RESTRICTION ENZYME

• Restriction Enzyme (RE)


  A type of nucleases protein that cut sugar-phosphate backbone of DNA strand (by hydrolysis) at very specific region known as restriction site

  
• Restriction Enzyme (RE)

  • Most RE found in bacteria
  • A natural counter-measure against viral infection
  • TWO types: EXOnucleases & ENDOnucleases
• Function of RE in Bacteria

  • Bacterial host DNA is protected due to RE specific methylation
  • An RE-methylase enzyme adds methyl group to its restriction site

  
• Classification of endonucleases (RE)

  • Type I
  • Type II
  • Type III
  • Type IV
• Type II RE


  • Cleave DNA at fixed position within or close to restriction site (palindromic sequence)
  • Generate reproducible fragments and predictable patterns

  
• Initial discovery: Endonuclease from Escherichia coli by Werner Arber and Stuart Linn

  1960

  
• First type II discovered: HindII from Haemophilus influenzae by Hamilton Smith


  1970
• Isoschizomers


  A pair of RE specific to the same recognition sequence
• Neoschizomers


  RE recognizes the same sequence but cuts differently
• How to name/write an RE

  Based on taxonomy of the organism in which they were discovered
• How does RE cleave the DNA

  1. RE bind to DNA strand non-specifically > Scan along the DNA

  2. RE recognise restriction site

  3. Cleave at sugar phosphate backbone hydrolysing phosphodiester bond (between 3' O-P 5')

  
• Palindromic sequence


  A sequence of nucleotide that is similar when read from forward and reverse

  
• Mirror-like palindromic


  A sequence that is similar from left and right of the same DNA strand

  
• Restriction site can be palindromic or mirror-like palindromic
• RE cleave DNA at specific restriction sites
• Type of restriction cut


  • Sticky ends
  • Blunt ends
• How much RE per µg Plasmid DNA/Linear DNA

  • Mostly depends on the type of RE/supplier suggestion
  • 1ug Plasmid DNA need 5U enzyme at 37℃/hour
  • 1ug Linear DNA need 1U enzyme at 37℃/hour

  
• Single digest

  Only 1 enzyme, single reaction time
• Double digest


  Use 2 enzymes, may use single reaction time or double reaction time
• When double/triple/multiple digesting, need to consider factors like enzyme activity, buffer, reaction time
• Incomplete digestion

  Not all plasmid cleaved by RE
• Star activity


  Specificity reduction or increase in cleavage at sites similar to the restriction site due to differences of a base (off-target cleavage)
• Optimal condition: enzyme cleave specifically, completely.

  Non-optimal condition: enzyme can cleave at similar site with single base difference or/and incompletely
• DIFFERENT ENZYME HAS DIFFERENT :

  Activity - pH, temperature , stability

  Buffer

  Reaction time
• Complete Digestion

  1. All plasmid cleaved by RE
  2. Cumulative size band equal to original plasmid size
  3. Bands may represent single / more fractions
• Incomplete Digestion

  1. Not all plasmid cleaved by RE
  2. Known as Single Band like Undigested plasmid band
• RELATIVE POSITIONS DNA BANDS FORM


  Uncut : NICKED | LINEAR | COVALENTLY BONDED | SUPERCOILED | CIRCULAR SINGLE STRANDED

  Cut : LINEAR