1 / 33

Tools

Tools. Enzymes Vectors Host DNA to be cloned. Enzymes. Nucleases Polymerases Ligases , Modifying enzymes Topoisomerase , . Exonucleases. Unit Enzyme 1ug DNA at 37C in 60 min in 50 ul reaction* ExonucleaseI ssDNA 3-5 ExonucleaseIII dsDNA 3-5

jui
Download Presentation

Tools

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Tools • Enzymes • Vectors • Host • DNA to be cloned

  2. Enzymes • Nucleases • Polymerases • Ligases, • Modifying enzymes • Topoisomerase,

  3. Exonucleases • Unit Enzyme 1ug DNA at 37C in 60 min in 50 ul reaction* ExonucleaseIssDNA 3-5 ExonucleaseIIIdsDNA 3-5 ExonucleaseVIIssDNA3-5 and 5-3 https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions

  4. How they cut Exo III

  5. Endonucleases • Dnase I ssDNA, dsDNA • DNA template degradation in transcription reactions • Removal of genomic DNA from RNA samples • DNase I footprinting • Nick Translation • Mung bean nucleases ssDNA • Removal of single-stranded extensions (3' and 5') to leave ligat able blunt ends • Transcriptional mapping • Cleavage of hairpin loops

  6. A Paternity Test

  7. Basic Structure of DNA to remember

  8. Isoschizomer Sph I (CGTAC^G) and Bbu I (CGTAC^G) Neo schizomer Sma I (CCC/GGG) and Xma I (C/CCGGG) Isocaudomer NheIG*CTAG C and AvrIIC*CTAG G C GATC*G G GATC*C

  9. Neo schizomer

  10. Double digestion Double digestion is a process in which we use two restriction enzymes to cut so that molecules do not snap back on itself or for orientation certainity

  11. RIBONUCLEASES RNase A Bovine pancreatic RNase A, Ranapipiens RNaseH RNase H family can be found in nearly all organisms, from archaea to bacteria and eukaryota. Rnase III Double stranded RNA degradation RNAi, microRNA

  12. Rnase based therapeutic for cancer Onconase down regulates microRNA expression through targeting microRNA precursors Cell Research (2012) 22:1199–1202. doi:10.1038/cr.2012.67; published online 24 April 2012

  13. Rnase H of HIV and HBV as an example 1- Structural Basis for the Inhibition of RNase H Activity of HIV-1 Reverse Transcriptase by RNase H Active Site-Directed Inhibitors. 2010 Journal of Virology

  14. Ligase

  15. Ligases and Ligastion T4 DNA ligase

  16. E.coli DNA polymerase I Nick translation

  17. Polymerase

  18. Klenow fragment Fill in reaction

  19. Bacteriophage T4 and T7 polymerase Bacteriophage T4 Active single-stranded 3'->5' exonuclease (ss DNA) (stronger than that of the Klenow fragment) Fill in Trimming back

  20. The T7 polymerase • Enzyme has very high proof reading and polymerization • The enzyme chemically or genetically modified • High processivity, and fast polymerase rate • Used in DNA sequencing

  21. Taq DNA polymerase Thermusaquaticus PCR optimization, Pfu DNA polymerase Pyrococcusfuriosus PCR (if DNA has to use in cloning)

  22. Terminal Deoxynuclotidyl Transferase Probe preparation tailing method

  23. AMV reverse transcriptase • HIV-1 reverse transcriptase from human immunodeficiency virus • M-MLV reverse transcriptase from theMoloney murine leukemia virus • AMV reverse transcriptase from the avian myeloblastosis virus

  24. RNA polymerases S6 RNA Polymerases T7RNA Polymerases

  25. Uses of different enzymes • Primer removal from PCR mixtures: Exo1 thermo • prior to PCR product sequencing (see Reference 2) • for one-tube "megaprimer" PCR mutagenesis (see​ Reference 3) • Removal of single-stranded DNA containing a 3'-hydroxyl terminus from nucleic acid mixtures • Assay for the presence of single-stranded DNA with a 3'-hydroxyl terminus (see​ Reference 4) http://www.thermoscientificbio.com/dna-and-rna-modifying-enzymes/exonuclease-i/

More Related