library can
be stored as a clear lysate at 4°C for months without significant loss in plaque-forming
activity [7].

Recently, a bacteriophage P1 cloning system has been developed which permits
cloning of DNA fragments as large as 100 kbp with an efficiency that is intermediate
between cosmids and yeast artificial chromosomes .

5. 
Scope
of Present Review

The extensive knowledge of the basic biology of lambda has permitted modifications
of its genome to suit the given experimental conditions. In the present review
we describe how the utility of lambda as a cloning vector rests essentially in its
intrinsic molecular organization. The following sections give an account of various
problems encountered in constructing lambda vectors and the strategies that have
been adopted to overcome them. A few commonly used vectors are described in detail,
taking into account their special values and limitations. The different methods
for screening and storage of genomic and cDNA libraries in lambda vectors are also
discussed.

6. 
Life
cycle 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 needed
before its applications for genetic manipulations are discussed. This section deals
with the basic biology of lambda.

The lambda virus particle contains a linear DNA of 48,502 bp with a single-stranded
5' extension of 12 bases at both ends; these extensions are complementary to each
other.

These ends are called cohesive ends or cos. During infection, the right
5' extension (cosR), followed by the entire genome, enters the host cell. Both
the cos ends are ligated by E. coli DNA ligase, forming a covalently closed circular
DNA 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 start
its replication; this results in the formation of multiple copies of the genome.
The protein components necessary for the assembly of mature phage particles are
synthesized by the coordinated expression of phage genes. Phage DNA is packaged
inside a coat, and the mature phages are released into the environment after cell
lysis. This mode of propagation is called the lytic cycle.

Alternatively, the phage genome may enter a dormant stage (prophage) by integrating
itself into a bacterial genome by site-specific recombination; during this stage
it is propagated along with the host in the subsequent progeny. This stage is termed
lysogeny. Changes in environmental and physiological conditions may activate the
prophage stage and trigger lytic events.

7. 
Phage
Lambda as a vector

Figure 6. Bacteriophage

The large genome size and complex genetic organization of lambda had posed
initial problems with its use as a vector. The problems, however, were surmounted
through the sustained efforts of researchers, and lambda has been developed into
an efficient vector.

The broad objectives in constructing various phage vectors are

-   the
presence of cloning sites only in the dispensable fragments,

-   the
capacity to accommodate foreign DNA fragments of various sizes,

-   the
presence of multiple cloning sites,

-   an
indication of incorporation of DNA fragments by a change in the plaque type,

-   the
ability to control transcription of a cloned fragment from promoters on the vector,

-   the
possibility of growing vectors and clones to high yield,

-   easy
and ready recovery of cloned DNA,

-   introduction
of 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 them
are discussed in this section. Manipulation of Restriction Sites The major obstacle
to the use of phage lambda as a cloning vector was essentially the presence of
multiple recognition sites for a number of restriction enzymes in its genome.

Initially, all attempts were directed toward minimizing the number of EcoRI
sites. Murray and Murray in 1974 were able to construct derivatives of lambda with
only one or two EcoRI sites. Similarly, Rambach and Toillais constructed lambda
derivatives with EcoRI sites only in the nonessential region of the genome by repeated
transfer on restrictive and nonrestrictive hosts . After several cycles of digestion,
packaging, and growth, phage derivatives with desirable restriction sites and full
retention of infectivity were obtained. All but one HindIII sites were removed
by recombination of known deletion mutants or substitutions. Recently, oligonucleotides
with specific sequences have been synthesized and introduced into the bacteriophage
lambda 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 production
of viable phage particles. The viability of the bacteriophage decreases when its
genome length is greater than 105% or less than 78% of that of wild-type lambda.
Genetic studies of specialized transducing bacteriophages showed, however, that
the 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 fragment
of 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 essential
for lytic growth. However, too much DNA cannot be deleted because there is a minimum
38-kbp requirement essential for efficient packaging. The de novo insertion of
DNA (even if heterogeneous) is essential for the formation of viable phages. This
constitutes a positive selection for recombinant phages carrying insertions. This
approach was successfully exploited in constructing recombinant phages carrying
E. coli and Drosophila melanogaster DNA [8].

7.2 Transfection of Recombinant Molecules

The problem of transfection of recombinant molecules constructed in vitro
was overcome by the successful in vitro assembly of viable and infectious phage
particles. 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 complementation
of two amber mutations. Two lambda lysogens, each carrying a single amber mutation
in a distinctly different gene, are induced and grown separately so that they can
synthesize the necessary proteins. Neither of the lysogens alone is capable of packaging
the phage DNA. The role of various phage products in DNA packaging has been studied
in detail[3]. The E protein is the major component of the bacteriophage head, and
in its absence all the viral capsid components accumulate. The D protein is involved
in the coupled process of insertion of bacteriophage DNA into the prehead precursor
and the subsequent maturation of the head. The A protein is required for the cleavage
of the concatenated precursor DNA at the cos sites. Two phage lysogens carrying
A and E or D and E mutations in the phage genome are induced separately, and cell
extracts 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 phage
DNA with recombinant molecules. In both the cell extracts, native phage DNA is also
present 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 of
regeneration of endogenous phages obtained in the library was partially overcome
by the use of b2-deleted prophages, which poorly excise out of the host chromosome
or by UV irradiation of packaging extracts.

Single-strain packaging.

Rosenberg have successfully developed a single-strain packaging system by
introducing deletion in the cos region of prophage, rendering the prophage DNA unpackagable
because cos is the packaging origin. Induction of the lysogen results in the intracellular
accumulation of all protein components needed for packaging.

However, packaging of phage DNA is prevented by the lack of cos sites on
the prophage DNA. On the other hand, exogenous DNA with cos sites is packaged efficiently
to produce an infectious bacteriophage particle. The single-strain system is superior
to 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 the
lysogen.

7.3 Biological Containment

The biological containment of recombinant phages is an important aspect
from the point of view of ethics and eventual biohazards. It is desirable that cloning
vectors and recombinants have poor survival in the natural environment and require
special laboratory conditions for their replication and survival. According to
Blattner, the lytic phages offer a natural advantage in this respect since the phage
and the sensitive bacteria coexist only briefly. A newly inserted segment may not
be compatible with E. coli metabolism for extended periods. To make the phage vectors
more safe, three amber mutations were introduced in its genome. The new vector
Xgt WES XC is safer because an amber suppressor host strain is a very rare occurrence
in the natural environment. Many vectors carry one of the amber mutations on the
genome so that they can be propagated only on an appropriate suppressor host.

8. 
Phage
vectors

Many phage vectors have been constructed in the recent past, each with its
own special features. There is no universal lambda vector which can fulfill all
the desired objectives of the cloning experiments.

The
choice of a vector depends mainly on

-   the
size of a DNA fragment to be inserted,

-   the
restriction enzymes to be used,

-   the
necessity for expression of the cloned fragment,

-   the
method 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

8.1 Replacement Vectors

Taking advantage of the maximum and minimum genome size essential for efficient
packaging and the presence of the nonessential central fragment, it is possible
to remove the stuffer fragment and replace it with a foreign DNA fragment in the
desired 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 physical
elimination of the nonessential region,

· 
ligation of the foreign DNA fragment between
the arms,

· 
in vitro packaging and infection.

The replacement vectors contain a pair of restriction sites to excise the
central stuffer fragments, which can be replaced by a desired DNA sequence with
compatible ends. The presence of identical sites within the stuffer fragment but
not in the arms facilitates the separation of the arms and the stuffer on density
gradient centrifugation. In many vectors, sets of such sites are provided on attached
polylinkers so that an insert can be easily excised. Two purified arms cannot be
packaged despite their being ligated to each other, because they fall short of
the minimum length required for packaging. This provides positive selection of
recombinants. The replacement vectors are convenient for cloning of large (in some
cases up to 24 kbp) DNA fragments and are useful in the construction of genomic
libraries of higher eukaryotes. Charon and EMBL are among the popular replacement
vectors.

8.2 Insertion Vectors

Because the maximum packagable size of lambda genome is 53 kb, small DNA
fragments can be introduced without removal of the nonessential (stuffer) fragment.
These vectors are therefore called insertion vectors. Cloning of foreign DNA in
these vectors exploits the insertional inactivation of the biological function,
which differentiates recombinants from nonrecombinants. Insertion vectors are particularly
useful in cloning of small DNA fragments such as cDNA.

AgtlO and Agtll are examples of this type of vector. In recent years a multitude
of lambda vectors have been constructed. Many innovative approaches have been used
to introduce desired properties into the vectors. The following section deals with
the strategies adopted for the construction of some of the commonly used vectors
and 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°C
in SM buffer containing 0.3% freshly distilled chloroform (94). The master stocks
of bacteriophage lambda are kept in 0.7% (vol/vol) dimethyl sulfoxide at -70°C
for long-term storage. Klinman and Cohen have developed a method for storage of
a phage library at -70°C by using top agar containing 30% glycerol.

Conclusion

In my work I determined
investigations 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. 
sequences
that permit the propagation of itself in bacteria (or in yeast for YACs) .

2. 
a cloning
site to insert foreign DNA; the most versatile vectors contain a site that can
be cut by many restriction enzymes .

3. 
a method
of selecting for bacteria (or yeast for YACs) containing a vector with foreign
DNA; uually accomplished by selectable markers for drug resistance .

Cloning vector - a DNA molecule that carries
foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell and
produces many copies of itself and the foreign DNA .

General Steps of
Cloning with Any Vector :

1. 
prepare
the vector and DNA to be cloned by digestion with restriction enzymes to generate
complementary ends ;

2. 
ligate
the foreign DNA into the vector with the enzyme DNA ligase;

3. 
introduce
the DNA into bacterial cells (or yeast cells for YACs) by transformation ;

4. 
select
cells containing foreign DNA by screening for selectable markers (usually drug
resistance);

Literature

1. 
Finbar Hayes The Function and Organization
of Plasmids// E. coli Plasmid Vectors Methods and Applications.- 2007.- vol.235
– pp. 1-18.

2. 
Mallory J. A. White and Wade A. Nichols Cosmid
Packaging and Infection of E. coli// E. coli Plasmid Vectors Methods and Applications.-
2007.- vol.235 – pp. 67-70

3. 
Tim S. Poulsen and Hans E. Johnsen BAC
End 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,
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