Cloning Vector : Features and their types - Practice Questions & MCQ

• The vectors are DNA molecules that can carry a foreign DNA segment and replicate inside the host cell. 
• Vectors may be plasmids, bacteriophages, YACs, BACs, etc.

Plasmid Vectors:

• Plasmids are the extra-chromosomal, self-replicating, and double-stranded closed and circular DNA molecules present in the bacterial cell. 
• The number of properties are specified by plasmids such as antibiotic and heavy metal resistance, nitro­gen fixation, pollutant degradation, bacteriocin and toxin production, colicin factors, etc.
• Plasmids have following advantages as a cloning vehicle:
  • It can be readily isolated from the cells.
  • It possesses a single restriction site for one or more restriction enzymes.
  • The insertion of foreign DNA does not alter the replication properties.
  • It can be reintroduced into the cell.

Bacteriophage Vectors:

• These are the viruses that attack the bacterial cells by injecting the DNA into the bacterial cells.
• The injected DNA is selectively replicated and expressed in the host bacterial cell resulting in a number of phages which burst out of the cell. 
• This ability of bacteriophages to transfer their DNA to the host cell makes them suitable as vectors for the artificial DNA.
• The bacteriophage has a linear DNA molecule, a single break will generate two fragments, foreign DNA can be inserted to generate chimeric phage particle. 

Lambda Phage Vectors:

• λ phages are viruses that can infect bacteria. The major advantage of the λ phage vector is its high transformation efficiency, about 1000 times more efficient than the plasmid vector.
• The extreme ends of the λ DNA are known as COS sites, each is single-stranded, 12 nucleotides long. 
• Because their sequences are complementary to each other, one end of λ DNA may base-pair with the other end of a different λ DNA, forming concatemers. 
• The two ends of a λ DNA may also bind together, forming a circular DNA. 
• In the host cell, the λ DNA circularizes because ligase may seal the join of the COS sites.

Cosmid:

• The cosmid vector is a combination of the plasmid vector and the COS site which allows the target DNA to be inserted into the λ head. 
• It has the following advantages:
  • High transformation efficiency.
  • The cosmid vector can carry up to 45 kb whereas plasmid and λ phage vectors are limited to 25 kb.

Artificial Chromosomes:

• Yeast Artificial Chromosome (YAC) or Bacterial Artificial Chromosome (BAC) vectors allow cloning of several hundred kb pairs which may represent the whole chromosome. 
• It can be cloned in yeast or bacteria by ligating them to vector sequences that allow their propagation as a linear artificial chromosome.
   

• Origin of replication (ori): 
  • This is a sequence from where replication starts and any piece of DNA, when linked to this sequence, can be made to replicate within the host cells. 
  • This sequence is also responsible for controlling the copy number of the linked DNA. 
  • So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin supports a high copy number.
• Selectable marker: 
  • In addition to ‘ori’, the vector requires a selectable marker, which helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants. 
  • Transformation is a procedure through which a piece of DNA is introduced in a host bacterium. 
  • Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are considered useful selectable markers for E. coli. 
• Cloning sites:
  • In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes. 
  • The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.