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 ExonucleaseI ssDNA 3-5 ExonucleaseIII dsDNA 3-5
Unit Enzyme
1ug DNA at 37C in 60 min in 50 ul reaction*
ExonucleaseI ssDNA 3-5
ExonucleaseIII dsDNA 3-5
ExonucleaseVII ssDNA and 5-3

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

13. A Paternity Test

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 certainty

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: /cr ; 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 Journal of Virology

22. Ligase

23. Ligases and Ligastion
T4 DNA ligase

24. What Ligase needs to make an efficient ligation
5 Phosphate is absolute requirement
If removed ligation can not take place
Adjusting the vector insert ratio 1:3 formula?
Contamination in DNA also influence ligation
efficiency

25. Need of Alkaline phosphatase (CIAP)
An enzyme that removes phosphatase from it substrate
Why we need to remove the phosphate if it absolutely required
How DNA is ligated after use of CIAP

26. Need of Alkaline phosphatase (CIAP)
An enzyme that removes phosphatase from it substrate
Why we need to remove the phosphate if it absolutely required
How DNA is ligated after use of CIAP

27. E.coli DNA polymerase I
Nick translation

28. Polymerase

29. Klenow fragment Fill in reaction

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

31. 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

32. Taq DNA polymerase Thermus aquaticus
PCR optimization,
Pfu DNA polymerase Pyrococcus furiosus
PCR (if DNA has to use in cloning)

33. Terminal De-oxynuclotidyl Transferase Probe preparation tailing method

35. AMV reverse transcriptase
HIV-1 reverse transcriptase from human immunodeficiency virus
M-MLV reverse transcriptase from the Moloney murine leukemia virus
AMV reverse transcriptase from the avian myeloblastosis virus

36. RNA polymerases
S6 RNA Polymerases
T7RNA Polymerases

37. Plasmid Isolation Solution 1 Solution 2 Solution 3 50 mM Tris pH 8
200 mM NaOH
3 M Potassium
10 mM EDTA
1 % SDS
Acetate pH 5.5
Re suspends the pellet and maintains the pH and ability to inhibit DNases
Breaks the cell wall to release the content. Denatures the DNA (Breaks H Bonds)
At nutralization The Chromo DNA aggregates being very large while plasmid can not as it is cccDNA

38. Alkaline de naturation and plasmid isolation

39. 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)