- A vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated or expressed. Cloning vector is a small DNA molecule capable of self-replication inside the host cell. Cloning vector is used for replicating donor DNA fragment within host cell. Vectors can replicate autonomously and typically include features to facilitate the manipulation of DNA as well as a genetic marker for their selective recognition.
- The different types of vectors available for cloning are plasmids, bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) and mammalian artificial chromosomes (MACs).
- The cloning vectors are limited to the size of insert that they can carry. Depending on the size and the application of the insert the suitable vector is selected for a particular purpose.
Characteristics of a cloning vectors
- It must be self-replicating inside host cell.
- Introduction of donor DNA fragment must not interfere with replication property of the vector.
- It must possess some marker gene such that it can be used for later identification of recombinant cell (usually an antibiotic resistance gene that is absent in the host cell).
- They should be easily isolated from host cell.
- it must be small in size
- It must possess restriction site for Restriction Endonuclease enzymes
- it must possess multiple cloning site
Types of cloning vectors used in gene cloning:
E. Bacterial Artificial Chromosome (BAC)
F. Yeast Artificial Chromosome (YAC)
G. Human Artificial Chromosome (HAC)
- It is isolated from E.coli
- Size: 4361 bp
- Cloning limit: 0.1-10 kb
- Marker gene: Ampicillin and Tetracycline resistant gene
- Restriction site for various restriction endonucleases
- Plasmids are extra chromosomal circular double stranded DNA replicating elements present in bacterial cells.
- Plasmids show the size ranging from 5.0 kb to 400 kb.
- Plasmids are inserted into bacterial cells by a process called transformation.
- Plasmids can accommodate an insert size of upto 10 kb DNA fragment.
- Generally plasmid vectors carry a marker gene which is mostly a gene for antibiotic resistance; thereby making any cell that contains the plasmid will grow in presence of the selectable corresponding antibiotic supplied in the media.
Advantages of using Plasmids as vectors:
- Easy to manipulate and isolate because of small size.
- More stable because of circular configuration.
- Replicate independent of the host.
- High copy number.
- Detection easy because of antibiotic-resistant genes.
Disadvantages of using Plasmids as vectors:
- Large fragments cannot be cloned.
- Size range is only 0 to 10kb.
- Example: lambda genome
- It is a phase (Virus) genome
- Inset size: 9-15 kb
- size: 48502 bp
- 1/3rd of the bacteriophage genome is non-essential, so that it can be cut, removed and replaced by donor DNA fragment during cloning
- The viruses that infect bacteria are called bacteriophage. These are intracellular obligate parasites that multiply inside bacterial cell by making use of some or all of the host enzymes.
- Bacteriophages have a very high significant mechanism for delivering its genome into bacterial cell. Hence it can be used as a cloning vector to deliver larger DNA segments.
- Most of the bacteriophage genome is non-essential and can be replaced with foreign DNA.
- Bacteriophages or phages are viruses which infect bacterial cells.
- The most common bacteriophages utilized in gene cloning are Phage λ and M13 Phage.
- A maximum of 53 kb DNA can be packaged into the phage.
- If the vector DNA is too small, it cannot be packaged properly into the phage.
Examples: Phage Lambda, M13 Phage, etc.
- Phage Lambda λ
- It has head, tail, and tail fibers.
- Its genome consists of 48.5 kb of DNA and 12 bp ss DNA which comprise of sticky ends at both the terminals. Since these ends are complementary, they are cohesive and also referred to as cos sites.
- Infection by λ phage requires adsorption of tail fibers on the cell surface, contraction of the tail, and injection of the DNA inside the cell.
- M13 Phage
- These vectors are used for obtaining single-stranded copies of the cloned DNA.
- They are utilized in DNA sequencing and in vitro mutagenesis.
- M13 phages are derived from filamentous bacteriophage M13. The genome of M13 is 6.4 kb.
- DNA inserts of large sizes can be cloned.
- From the double-stranded inserts, pure single-stranded DNA copies are obtained.
Types of Phage Vectors
There are 2 types of phage vectors:
- Insertion vectors- these contain a particular cleavage site where the foreign DNA of up to 5-11 kb can be inserted.
- Replacement vectors- the cleavage sites flank a region which contains genes not necessarily important for the host, and these genes can be deleted and replaced by the DNA insert.
Advantages of using Phage Vectors
- They are way more efficient than plasmids for cloning large inserts.
- Screening of phage plaques is much easier than identification of recombinant bacterial colonies.
Phagemids or Phasmid
- They are prepared artificially.
- Phasmid contains the F1 origin of replication from F1 phage.
- They are generally used as a cloning vector in combination with M13 phage.
- It replicates as a plasmid and gets packaged in the form of single-stranded DNA in viral particles.
Advantages of using Phagemids:
- They contain multiple cloning sites.
- An inducible lac gene promoter is present.
- Blue-white colony selection is observed.
Example: super COS1
- size: 7900 bp
- It has combined feature of both phase and plasmid
- Cloning limit: 30-50 kb
- Cosmids are plasmids.
- They are capable of incorporating the bacteriophage λ DNA segment. This DNA segment contains cohesive terminal sites (cos sites).
- Cos sites are necessary for efficient packaging of DNA into λ phage particles.
- Large DNA fragments of size varying from 25 to 45 kb can be cloned.
- They are also packaged into λ This permits the foreign DNA fragment or genes to be introduced into the host organism by the mechanism of transduction.
Advantages of using cosmids as vectors:
- They have high transformation efficiency and are capable of producing a large number of clones from a small quantity of DNA.
- Also, they can carry up to 45 kb of insert compared to 25 kb carried by plasmids and λ.
Disadvantages of using cosmids as vectors:
- Cosmids cannot accept more than 50 kb of the insert.
Bacterial artificial chromosomes (BACs)
- Example: pUvBBAC
- It is artificially synthesized plasmid
- size: 11827 bp
- It is modification of bacterial F-plasmid
- Cloning limit: 35-300 kb
- Marker gene: chloramphenicol resistant gene and lactose metabolizing gene (LacZ)
- BACs basically have marker like sights such as antibiotic resistance genes and a very stable origin of replication (ori) that promotes the distribution of plasmid after bacterial cell division and maintaining the plasmid copy number to one or two per cell.
- BACs are basically used in sequencing the genome of organisms in genome projects (example: BACs were used in human genome project).
- Several hundred thousand base pair DNA fragments can be cloned using BACs.
Advantages of BACs:
- They are capable of accommodating large sequences without any risk of rearrangement.
- BACs are frequently used for studies of genetic or infectious disorders.
- High yield of DNA clones is obtained.
Disadvantages of BACs:
- They are present in low copy number.
- The eukaryotic DNA inserts with repetitive sequences are structurally unstable in BACs often resulting in deletion or rearrangement.
Yeast artificial chromosomes (YACs)
- Example: pYAC3
- It is an artificial chromosome having yeast centromere isolated from Saccharomyces cerevisiae and ligated to bacterial plasmid
- size: 11400 bp
- It has telomere sequence
- Marker: similar as for identification of yeast cell
- Cloning limit: 100-1000 kb
- YACs are yeast expression vectors.
- Mostly YACs are used for cloning very large DNA fragments and for the physical mapping of complex genomes.
- YACs have an advantage over BACs in expressing eukaryotic proteins that require post translational modifications.
- But, YACs are known to produce chimeric effects which make them less stable compared to BACs.
- A large DNA insert of up to 200 kb can be cloned.
- They are used for cloning inside eukaryotic cells. These act as eukaryotic chromosomes inside the host eukaryotic cell.
- It possesses the yeast telomere at each end.
- Both yeast and bacterial cells can be used as hosts.
Advantages of using YACs:
- A large amount of DNA can be cloned.
- Physical maps of large genomes like the human genome can be constructed.
Disadvantages of using YACs:
- Overall transformation efficiency is low.
- The yield of cloned DNA is also low.
Human artificial chromosome (HAC)
- Human artificial chromosomes are artificially synthesized.
- They are utilized for gene transfer or gene delivery into human cells.
- It can carry large amounts of DNA inserts.
- They are used extensively in expression studies and determining the function of the human chromosomes.
Advantages of using HACs:
- No upper limit on DNA that can be cloned.
- it avoids the possibility of insertional mutagenesis.