Lecture 3 Introduction to recombinant DNA Technology

Tools Enzymes Vectors Host DNA to be cloned

Enzymes Nucleases Polymerases Ligases, Modifying enzymes Topoisomerase,

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 3-5 and 5-3 https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions

How they cut Exo III

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

A Paternity Test

Basic Structure of DNA to remember

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

Neo schizomer

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

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

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

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

Ligase

Ligases and Ligastion T4 DNA ligase

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

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

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

E.coli DNA polymerase I Nick translation

Polymerase

Klenow fragment Fill in reaction

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

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

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

Terminal De-oxynuclotidyl Transferase Probe preparation tailing method

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

RNA polymerases S6 RNA Polymerases T7RNA Polymerases

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

Alkaline de naturation and plasmid isolation

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/