abe workshop june 13, 2006 orientation lab safety and restriction enzymes kabi neupane, ph.d....
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![Page 1: ABE Workshop June 13, 2006 Orientation Lab Safety and Restriction Enzymes Kabi Neupane, Ph.D. Leeward Community College](https://reader030.vdocuments.net/reader030/viewer/2022032723/56649cfd5503460f949cddb4/html5/thumbnails/1.jpg)
ABE Workshop June 13, 2006
Orientation Lab Safety and Restriction Enzymes
Kabi Neupane, Ph.D.
Leeward Community College
![Page 2: ABE Workshop June 13, 2006 Orientation Lab Safety and Restriction Enzymes Kabi Neupane, Ph.D. Leeward Community College](https://reader030.vdocuments.net/reader030/viewer/2022032723/56649cfd5503460f949cddb4/html5/thumbnails/2.jpg)
ABE Workshop June 13, 2006
Objectives
• Familiarize with laboratory safety
• Learn about Restriction enzymes
• Perform a restriction digestion of Arabidopsis genomic DNA
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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
![Page 4: ABE Workshop June 13, 2006 Orientation Lab Safety and Restriction Enzymes Kabi Neupane, Ph.D. Leeward Community College](https://reader030.vdocuments.net/reader030/viewer/2022032723/56649cfd5503460f949cddb4/html5/thumbnails/4.jpg)
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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ABE Workshop 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
![Page 6: ABE Workshop June 13, 2006 Orientation Lab Safety and Restriction Enzymes Kabi Neupane, Ph.D. Leeward Community College](https://reader030.vdocuments.net/reader030/viewer/2022032723/56649cfd5503460f949cddb4/html5/thumbnails/6.jpg)
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
![Page 7: ABE Workshop June 13, 2006 Orientation Lab Safety and Restriction Enzymes Kabi Neupane, Ph.D. Leeward Community College](https://reader030.vdocuments.net/reader030/viewer/2022032723/56649cfd5503460f949cddb4/html5/thumbnails/7.jpg)
ABE Workshop June 13, 2006
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.
EcoRI
HindIII- OH 3’
5’ P -
- P 5’
3’ OH -
![Page 8: ABE Workshop June 13, 2006 Orientation Lab Safety and Restriction Enzymes Kabi Neupane, Ph.D. Leeward Community College](https://reader030.vdocuments.net/reader030/viewer/2022032723/56649cfd5503460f949cddb4/html5/thumbnails/8.jpg)
ABE Workshop June 13, 2006
Blunt End Cutters
AluI
HaeIII
• Some restriction enzymes cut DNA at opposite base
• They leave blunt ended DNA fragments
• These are called blunt end cutters
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ABE Workshop June 13, 2006
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’
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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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ABE Workshop 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
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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 C T G C A * G
Sma I Serratia marcescens C C C * G G G
Taq I Thermophilus aquaticus T * C G A
Xma I Xanthamonas malvacearum C * C C G G G
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ABE Workshop June 13, 2006
Cloning Vectors
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ABE Workshop June 13, 2006
Typical Restriction Digest
Sterile, deionized water 16.3 µlRE 10X Buffer 2.0 µlAcetylated BSA, 10µg/µl 0.2 µlDNA, 1µg/µl 1.0 µlMix by pipetting, then add:Restriction Enzyme, 10u/µl 0.5 µlFinal volume 20.0 µl
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ABE Workshop June 13, 2006
How does it Look after Restriction Digestion?
Plasmid DNA Digest Genomic DNA Digest