Transcript Orientation Lab Safety and Restriction Enzymes

Orientation Lab Safety and
Restriction Enzymes
Kabi Neupane, Ph.D.
Leeward Community College
ABE Workshop
June 13, 2006
Objectives
• Familiarize with laboratory safety
• Learn about Restriction enzymes
• Perform a restriction digestion of
Arabidopsis genomic DNA
ABE Workshop
June 13, 2006
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
ABE Workshop
June 13, 2006
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.
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June 13, 2006
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
ABE Workshop
June 13, 2006
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
ABE Workshop
June 13, 2006
Sticky End Cutters
• Most restriction enzymes make staggered cuts
• Staggered cuts produce single stranded “stickyends”
• DNA from different sources can be spliced easily
because of sticky-end overhangs.
HindIII
EcoRI
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June 13, 2006
Blunt End Cutters
• Some restriction enzymes cut DNA at
opposite base
• They leave blunt ended DNA fragments
• These are called blunt end cutters
AluI
HaeIII
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June 13, 2006
Recognition Sequences
• Many restriction sequences are palindromic.
For example,
5’ GAATTC 3’
3’ CTTAAG 5’
(Read the same in the opposite direction (eg. madam, race car…)
• Each restriction enzyme always cuts at the
same recognition sequence.
ABE Workshop
June 13, 2006
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.
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June 13, 2006
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
ABE Workshop
June 13, 2006
How restriction enzymes are
named?
Enzyme
Organism from which derived
Target sequence
(cut at *)
5' -->3'
Bam HI
Bacillus amyloliquefaciens
G* G A T C C
Eco RI
Escherichia coli RY 13
G* A A T T C
Hind III
Haemophilus inflenzae Rd
A* A G C T T
Mbo I
Moraxella bovis
*G A T C
Pst I
Providencia stuartii
CTGCA*G
Sma I
Serratia marcescens
CCC*GGG
Taq I
Thermophilus aquaticus
T*CGA
Xma I
Xanthamonas malvacearum
C*CCGGG
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June 13, 2006
Cloning Vectors
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June 13, 2006
Typical Restriction Digest
Sterile, deionized water
RE 10X Buffer
Acetylated BSA, 10µg/µl
DNA, 1µg/µl
Mix by pipetting, then add:
Restriction Enzyme, 10u/µl
Final volume
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16.3 µl
2.0 µl
0.2 µl
1.0 µl
0.5 µl
20.0 µl
June 13, 2006
How does it Look after Restriction
Digestion?
Plasmid DNA Digest
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Genomic DNA Digest
June 13, 2006