Vectors and Restriction Enzymes-II PDF

VECTORS AND RESTRICTION ENZYMES-II References Clark D. P. and N. J. Pazdernik, 2009. Biotechnology-Applying the Genetic Revolution, Elsevier Inc. Green M. R. and J. Sambrook, 2012. Molecular Cloning: A Laboratory Manual, 4rth Ed....

VECTORS AND RESTRICTION ENZYMES-II References Clark D. P. and N. J. Pazdernik, 2009. Biotechnology-Applying the Genetic Revolution, Elsevier Inc. Green M. R. and J. Sambrook, 2012. Molecular Cloning: A Laboratory Manual, 4rth Ed. Vol. 1-3. Cold Spring Harbor Laboratory Press. Henry R. J., 2005. Practical Applications of Plant Molecular Biology. Chapman & Hall, London. 2 Learning Goals Cloning Vectors Plasmid Vectors and their properties Host Organisms Restriction Endonucleases Types Mode of action Example 3 Hosts for Cloning Vectors Ideal Capable of rapid growth in inexpensive medium Nonpathogenic hosts Capable of incorporating DNA should Genetically stable in culture Equipped with appropriate be enzymes to allow replication of the vector  Bacteria Eukaryote Escherichia coli Bacillus subtilis Saccharomyces cerevisiae Well-developed Easily transformed Well-developed genetics Nonpathogenic genetics Many strains Naturally secretes Nonpathogenic available proteins Can process mRNA Best known Endospore formation and proteins bacterium simplifies culture Easy to grow Potentially Genetically unstable Plasmids unstable pathogenic Genetics less Will not replicate Periplasm traps developed than most bacterial proteins in E. coli plasmids Advantages Disadvantages What determines the choice vector? insert size  vector size  restriction sites  copy number  cloning efficiency  ability to screen for inserts  ENZYME/RESTRICTION ENDONUCLEASE Enzymes that Especial class Naturally Discovered in Chop up the cut DNA at or of enzymes that Often called produced by the late 1970s viral nucleic near specific cleave (cut) restriction Essential tools bacteria that by Werner acids and recognition DNA at a endonucleases for recombinant use them as a Arber, Hamilton protect a nucleotide specific unique (Because they DNA defense Smith, and bacterial cell by sequences internal cut within the technology. mechanism Daniel hydrolyzing known as location along molecule). against viral Nathans. phage DNA. restriction sites. its length. infection. 8 Restriction Enzymes Restriction enzymes types Type 1: enzymes recognize DNA sequences but cut the DNA in random sites that can be as far as 1,000 base pairs away from the recognized site Type 2: enzymes recognize and cut within the recognized site Type 3: enzymes recognize sequences but cut at a different location within 25 base pairs of the recognized site Type II restriction endonucleases- target sites 4-8 bp in length 9 10  Restriction site 5 3 GAATTC DNA CTTAAG 3 5 1 Restriction enzyme cuts sugar-phosphate backbones. 3 5 5 3 5 Sticky 3 3 5 end 5 3 2 DNA fragment added 3 from another molecule 5 cut by same enzyme. Base pairing occurs. 5 3 5 3 5 3 G AATT C G AATT C C TTAA G C TTAA G 3 53 5 3 5 One possible combination 3 DNA ligase seals strands 5 3 3 Recombinant DNA molecule 5 Enzyme Recognition Site Each enzyme digests (cuts) DNA at a specific sequence = restriction site Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC) Generates 5 prime overhang Overhang 12 Mode of Action of Restriction Enzymes 5' overhangs: The enzyme cuts asymmetrically within the recognition site such that a short single-stranded segment extends from the 5' ends. BamHI cuts in this manner. 3' overhangs: asymmetrical cutting within the recognition site, but the result is a single-stranded overhang from the two 3' ends. KpnI cuts in this manner. Blunts: Enzymes that cut at precisely opposite sites in the two strands of DNA generate blunt ends without overhangs. SmaI is an example of an enzyme that generates blunt 13 14 https://enzymefinder.neb.com/ 15  Restriction maps A map showing positions of restriction sites in a DNA sequence 16 17 THANK YOU ANY QUESTION??