ABE Workshop June 13, 2006

1
Orientation Lab Safety and Restriction Enzymes

• Kabi Neupane, Ph.D.
• Leeward Community College

2
Objectives

• Familiarize with laboratory safety
• Learn about Restriction enzymes
• Perform a restriction digestion of Arabidopsis
  genomic DNA

3
Laboratory Safety

• Emergency procedures
• Eye wash stations
• Locate both eye wash stations
• Personal safety
• Lab coats, gloves, goggles
• Chemical safety
• Material Safety Datasheets (MSDS)
• Red binder located on the back
• Biological safety

4
Enzymes

• Enzymes are proteins
• biological catalysts ? help drive biochemical
  reactions
• Enzyme names end with an ase (eg., endonuclease)
• Bacteria have evolved a class of enzymes that
  destroy foreign DNA (eg. Virus DNA).
• protect bacteria from bacteriophages (Viruses).
• Bacteriophages cannot multiply if their DNA is
  destroyed by the host.

5
Restriction Endonucleases

• Restriction endonucleases RESTRICT viruses
• Viral genome is destroyed upon entry
• Restriction endonuclease Restriction enzymes
• Endo (inside), nuclease (cuts nucleic acid)
• Restriction endonuclease recognizes a short and
  specific DNA sequence and cuts it from inside.
• The specific DNA sequence is called recognition
  sequence

6
Restriction Enzyme Use

• Discovery of enzymes that cut and paste DNA make
  genetic engineering possible.
• Restriction enzyme cuts DNA and generates
  fragments
• Ligase joins different DNA fragments
• DNA fragments from different species can be
  ligated (joined) to create Recombinant DNA

7
Sticky End Cutters

• Most restriction enzymes make staggered cuts
• Staggered cuts produce single stranded
  sticky-ends
• DNA from different sources can be spliced easily
  because of sticky-end overhangs.

8
Blunt End Cutters

• Some restriction enzymes cut DNA at opposite base
• They leave blunt ended DNA fragments
• These are called blunt end cutters

9
Recognition Sequences

• Many restriction sequences are palindromic. For
  example,
• (Read the same in the opposite direction (eg.
  madam, race car)
• Each restriction enzyme always cuts at the same
  recognition sequence.

5 GAATTC 3 3 CTTAAG 5
10
Protection of Self DNA

• Bacteria protect their self DNA from restriction
  digestion by methylation of its recognition site.
• Methylation is adding a methyl group (CH3) to
  DNA.
• Restriction enzymes are classified based on
  cognition sequence and methylation pattern.

11
Classification

• Type I and III
• Large enzyme complex
• Recognition site is away from the site where the
  DNA is cleaved
• Methylation and restriction done by the same
  enzyme
• Type IV
• Only methylated DNA is cleaved
• Type II
• Recognition and cleavage site are same or close.
• Restriction and methylation enzymes are different
• Exclusively used in laboratories

12
How restriction enzymes are named?
13
Cloning Vectors
14
Typical Restriction Digest
Sterile, deionized water 16.3 µl RE 10X Buffer
2.0 µl Acetylated BSA, 10µg/µl 0.2 µl DNA,
1µg/µl 1.0 µl Mix by pipetting, then
add Restriction Enzyme, 10u/µl 0.5 µl Final
volume 20.0 µl
15
How does it Look after Restriction Digestion?
Plasmid DNA Digest
Genomic DNA Digest