An Overview of Recombinant DNA Technology

• The methods used for manipulating nucleic acid/ genome (DNA) of an organism are collectively referred to as Recombinant Dna (rDNA) technology or genetic engineering.
• The fundamental theme of rDNA technology is the isolation and propagation of a desired DNA molecule (gene) from a source with an aim to have its product in ample quantity. This technique is called gene cloning.

•  rDNA technology is a two-component system: a compatible host and a vector combination, where the vector provides essential sequences required for its replication in a compatible host which provides various replication functions. 
• The cloning vector should be small in size and have an origin of replication or ori site, unique restriction sites and selectable marker. 
• Plasmids are circular, extra-chromosomal double stranded DNA (dsDNA) capable of autonomous replication. 
• The bacteriophages Lambda (λ) and M13 are the two most common phages whose genomes have been frequently used to make cloning vectors for E. coli host. 
• The bacteriophage lambda, a bacterial virus that infects E. coli, has been widely used as a cloning vector. 
• Typical vectors coming out of lambda genome fall into two broad classes, namely ‘insertion vectors’ and ‘replacement vectors’.
•  M13 is a filamentous bacteriophage of E. coli having genome consisting 6.4kb long circular DNA packaged in a tubular capsid. 
•  An example of M13 based vector for E. coli is M13mp18 which facilitates blue/white selection of recombinants. 
• Cosmids are a type of hybrid (combination) vector that replicate like a plasmid but can be packaged in vitro into lambda phage coats.
• A typical cosmid has replication functions, unique restriction endonuclease sites, and selective markers contributed by plasmid DNA, combined with a lambda DNA segment that includes the joined cohesive ends (cos sites). 
• Phasmids are true combination vectors between phage and plasmid. They are linear duplex DNAs whose ends are lambda segments that contain all the genes required for a lytic infection and the middle segment is linearised plasmid. 
• Among eukaryotic host vector system, the most common is the baker’s yeast, Saccharomyces cerevisiaes, from which YAC’s have been derived through genetic engineering.
• A YAC cloning vector consists of two copies of a yeast telomeric sequence (telomeres are the sequences at the ends of chromosomes), a yeast centromeric sequence, a yeast ARS (an autonomously replicating sequence) and appropriate selectable markers.