DNA Enzymes Ligation enzymes Restriction enzymes Polymerizase enzyme

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DNA Enzymes • • • Ligation enzymes Restriction enzymes Polymerizase enzyme Strand-displacing polymerases Helicase

DNA Enzymes • • • Ligation enzymes Restriction enzymes Polymerizase enzyme Strand-displacing polymerases Helicase enzymes Typically these enzymes are Proteins discovered in cells. Obtainable from Scientific supply companies.

DNA Hybridization Sticky ends DNA ligase DNA restriction enzyme www. cs. duke. edu/~reif/paper/peng/. .

DNA Hybridization Sticky ends DNA ligase DNA restriction enzyme www. cs. duke. edu/~reif/paper/peng/. . . /talk. Walker. Design. ppt

Ligation: Ligase – “to bind” or “to glue together” T 4 DNA Ligase –

Ligation: Ligase – “to bind” or “to glue together” T 4 DNA Ligase – a single polypeptide, MW ~ 86, 000 daltons, (p. H 7. 5 – 8. 0, 10 m. M Mg 2+, DTT, Na. Cl 200 m. M to stop reaction) Self-assembled DNA Nanostructures and DNA Devices, Nanofabrication Handbook, Taylor and Francis 2012, with Nikhil Gopalkrishnan, Thom La. Bean and John Reif http: //www. bio. miami. edu/dana/pix/phosphodiester. jpg

DNA Hybridization & Enzyme Ligation activity Sticky ends DNA ligase DNA restriction enzyme www.

DNA Hybridization & Enzyme Ligation activity Sticky ends DNA ligase DNA restriction enzyme www. cs. duke. edu/~reif/paper/peng/. . . /talk. Walker. Design. ppt http: //www. angelfire. com/sc 3/toxchick/molbiolab 01. html

Restriction Enzymes Self-assembled DNA Nanostructures and DNA Devices, Nanofabrication Handbook, Taylor and Francis 2012,

Restriction Enzymes Self-assembled DNA Nanostructures and DNA Devices, Nanofabrication Handbook, Taylor and Francis 2012, with Nikhil Gopalkrishnan, Thom

Exonucleases & Endonucleases • Exonucleases • Endonucleases • Restriction Endonucleases – Type I –

Exonucleases & Endonucleases • Exonucleases • Endonucleases • Restriction Endonucleases – Type I – cut elsewhere of recognition sites – Type II – cut within recognition sites http: //www. eplantscience. com/index_files/biotechnology/Genes%20&%20 Genetic%20 Engineer ing/Tools%20 of%20 Genetic%20 Engineering/biotech_enzymes. php

Restriction Enzymes http: //www. scq. ubc. ca/restriction-endonucleases-molecular-scissors-for-specifically-cutting-dna/

Restriction Enzymes http: //www. scq. ubc. ca/restriction-endonucleases-molecular-scissors-for-specifically-cutting-dna/

Restriction enzyme action Sticky ends DNA ligase DNA restriction enzyme www. cs. duke. edu/~reif/paper/peng/.

Restriction enzyme action Sticky ends DNA ligase DNA restriction enzyme www. cs. duke. edu/~reif/paper/peng/. . . /talk. Walker. Design. ppt http: //www. angelfire. com/sc 3/toxchick/molbiolab 01. html

Restriction Enzymes Some restricton enzymes produce "sticky" ends: Other restriction enzyme’s cleavage produces "blunt"

Restriction Enzymes Some restricton enzymes produce "sticky" ends: Other restriction enzyme’s cleavage produces "blunt" ends: http: //www. scq. ubc. ca/restriction-endonucleases-molecular-scissors-for-specifically-cutting-dna/

Restriction Endonucleases • Nicking Enzymes • Restriction Enzymes

Restriction Endonucleases • Nicking Enzymes • Restriction Enzymes

DNAzymes Montypython. scs. uiuc. edu

DNAzymes Montypython. scs. uiuc. edu

Polymerization • Denaturation of target (template) – Usually 95 o. C • Annealing of

Polymerization • Denaturation of target (template) – Usually 95 o. C • Annealing of primers – Temperature of annealing is dependent on the G+C content – May be high (no mismatch allowed) or low (allows some mismatch) stringency • Extension (synthesis) of new strand Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Polymerization Thermo. Fischer Scientic

Polymerization Thermo. Fischer Scientic

Polymerization Khan Acadamy

Polymerization Khan Acadamy

Polymerization Khan Acadamy

Polymerization Khan Acadamy

Discovery of Thermostable DNA Polymerases: Yellowstone National Park Donna C. Sullivan, Division of Infectious

Discovery of Thermostable DNA Polymerases: Yellowstone National Park Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Discovery of Thermostable DNA Polymerases: Deep Sea Vents Donna C. Sullivan, Division of Infectious

Discovery of Thermostable DNA Polymerases: Deep Sea Vents Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Thermostable Polymerases Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Thermostable Polymerases Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Thermostable Polymerases • Taq: Thermus aquaticus (most commonly used) – Sequenase: T. aquaticus YT-1

Thermostable Polymerases • Taq: Thermus aquaticus (most commonly used) – Sequenase: T. aquaticus YT-1 – Restorase (Taq + repair enzyme) • Tfl: T. flavus • Tth: T. thermophilus HB-8 • Tli: Thermococcus litoralis • Carboysothermus hydrenoformans (RT-PCR) • P. kodakaraensis (Thermococcus) (rapid synthesis) • Pfu: Pyrococcus furiosus (fidelity) – Fused to DNA binding protein for processivity Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Strand Displacement Polymerases Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Strand Displacement Polymerases Donna C. Sullivan, Division of Infectious Diseases, University of Mississippi

Helicase Enzymes • Helicase enzymes are motor proteins that moving along a DNA double

Helicase Enzymes • Helicase enzymes are motor proteins that moving along a DNA double helix to denature its structure (unwind the double helix) independent of temperature. • In particularly, helicase enzymes directionally break hydrogen bonds between base pairing in DNA double helix. • Animation of Helicase Unwinding the DNA Double Helix: https: //study. com/academy/lesson/how-helicase-unwinds-the-dna-double-helix-inpreparation-for-replication. html http: //click 4 biology. info/c 4 b/3/chem 3. 4. htm http: //www. pdbj. org/eprots/index_en. cgi? PDB%3 A 3 BEP

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