1. Lecture 3 Introduction to recombinant DNA Technology

2. Tools  Enzymes  Vectors  Host  DNA to be cloned

3. Enzymes  Nucleases  Polymerases  Ligases,  Modifying enzymes  Topoisomerase,

4. Exonucleases Unit Enzyme 1ug DNA at 37C in 60 min in 50 ul reaction* ExonucleaseIssDNA3-5 ExonucleaseIIIdsDNA3-5 ExonucleaseVIIssDNA 3-5 and 5-3 https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions

5. How they cut Exo III

6. 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

14. Basic Structure of DNA to remember

15. Iso schizomer Sph I (CGTAC^G) and Bbu I (CGTAC^G) Neo schizomer Sma I (CCC/GGG) and Xma I (C/CCGGG) Iso caudomer NheI G*CTAG C and AvrII C*CTAG G C GATC*G G GATC*C

16. Neo schizomer

17. 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

19. RIBONUCLEASES RNase A Bovine pancreatic RNase A, Rana pipiens 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

20. 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

21. 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

22. Ligase

23. Ligases and Ligastion T4 DNA ligase

24. E.coli DNA polymerase I Nick translation

25. Polymerase

26. Klenow fragment Fill in reaction

27. 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

28. 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

29. Taq DNA polymeraseThermus aquaticus PCR optimization, Pfu DNA polymerasePyrococcus furiosus PCR (if DNA has to use in cloning)

30. Terminal Deoxynuclotidyl TransferaseProbe preparation tailing method

32. AMV reverse transcriptase HIV-1 reverse transcriptase from human immunodeficiency virushuman immunodeficiency virus M-MLV reverse transcriptase from the Moloney murine leukemia virusMoloney murine leukemia virus AMV reverse transcriptase from the avian myeloblastosis virusavian myeloblastosis virus

33. RNA polymerases S6 RNA Polymerases T7RNA Polymerases

34. 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/