1. ABE Workshop June 13, 2006 Orientation Lab Safety and Restriction Enzymes Kabi Neupane, Ph.D. Leeward Community College

2. ABE Workshop June 13, 2006 Objectives Familiarize with laboratory safety Learn about Restriction enzymes Perform a restriction digestion of Arabidopsis genomic DNA

3. 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

4. ABE Workshop June 13, 2006 Enzymes Enzymes are proteins –biological catalysts  help drive biochemical reactions Enzyme names end with an ase (eg., endonucle ase ) 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. 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

6. 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

7. 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 -

8. 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

9. 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’

10. 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 (CH 3 ) to DNA. Restriction enzymes are classified based on cognition sequence and methylation pattern.

11. 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

12. ABE Workshop June 13, 2006 How restriction enzymes are named? EnzymeOrganism from which derived Target sequence (cut at *) 5' -->3' Bam HIBacillus amyloliquefaciensG* G A T C C Eco RIEscherichia coli RY 13G* A A T T C Hind IIIHaemophilus inflenzae RdA* A G C T T Mbo IMoraxella bovis*G A T C Pst IProvidencia stuartiiC T G C A * G Sma ISerratia marcescensC C C * G G G Taq IThermophilus aquaticusT * C G A Xma IXanthamonas malvacearumC * C C G G G

13. ABE Workshop June 13, 2006 Cloning Vectors

14. ABE Workshop June 13, 2006 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. ABE Workshop June 13, 2006 How does it Look after Restriction Digestion? Plasmid DNA Digest Genomic DNA Digest