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Cloning Vector Types and Characteristics

In this cloning vector types and characteristics post we have briefly explained about cloning vector features and  types and characteristics of cloning vectors.

cloning Vector Features and Characteristics

Vectors are the DNA molecules, which carry a foreign DNA fragment to be cloned. They are cloning DNA vehicles, examples of which are Plasmids, Bacteriophages, cosmids, phagemids and artificial chromosomes.

The vector types differ in the molecular properties they have and in the maximum size of DNA that can be cloned into each.

Vectors Characteristics

Self-replicating, multiple copies.

Replication origin site.

Cloning DNA site.

Selectable marker gene.

Low molecular weight.

Easily isolates and purifies.

Easily isolates into host cells.

Types of Vectors

Vectors can be classified into different groups depending on the purpose of the process and the type of particles used in the process. The following are the commonly studied group of vectors that are used for different purposes;

1. Bacterial Plasmid

Bacterial plasmids are extra chromosomal elements that replicate autonomously in cells. Their DNA is circular and double stranded and carries sequences required for plasmid replication (ori sequence) and for the plasmid’s other functions. Example: Streptomyces species, Borellia burgdorferi).

The size of plasmids varies from 1to 500 kb. Plasmids were the first cloning vectors. DNA fragments of about 570kb are efficiently cloned in plasmid cloning vectors. Plasmids are the easiest to work with.

They are easy to isolate and purify, and they can be reintroduced into a bacteria by transformation. Naturally occurring plasmid vectors rarely possess all the characteristics of an ideal vector.

Hence plasmid cloning vectors are derivatives of natural plasmids and are “engineered” to have features useful for cloning DNA.

Examples

pBR 322 (plasmid discovered by Bolivar and Rodriguez 322) and pUC 19 (plasmid from University of California).

Herbert Boyer and Stanley Cohen in 1973 showed it was possible to transplant DNA segments from a frog into a strain of Escherichia coli using pSC101, a genetically modified plasmid, as the vector.

The work laid the foundation for the birth of Genetech, the first company dedicated to commercialization of recombinant DNA.

Features

It has a high copy number; so many copies of a cloned piece of DNA can be generated readily. It has amp R (ampicillin resistant) selective marker

It has a number of unique restriction sites clustered in one region, called a multiple cloning site (MCS) or polylinker.

The MCS is inserted into part of the E.coli β – galactosidase (lac Z+ ) gene. Figure illustrates how a piece of DNA can be inserted into a plasmid cloning vector such as pUC19.

Vector

(a) PUC19 (b) PBR322

Insertion of a piece of DNA into the plasmid cloning vector pUC19 to produce a recombinant DNA molecule.

2. Bacteriophage

They are viruses that replicate within the bacteria. A phage can be employed as vector since a foreign DNA can be spliced into phage DNA, without causing harm to phage genes.

The phage will reproduce (replicate the foreign DNA) when it infects bacterial cell. Both single and double stranded phage vectors have been employed in recombinant DNA technology.

Derivatives of phage can carry fragments up to about 45 kb in length. PI bacteriophage can carry fragments up to 95 kb.

Structure of phage λ

Phage λ consists of a head and a tail (both proteins). The DNA, located in the head, is a linear molecule of about 50 kb. At each end of the DNA, there are single stranded extensions called cohesive (cos) ends.

On attachment with tail to E.coli, phage injects its DNA into the cell. Inside E.coli the phage linear DNA cyclizes and gets ligated through cos ends to form a circular DNA . The phage DNA has two fates – lytic cycle and lysogenic cycle.

Life cycle of phage λ

Only about 50% of phage λ DNA is necessary for its multiplication and other functions. Thus, as much as 50% (i.e. up to 20 kb) of the phage DNA can be replaced by a donor DNA for use in cloning experiments.

However, several restriction sites are present on phage which is not by itself a suitable vector. The λ based phage vectors are modifications of the natural phage with much reduced number of restriction sites.

The main advantage of using phage vectors is that foreign DNA can be packed into the phage (invitro packaging), the latter in turn can be injected into the host cell very effectively (Note: no transformation is required).

Cloning using a λ phage

3. Cosmids

Cosmids are the vectors possessing the characteristics of both plasmid and bacteriophage. Cosmids can be constructed by adding a fragment of phage λ DNA including cos site, to plasmids.

Once inside the host cell, cosmids behave just like plasmids and replicate. The advantage with cosmids is that they carry larger fragments of foreign DNA (35–45 kb) compared to plasmids.

4. Phagemids

Phagemids are the combination of plasmid and phage and can function as either plasmid or phage. Since they posses functional origins of replication of both plasmid and phage λ they can be propagated (as plasmid or phage) in appropriate E.coli.

5. Artificial chromosome Vectors

Artificial chromosomes are cloning vectors that can accommodate very large pieces of DNA, producing recombinant DNA molecules resembling small chromosomes.

a. YAC

Yeast artificial chromosome YAC is a synthetic DNA that can accept large fragments of foreign DNA between 0.2 to 2.0 Mb (particularly human DNA). In addition to origin of replication sequence and selectable markers they possess centromeric and telomeric regions, and therefore the recombinant DNA can be maintained like a yeast chromosome.

b. BACs

Bacterial Artificial chromosomes (BACs) : BACS can accept DNA inserts around 300 kb. A major part of the sequencing of human genome has been accomplished by using a library of BAC recombinant. BACS are vectors containing the origin replication of a natural plasmid called the F factor, a MCS, a selectable marker and often some other features.

6. Plasmid shuttle Vectors

The plasmid vectors that are specifically designed to replicate in two or more different host organisms (say in E.coli and yeast) are referred to as shuttle vectors. The origins of replication for two hosts are combined in one plasmid.

7. Expression vectors

An expression vector is a cloning vector containing the regulatory sequences (promoter sequence) necessary to allow the transcription and translation of a cloned gene or genes.

Expression vectors are essentially derivatives of the plasmid cloning vectors used in the host. They are used to produce the protein encoded by a cloned gene in the transformed host.

For example, the biotechnology industry produces pharmaceutically active proteins with the use of expression vectors and the appropriate host.

Applications

1. Cloning vectors are the most common type of vector used to introduce foreign DNA into host cells for a variety of applications.

2. One of the most important uses of vectors is to create designed organisms that perform a certain purpose, such as engineering E. coli bacteria to produce insulin.

3. Vectors can be used to isolate a specific gene sequence within a genome and utilise DNA sequencing to determine its nucleotide sequence.

4. It also aids in the identification of control and regulatory sequences in genomes for the purposes of research and analysis.

5. Cloning vectors can be used to investigate protein structure, function, and synthesis in many organisms.

6. Phage therapy is a type of treatment in which bacteriophage vectors are used to treat various bacterial infections in people and other animals.

7. Vectors can also be used to detect mutations in various sections of DNA sequences, as well as to diagnose gene abnormalities in illnesses.

Further Readings

Reference

Cloning Vector Types and Characteristics

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