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.
Host Vector System
- 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.