Vector: Definition, Properties and types & examples. - MyTecNika

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Vector: Definition, Properties and types & examples.

 VECTOR

A Vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell and into which the DNA fragment to be cloned (called DNA insert) is integrated for cloning. Therefore, a vector must have an origin of DNA replication (denoted as ori) that functions in the host cell. Any extra-chromosomal small genome, e.g., plasmid, phage or virus, may be used as a vector.

Properties of A Good Vector

A good vector must have the following properties.

1. It should be able to replicate autonomously. When the objective of cloning is to obtain a large number of copies of the DNA insert, the vector replication must be under relaxed control so that it can generate multiple copies of itself in a single host cell.

2. A vector should be ideally less than 10 kb in size because large DNA molecules are broken during purification procedure. In addition, large vectors present difficulties during various manipulations required for gene cloning

3. The vector should be easy to isolate and purify.

4. It should be easily introduced into the host cells, i.e., transformation of the host with the vector should be easy.

5.The vector should have suitable marker genes that allow easy detection and/or selection of the transformed host cells.

6. When the objective is gene transfer, it should have the ability to integrate either itself or the DNA insert it carries into the genome of the host cell.

7. The cells transformed with such vector molecules that contain the DNA insert (recombinant DNA) should be identifiable or selectable from those transformed by the vector molecules only.& A vector should contain unique target sites for as may restriction enzymes as possible into which the DNA insert can be integrated without disrupting an essential function.

9. When expression of the DNA insert is desired, the vector should contain at least suitable control elements, e.g., promoter, operator and ribosome binding sites; several other features may also be important.

 It should be kept in mind that (

1) the DNA molecules used as vectors have coevolved with their specific natural host species, and hence are adapted to function well in them and in their closely related species. Therefore, the choice of vector depends largely on the host species into which the DNA insert or gene is to be cloned. In addition,

 (2) most naturally occurring vectors do not have all the required functions; therefore, useful vectors have been created by joining together segments performing specific functions (called modules) from two or more natural entities. A brief description of some of the important vectors used in different hosts is given below.

 Cloning And Expression Vectors

All vectors used for propagation of DNA inserts in a suitable host are called cloning vectors. But when a vector is designed for the expression of, i.e., production of the protein specified by, the DNA insert, it is termed as expression vector. As a rule, such vectors contain at least the regulatory sequences, i.e., promoters, operators, ribosomal binding sites, etc., having optimum function in the chosen host. It is desirable that all cloning vectors have relaxed replication control so that they can produce multiple copies per host cell.

When an eukaryotic gene is to be expressed in a prokaryote, the eukaryotic coding sequence has to be placed after prokaryotic promoter and ribosome binding site, since the regulatory sequences of eukaryotes are not recognised in prokaryotes. In addition, eukaryotic genes, as a rule, contain introns (noncoding regions) present within their coding regions. These introns must be removed to enable the proper expression of eukaryotic genes since prokaryotes lack the machinery needed for their removal from the RNA transcripts. When eukaryotic genes are isolated as cDNA, they are intron-free and suitable for expression in prokaryotes.

Several strategies have been attempted for the construction of expression vectors using regulatory sequences of the appropriate hosts. These approaches may be grouped into the following two broad categories.

1. Construction of vectors allowing the synthesis of fusion proteins comprising amino acids encoded by a sequence in the vector and those encoded by the DNA insert (translational fusion).

2. Development of vectors permitting the synthesis of pure proteins encoded exclusively by the DNA inserts (transcriptional fusion).