Restriction-enzymes-final.ppt
- 2. restriction enzymes ,Also called restriction Endonucleses 1962: “molecular scissors” discovered in in bacteria E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA 3,000 enzymes have been identified, around 200 have unique properties, many are purified and available commercially
- 3. • Molecular scissors that cut double stranded DNA molecules at specific points. • Found naturally in a wide variety of prokaryotes
- 4. Discovery • Arbor and Dussoix in 1962 discovered that certain bacteria contain Endonucleases which have the ability to cleave DNA. • In 1970 Smith and colleagues purified and characterized the cleavage site of a Restriction Enzyme. • Werner Arbor, Hamilton Smith and Daniel Nathans shared the 1978 Nobel prize for Medicine and Physiology for their discovery of Restriction Enzymes
- 5. Restriction Enzymes • Restriction enzymes are endonucleases (Endo (inside), nuclease(cuts nucleic acid), which catalyze the cleavage of the phosphodiester bonds within both strands of DNA. • They require Mg+2 for activity and generate a 5 prime (5') phosphate and a 3 prime (3') hydroxyl group at the point of cleavage.
- 6. • The distinguishing feature of restriction enzymes is that they only cut at very specific sequences of bases. This specific DNA sequence is called recognition sequence. Restriction enzymes are traditionally classified according to the subunit composition, cleavage position, sequence-specificity and cofactor requirements.
- 7. • A restriction enzyme requires a specific double stranded recognition sequence of nucleotides to cut DNA. • Recognition sites are usually 4 to 8 base pairs in length. • Cleavage occurs within or near the site.
- 8. • Restriction enzymes are named according to the organism from which they are isolated. • This is done by using the first letter of the genus followed by the first two letters of the species and additional letter or number represent the strain or serotypes. • Only certain strains or sub-strains of a particular species may produce restriction enzymes. Naming of Restriction enzymes
- 9. Mechanism of Action • Restriction Endonuclease scan the length of the DNA , binds to the DNA molecule when it recognizes a specific sequence and makes one cut in each of the sugar phosphate backbones of the double helix – by hydrolyzing the phoshphodiester bond. Specifically,the bond between the 3’ O atom and the P atom is broken.
- 10. Named for bacterial genus, species, strain, and type Example: EcoR1 Genus: Escherichia Species: coli Strain: R Order discovered: 1
- 11. Example of RE naming: HindII: Restriction enzyme was isolated from Haemophilus influenzae serotype d • There are hundreds of different REs from different microorganisms. • Different restriction enzymes have different recognition sequences. • This makes it possible to create a wide variety of different gene fragments.
- 12. Examples: Enzyme Organism from which derived Target sequence (cut at *) 5' -->3' Bam HI Bacillus amyloliquefaciens G* G A T C C Eco RI Escherichia coli RY 13 G* A A T T C Hind III Haemophilus inflenzae Rd A* A G C T T Mbo I Moraxella bovis *G A T C Pst I Providencia stuartii C T G C A * G Sma I Serratia marcescens C C C * G G G Taq I Thermophilus aquaticus T * C G A Xma I Xanthamonas malvacearum C * C C G G G
- 13. Restriction enzymes recognize specific 4-8 bp sequences Some enzymes cut in a staggered fashion - “sticky ends” EcoRI 5’…GAATTC…3’ 3’…CTTAAG…5’ Some enzymes cut in a direct fashion – “blunt ends” PvuII 5’…CAGCTG…3’ 3’…GTCGAC…5’
- 17. Many recognition sequences are palindromic. For example, 5’ GAATTC 3’ 3’ CTTAAG 5’ palindromic: read the same in the opposite direction
- 18. Sticky and Blunt end cutters Not all restriction endonucleases cut symmetrically and leave blunt ends. Many endonucleases cleave the DNA backbones in positions that are not directly opposite each other or can make staggered cuts, which produce single stranded “sticky-ends” DNA from different sources can be spliced easily because of these sticky-end overhangs.
- 19. Example of RE that produce sticky end cutters:
- 20. Some restriction enzymes cut DNA at opposite base. They leave blunt ended DNA fragments These are called blunt end cutters HaeIII
- 21. Types of Restriction Enzymes Cleavage site Location of methylase Examples Type I Random Around 1000bp away from recognition site Endonuclease and methylase located on a single protein molecule EcoK I EcoA I CfrA I Type II Specific Within the recognition site Endonuclease and methylase are separate entities EcoR I BamH I Hind III Type III Random 24-26 bp away from recognition site Endonuclease and methylase located on a single protein molecule EcoP I Hinf III EcoP15 I
- 22. Isoschizomers and Neochischizomers • Two Restriction enzymes that have the same recognition sequence as well as the same cleavage site are Isoschizomers. For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other • Restriction enzymes that have the same recognition sequence but cleave the DNA at a different site within that sequence are Neochizomers. Eg:SmaI and XmaI C C C G G G C C C G G G G G G C C C G G G C C C Xma I Sma I
- 23. Restriction Enzyme Uses 1. Recombinant DNA technology *Discovery of enzymes that cut and paste DNA make genetic engineering possible. *Restriction enzyme cuts DNA and generates fragments *Ligase joins different DNA fragments *DNA fragments from different species can be ligated (joined) to create Recombinant DNA
- 25. 2. Cloning Replicates a sequence inserted into a host cell 3. DNA restriction mapping The location of the restriction enzyme cleavage sites on the DNA molecule.
- 27. • Restriction enzyme is part of the cell’s restriction-modification system in bacteria. • The phenomenon of restriction modification in bacteria is a small scale immune system for protection from infection by foreign DNA. • Bacteria can protect themselves only after foreign DNA has entered their cytoplasm. Biological Function
- 28. • Restriction Enzymes can be used to generate a restriction map. This can provide useful information in characterizing a DNA molecule.
- 29. Restriction Fragment Length Polymorphism is a tool to study variations among individuals & among species
- 30. Why don’t bacteria destroy their own DNA with their restriction enzymes?
- 31. Unit Determination Assay • One unit of restriction endonuclease is defined as the amount of enzyme required to digest one microgram of the appropriate substrate DNA completely in 60 minutes under the conditions specified for that enzyme.
- 32. Set up of a restriction enzyme reaction • A RE reaction contains the DNA to be analyzed, • A restriction enzyme, • A restriction enzyme buffer mix. –contains a buffering agent to maintain constant pH, –and Mg++ (from MgCl2) as a necessary cofactor for enzyme activity.
- 33. Digestion by Restriction Enzyme • Measure the DNA concentration – Use the Nano-drop spectrophotometer to measure the concentration of DNA, this is used to determine the amount of HinfI restriction enzyme to be used. Digestion of DNA • Mix the following components in a clean microtube. • Mix gently and spin down for a few seconds. • Incubate at 37°C for 16 hours.