Vectors for moleculars cloning

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].

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