restriction enzymes

Examples of restriction enzymes in the following topics:

• Molecular Products from Microbes

  • Restriction enzymes are a specific class of enzymes isolated from various bacteria and archaea, in which they grow naturally as a means of protection against viral infection.
  • The enzymes have the ability to recognize foreign DNA and cut it up.
  • The isolation of approximately 3000 restriction enzymes has allowed molecular biologists to utilize them in processes such as cloning and the production of recombinant DNA .
  • An example of a specific restriction enzyme, EcoRI, which exhibits the ability to target specific sequences within DNA.
  • Describe how Taq polymerase, restriction enzymes and DNA ligase are used in molecular biology

• Recombinant DNA Technology

  • The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted.
  • The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with that at the ends of the foreign DNA.
  • Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme, for example EcoRI.
  • The purified DNA is then treated with a restriction enzyme to generate fragments with ends capable of being linked to those of the vector.
  • If necessary, short double-stranded segments of DNA (linkers) containing desired restriction sites may be added to create end structures that are compatible with the vector.

• Obtaining DNA

  • The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted.
  • The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with that at the ends of the foreign DNA.
  • Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme.
  • DNA prepared from the vector and foreign source are treated with restriction enzymes to generate fragments with ends capable of being linked to those of the vector and they are simply mixed together at appropriate concentrations and exposed to an enzyme (DNA ligase) that covalently links the ends together.
  • The former can therefore be amplified and screened for the presence of the gene of interest in the cloning vector by restriction digest analysis.

• Vectors for Genomic Cloning and Sequencing

  • Modern plasmids generally have many more features, notably including a "multiple cloning site" which includes nucleotide overhangs for insertion of an insert, and multiple restriction enzyme consensus sites to either side of the insert.
  • The vectors can be extracted from the bacteria, and the multiple cloning sites can be cut by restriction enzymes to excise the hundredfold or thousandfold amplified insert.

• Enzymes Used in Industry

  • Synthetic molecules, called artificial enzymes, also display enzyme-like catalysis.
  • Many drugs and poisons are enzyme inhibitors.
  • In addition, some household products use enzymes to speed up biochemical reactions (e.g., enzymes in biological washing powders break down protein or fat stains on clothes; enzymes in meat tenderizers break down proteins into smaller molecules, making the meat easier to chew).
  • Industrially-produced barley enzymes are widely used in the brewing process to substitute for the natural enzymes found in barley.
  • In molecular biology, restriction enzymes, DNA ligase, and polymerases are used to manipulate DNA in genetic engineering, important in pharmacology, agriculture and medicine, and are essential for restriction digestion and the polymerase chain reaction.

• DNA Sequencing of Insertion Sites

  • Also, the length of each restriction DNA fragment containing a target sequence must be determined by Southern hybridization followed by sub-genomic fractioning before intramolecular ligation and PCR amplification.
  • It involves cutting genomic DNAs with a restriction enzyme, ligating vectorettes to the ends, and amplifying the flanking sequences of a known sequence using primers derived from the known sequence along with a vectorette primer.

• Extremely Halophilic Archaea

  • In archaea, this adaptation is restricted to the the extremely halophilic family Haloarchaea (often known as Halobacteriaceae).
  • To use this method, the entire intracellular machinery - including enzymes, structural proteins, and charged amino acids that allow the retention of water molecules on their surfaces - must be adapted to high salt levels.

• Cofactors and Energy Transitions

  • These proteins are commonly enzymes.
  • An inactive enzyme without the cofactor is called an apoenzyme, while the complete enzyme with cofactor is the holoenzyme.
  • Some enzymes or enzyme complexes require several cofactors.
  • An exception to this wide distribution is a group of unique cofactors that evolved in methanogens, which are restricted to this group of archaea.
  • Each class of group-transfer reaction is carried out by a particular cofactor, which is the substrate for a set of enzymes that produce it and a set of enzymes that consume it.

• Polyketide Antibiotics

  • Secondary metabolites are often restricted to a narrow set of species within a phylogenetic group.
  • They are broadly divided into three classes: type I polyketides (often macrolides produced by multimodular megasynthases), type II polyketides (often aromatic molecules produced by the iterative action of dissociated enzymes), and type III polyketides (often small aromatic molecules produced by fungal species).
  • Polyketides are synthesized by one or more specialized and highly complex polyketide synthase (PKS) enzymes.

• Synthesizing DNA

  • Whereas enzymes synthesize DNA and RNA in a 5' to 3' direction, chemical oligonucleotide synthesis is carried out in the opposite, 3' to 5' direction.
  • They are most commonly used as antisense oligonucleotides, small interfering RNA, primers for DNA sequencing and amplification, probes for detecting complementary DNA or RNA via molecular hybridization, tools for the targeted introduction of mutations and restriction sites, and for the synthesis of artificial genes.