Tools Enzymes

Vectors

Host

DNA to be cloned

Enzymes

Nucleases

Polymerases

Ligases,

Modifying enzymes Topoisomerase,

Exonucleases

• Unit Enzyme1ug DNA at 37C in 60 min in 50 ul reaction*

ExonucleaseI ssDNA 3-5ExonucleaseIII dsDNA 3-5ExonucleaseVII ssDNA 3-5 and 5-3

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

How they cutExo 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 digestionDouble 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

RIBONUCLEASESRNase ABovine pancreatic RNase A, Rana pipiens

RNaseHRNase H family can be found in nearly all organisms, from archaea to bacteria and eukaryota.

Rnase IIIDouble stranded RNA degradationRNAi, 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 ligationefficiency

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 aquaticusPCR optimization,

Pfu DNA polymerase Pyrococcus furiosus

PCR (if DNA has to use in cloning)

Terminal De-oxynuclotidyl TransferaseProbe 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 polymerasesS6 RNA Polymerases

T7RNA Polymerases

Plasmid Isolation

Solution 1 Solution 2 Solution 350 mM Tris pH 8 200 mM NaOH 3 M Potassium10 mM EDTA 1 % SDS Acetate pH 5.5Re 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 thermoprior 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 mixturesAssay 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/