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Vectors are DNA molecules that are used as a vehicle to carry foreign DNA fragments into other cells where they can replicate and/or express. Among these most commonly used vector is a plasmid.
Characteristics of vectors:
- Self replicating, multiple copies.
- Replication origin site.
- Cloning site.
- Selectable marker gene.
- Low molecular weight.
- Easily isolates and purifies.
- Easily isolates into host cells.
The molecular cloning process involves the following steps:
Restriction enzymes – helps to cut the DNA of interest.
Ligation – DNA of interest is pasted to the vector DNA with the help of ligase.
Transformation – helps in the introduction of vector DNA into the host cell often by bacteria or yeast. Then the host cells copy the vector DNA along with their own DNA, creating multiple copies of interested DNA.
Isolation – the vector DNA is then isolated (separate )from the host cell and then purify.
Classification of vectors:
a) Cloning vectors – use to clone foreign DNA.
b) Artificial vectors – they are engineered vectors so that any foreign DNA can be transcript in RNA and translate into proteins.
Important features of cloning vectors:-
- Ability to replicate into host cells.
- Unique restriction enzyme sites for insertional cloning.
- Genetic marker to select for host cells containing the vectors.
- Low molecular weight.
Figure: Schematic representation of the pBR322 plasmid, one of the first plasmids widely used as a cloning vector. The pUC plasmid has a high copy number, contains a multiple cloning site (polylinker), a gene for ampicillin antibiotic selection, and can be used for the blue-white screen. Image Source: Wikipedia
Types of vectors:
1) Plasmids- extrachromosomal, double-stranded DNA molecules, circular, self-replicating. Contain origin of replication, allowing for replication-independent and found in prokaryotes.
Classification if plasmids
a) Fertility plasmid- fertility plasmid is also known as F plasmid, contains a transferred gene that allows genes to be transferred from one cell to another through conjugation.
E.g. F plasmid of Escherichia coli
b) Col plasmid- col plasmid contains genes that make bacteriocins (also known as aa colicins), which are protein that kills other bacteria and thus defends the host bacterim.
E.g. ColE1 of Escherichia coli
c) Resistance plasmid – resistance plasmid or R plasmid contains genes that help the bacteria cell defend against the environment, factors such as poisons or antibiotics.
E.g. RP4 in Pseudomonas
d) Degradative plasmid- degradative plasmid helps the host bacterium to digest compounds that are commonly found in nature, such as camphor, xylene, toluene. These plasmids contain the gene for special enzymes that break down specifics compound.
E.g. Tol of Pseudomonas putida
e) Virulence plasmids- when a virulence plasmid is inside the bacterium, it turns that plasmid into pathogen which is an agent of antigens.
E.g. Ti plasmid of Agrobacterium tumefaciens
2) Bacteriophage lambda- phage lambda is a bacteriophage or phage i.e. bacterial virus that uses E. coli as a host cell. Its structure is as a typical phage- head, tail, fibers. Lamda viral genome contains 485kb linear DNA with 12base ssDNA. Sticky end at both ends and ends is complementary in sequence and can hybridize to each other.
E.g. phage lambda, M13phage
3) Cosmid- the Cosmid vectors are the combination of plasmid vector and the cos site which allows the target DNA to be inserted into the lambda head.
4) Artificial chromosomes- artificial chromosomes are DNA molecules or fragments assembled in vitro from define constituents, which guarantee stable maintenance of large DNA fragments with the properties of natural chromosomes. Useful for genome sequencing programs, functional characteristics of entire genome regions, and for transduction of large DNA segment into human and non-human mammalian cells.
Types of artificial chromosomes:
a) Bacterial artificial chromosome (BAC)
b) Yeast artificial chromosome(YAC)
- 5% – https://wikimili.com/en/Cloning_vector
- 4% – https://www.slideshare.net/ag1805x/artificial-vectors
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