Recombinant DNA Technology: Restriction Endonucleases

Restriction Enzymes

• Restriction enzymes, also known as restriction endonucleases, are enzymes primarily isolated from bacteria that cleave double-stranded DNA (dsDNA).
• They are used by prokaryotes to destroy foreign genetic material, such as viruses, in defense.

Characteristics of Restriction Endonucleases

• Restriction endonucleases target dsDNA with high precision to specific nucleotide sequences (target sequence).
• Some restriction endonucleases are isoschizomers, which recognize and cleave the same target sequence of dsDNA.
• These enzymes generate fragments with 5’-PO4 and 3’-OH endpoints.

Classification of Restriction Endonucleases

• Type I restriction endonucleases:
  • Require ATP to hydrolyze DNA
  • Cause chemical modifications of DNA by methylation
  • Cleave DNA randomly
• Type II restriction endonucleases:
  • Do not require ATP for hydrolytic activity
  • Do not modify DNA by methylation
  • Recognize and cleave dsDNA at specific target sequences
  • Recognition sequence consists of 4-6 base-pairs, with a twofold axis of symmetry
  • Can be cleaved into blunt ends or sticky-ends
  • Example: EcoRI, which recognizes the hexanucleotide sequence GAATTC
• Type III restriction endonucleases:
  • Require ATP to hydrolyze DNA
  • Cause chemical modification of DNA by methylation
  • Recognize and cleave dsDNA at specific target sequences

Naming of Restriction Endonucleases

• Restriction endonucleases are written in italics (3 letter code)
• The first letter (Capital letter) indicates the genus of the organism
• The second and third letter represents an abbreviation of the species
• Prokaryotes with more than one restriction enzyme are also provided a number

Restriction Mapping

• A method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints
• Relies on the use of restriction enzymes
• After a DNA segment has been digested, the resulting fragments can be examined using gel electrophoresis, which separates pieces of DNA according to their size

Cloning and Expression Vectors

• Why genetically modify organisms?
  • To create disease, drought, and pest resistant crops that are nutritionally enhanced
  • To create modified animal models for research regarding cancer, obesity, heart diseases, etc.
  • To create modified mosquitoes to prevent diseases such as malaria
  • To produce drugs, such as insulin, hormones, vaccines, and anti-cancer drugs

Cloning Vectors

• What does it mean to clone?
  • A clone is a population of identical organisms derived from a single parental organism
• Plasmids and bacteriophages are used as cloning vectors
• Vector is used to indicate a vehicle or carrier of a gene/genes

Plasmids

• Extra chromosomal DNA – circular
• Bacteria often have plasmids, which are circular loops of DNA
• Bacteria can also take in new plasmids
• Plasmids have three common features:
  • Replicator (origin of replication)
  • Selectable marker (gene referring resistance to an antibiotic)
  • Cloning site (sequence of one or more restriction sites)

Recombinant/Chimeric Plasmids

• Foreign DNA fragment inserted into plasmid using RE digestion or other cloning method
• The polylinker or MCS (multiple cloning sites) contains various restriction sites and long stretches of nucleotides enabling transcription and translation

Bacteriophages

• Bacteria infecting viruses
• Plasmids can only accommodate a fragment of up to 10 kb
• Bacteriophages, such as bacteriophage λ, can be used to clone fragments of up to 40 kb
• These recombinant bacteriophages replicate in E. coli as plasmids

Shuttle Vectors

• A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species
• Therefore, DNA inserted into a shuttle vector can be replicated in two different host species