Cloning Vector - Definition, Features and Types

Aug 03, 2023, 16:45 IST

Using genetic vectors, foreign DNA may be introduced into recipient cells.

A vector is a DNA molecule used in molecular cloning to intentionally transport foreign genetic information into a different cell so that it can be duplicated and/or expressed.

Vectors can multiply on their own and often have characteristics that make it easier to manipulate DNA as well as a genetic marker for selective identification.

Plasmids, bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and mammalian artificial chromosomes are among the vectors that may be used for cloning. (MACs).

The size of the insert that the cloning vectors can carry is constrained. The best vector is chosen for a given purpose depending on the insert's size and use.

Cloning Vectors

A vector in molecular biology is a DNA molecule that introduces foreign genetic information into a different cell. Vectors come in four main categories: plasmids, viral vectors, cosmids, and artificial chromosomes. A multi-cloning site, an origin of replication, and a selectable marker are features all designed vectors have.

• A transgene insert plus a longer sequence that acts as the vector's "backbone" make up the vector, typically a DNA sequence. It is usually the function of a vector to isolate, multiply, or express the insert in the target cell when genetic information is transferred to another cell.
• Expression vectors (expression constructs) are vectors designed specifically to express the transgene in the target cell. They typically have a promoter sequence that activates transgene expression.
• Contrary to expression vectors, simpler vectors known as transcription vectors can only replicate in a target cell but cannot express themselves. The insert of expression vectors is amplified using means of transcription vectors.
• Although the insertion of a viral vector is frequently referred to as transduction, it is typically called transformation for bacterial cells and transfection for eukaryotic cells.
• A double-stranded, often circular DNA sequence called plasmid may autonomously replicate inside target host cells.

Also Check - Climate Factor Changes

Cloning Vector Characteristics

All vectors are carrier DNA molecules, regardless of the kind of vector chosen.

These carrier molecules generally need to share a few characteristics, like

• It needs to reproduce on its own inside the host cell.
• It must have a special restriction site for restriction enzymes.
• The introduction of donor DNA fragments must not hamper the vector's ability to replicate itself.
• It must have a marker gene, often an antibiotic resistance gene missing from the host cell, that may be used to identify the recombinant cell later.
• It should be simple to extract from the host organism.

Also Check - Classification Of Animal Kingdom

Origin of replication (Ori) in the host cell

• To reproduce inside the host cell, a vector must include the required nucleic acid sequences.
• Using the host cell's replication apparatus, the vector's replication begins at the Ori sequence. (DNA polymerase and other enzymes).
• The majority of the time, ori sequences have more A and T nucleotides than G and C. In comparison to G-C pairings, A-T pairs have two hydrogen bonds. As a result, the DNA becomes single-stranded, which is necessary for efficient DNA replication, thanks to AT-rich sequences in the ori region.

Copy Number

• The number of copies of a vector molecule kept in the host cell is the copy number. This is inextricably linked to the vector's capacity for replication.
• Replication from a vector's ori is regulated by relaxed and rigorous mechanisms.
• In general, vectors with relaxed replication control have a higher copy number than those with strict control.
• With the relaxed process, the host cell's replication apparatus is not required to start the vector's replication. Host machinery is only needed during the elongation and termination phases.
• Stringent control is characterised by the requirement of the host's replication apparatus for vector replication to begin.

Sites for Multiple Clones (MCS)

• Multiple cloning sites are the crucial component of a plasmid cloning vector. (MCS). The vector molecule must have the proper sequences that simplify incorporating an insert fragment. This is what the MCS does. A multiple cloning site is a brief section of DNA on a vector with several restriction enzyme cut sites. For the identification and operation of restriction enzymes, these sites are crucial.
• Multiple restriction endonuclease cleavage sites are most frequently seen in the MCS. FoI and the vector are digested with the same restriction endonucleases to clone FoI into the vector.
• This causes the vector and the FoI that is supposed to be put into it to have "sticky ends."
• The DNA ligase enzyme is then used to combine the complementarily digested vector-insert pair.

Selectable Marker

It is necessary to establish if the vector is present or not within the host cell. It is crucial that such a confirmation of vector presence in the host cells may be performed quickly by utilising a visually distinct phenotype.

For this reason, the vector carries at least one gene that gives the host cells a unique phenotype. It is simple to compare host cells with this gene to those without it.

These selectable markers, also known as marker genes in the vector, are frequently antibiotic-resistance cassettes.

Only host cells with the vector will thrive (positive selection) when plated on a medium containing the selection pressure (the matching antibiotic), whereas host cells without the vector will perish.

Sequence components required for the expression of cloned DNA fragment

A promoter sequence is necessary on the vector to stimulate transcription of the cloned FoI using the host cell's transcriptional machinery. The FoI may be cloned downstream of the promoter owing to the vector backbone's construction. A section of DNA known as a promoter binds the RNA polymerase to start transcription.

• The elements of a sequence for translation

To enable the effective translation of the FoI mRNA, the vector backbone must have the appropriate genetic sequence elements if it is intended to produce the protein product of the FoI cloned on the vector. The most basic needs are:

• Start codon for the ribosome binding site (RBS)

These critical areas must be properly planned for the host cell. For the bacterial ribosome machinery to efficiently drive the translation of the FoI mRNA product, the vector must contain the Shine Dalgarno sequence if the host cell is a bacterium.

Additionally, depending on the experiment's needs, the vectors may include the right signal sequences to make it easier to add particular amino acid sequences to the finished protein product of the cloned FoI.

Cloning Vector Types

Plasmids

Plasmids are circular double-stranded DNA replicating elements with an additional chromosomal component found in bacterial cells.

• The size of plasmids ranges from 5.0 kb to 400 kb.
• Transformation is the process through which plasmids are introduced into bacterial cells.
• Plasmids can hold a DNA fragment with an insert size of up to 10 kb.
• Plasmid vectors often contain a marker gene that is primarily a gene for antibiotic resistance. As a result, any cell that carries the plasmid will grow in the presence of the selectable matching antibiotic in the culture.

Bacteriophage

• The term "bacteriophage" refers to viruses that infect bacteria. These obligate intracellular parasites feed on part or all of the host enzymes to grow inside the bacterial cell.
• Bacteriophages are very effective in introducing their genome into bacterial cells.
• The majority of the bacteriophage genome is non-essential and may be replaced with foreign DNA, therefore, it can be utilised as a cloning vector to transport bigger DNA segments.
• It is possible to alter DNA fragments up to 20 kb in size using bacteriophage as the vector.

Bacterial Artificial Chromosomes (BACs)

• Bacterial artificial chromosomes (BACs) are small plasmids that may be used to clone DNA pieces that are 75 to 300 kb in size.
• The key distinguishing characteristics of BACs include marker-like features, such as antibiotic resistance genes, and a very stable origin of replication (ori), which encourages the dispersion of plasmid following bacterial cell division and keeps the plasmid copy number to one or two per cell.
• The main use of BACs in genome projects is the sequencing of organisms' genomes.
• BACs may be used to clone DNA segments of a few hundred thousand base pairs.

Yeast Artificial Chromosomes (YACs)

A large DNA segment with sizes ranging from 100 kb to 3000 kb can be cloned using YACs, which are yeast expression vectors.

Compared to BACs, YACs have an advantage in expressing eukaryotic proteins that need post-translational modifications. However, YACs are known to produce chimeric effects, making them less stable than BACs. They are typically used for cloning huge DNA fragments and physically mapping complex genomes.

Human Artificial Chromosomes (HACs)

• Mammalian or human artificial chromosomes, often known as HACs or MACs, are still being developed.
• HACs are tiny chromosomes that can substitute for a human cell population's missing chromosome.
• The size of HACs, which contain novel genes discovered by human researchers, ranges from 6 to 10 Mb.
• HACs may be utilised as carriers for introducing novel genes. They can also be used to examine gene expression and understand the role of mammalian chromosomes in the mammalian system.

The Utilisation of Vectors

Because of how easy, affordable, and quick the procedure is, the use of vectors in molecular biology and genetic engineering has grown. Major uses of vectors in molecular biology include the following:

• The most crucial class of vectors utilised for introducing foreign DNA into host cells for various applications is the cloning vector family.
• One of the most significant uses of vectors is the creation of designed organisms with a specific purpose, such as altering E. coli bacteria to produce insulin.
• Vectors can be used to extract a specific gene sequence from a genome so that DNA sequencing can be used to identify its nucleotide sequence.
• Additionally, it aids in identifying control and regulatory sequences in genomes for research and analysis.
• Cloning vectors can be used to investigate the composition, function, and synthesis of proteins in various species.
• Phage therapy is a type of medicine that treats various bacterial diseases in people and other animals by using bacteriophage vectors.
• Aside from detecting mutations in specific DNA sequences, vectors may also be used to diagnose gene abnormalities linked to certain illnesses.
• Clinical microbiology has exploited recombinant DNA technology in various ways, including recombinant antigens, vaccinations, and diagnostic probes.
• To detect illnesses like HIV, HCV, and CMV, recombinant antigens have been created using cloning procedures.
• One of the elements in molecular biology known as vectors allow for various investigations on cell structure, nucleic acid content, and genetic engineering methods.

Cloning Vector: FAQs

Q1. Does pBR322 function as a cloning vector?

Ans. The plasmid cloning vector pBR322 DNA is used in E coli. The molecule is a double-stranded circular with 4,361* base pairs. Chloramphenicol can amplify the genes in pBR322 that confer resistance to tetracycline and ampicillin.

Q2. What does "cloning in a vector" mean?

Ans. The foreign DNA is replicated and expressed using the host cell machinery. It multiplies one copy of DNA into several copies.

Q3. What are some examples of cloning vectors?

Ans. Cloning vectors are small genomes into which foreign DNA may be inserted and transferred to a host cell for cloning. Examples include cosmids, bacteriophages, and plasmids.

Q4. Do cloning vectors include cosmids?

Ans. A hybrid plasmid known as a cosmid contains the cos gene from the Lambda phage. They are frequently employed in genetic engineering as a cloning vector. One may create genomic libraries using cosmids. Collins and Hohn introduced them in 1978.