Cloning and expression of vectors pdf

  • The genomes of even the simplest cells are much too large to directly analyze in detail at the molecular level and the problem is compounded for complex organisms.
  • The human genome contains about 6 × 109 base pairs (bp) in the 23 pairs of chromosomes.
  • Cleavage of human DNA with restriction enzymes that produce about one cut for every 3000 base pairs yields some 2 million fragments, far too many to separate from each other directly.
  • This obstacle to obtaining pure DNA samples from large genomes has been overcome by recombinant DNA technology.
  • With this method, any gene can be purified.
  • Its sequence determined, the functional regions of the sequence explored by altering it in planned ways and reintroducing the DNA into cells and into whole organisms.
  • The recombinant DNA technology is the preparation of large numbers of identical DNA molecules.
  • A DNA fragment of interest is linked through standard 3′ → 5′ phosphodiester bonds to a vector DNA molecule, which can replicate when introduced into a host cell.
  • When a single recombinant DNA molecule, composed of a vector plus an inserted DNA fragment, is introduced into a host cell, the inserted DNA is reproduced along with the vector, producing large numbers of recombinant DNA molecules that include the fragment of DNA originally linked to the vector.