Restriction enzyme

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    06-Aug-2015

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<ol><li> 1. Restriction endonucleasesest ct o e do uc eases and their applications </li><li> 2. Hi t f t i tiHistory of restriction endonucleases and its rolee do uc eases a d ts o e in establishing molecular bi lbiology </li><li> 3. Restriction enzymes Over 10,000 bacteria species have been screened for restriction enzymes O 2 500 t i ti h b f d Over 2,500 restriction enzymes have been found Over 250 distinct specificities Occasionally enzymes with novel DNA sequence Occasionally enzymes with novel DNA sequence specificities are still found while most now prove to be duplicates (isoschizomers) of already di d ifi itidiscovered specificities. </li><li> 4. Restriction Enzyme Function It is generally believed that the biological function of restriction enzymes is to protect cells from foreign DNA. Infecting DNA is cleaved (restricted) by the restriction enzyme(s) preventing it f f ll li ti dfrom successfully replicating and parasitizing the cell. </li><li> 5. Why the bacteria does not kill itself? The Restriction Enzyme Modification Systems if everything gets cleaved, how come the bacteria does not kill itself? Usually, organisms that make restriction enzymes also make a companion modification enzyme (DNA methyltransferase) that protects their own( y ) p DNA from cleavage. These enzymes recognize the same DNAy g sequence as the restriction enzyme they accompany, but instead of cleaving the sequence, they disguise it by methylating one of the bases in h DNA t deach DNA strand. </li><li> 6. Classification of Restriction enzymesenzymes Class I Class II (93%) Class III Restriction-methylase on the same subunit Homo-dimers, methylase on a separate subunit Restriction-methylase on the same subunit p ATP-dependent Mg++ dependent ATP-dependent Binds to DNA recognition site and t DNA d l recognize symmetric DNA sequences and l ithi th Cut the DNA at the recognition site and th di i t fcuts DNA randomly - any DNA as long as it comes in contact cleave within the sequences then dissociate from the DNA </li><li> 7. Type II Restriction enzymes are endonucleases that cut DNA at specific sites, and are most useful for molecular biology research </li><li> 8. Type II Restriction enzymes Recognition sitesRecognition sites are P li d iPalindroimes: 121 IFFI ABAIFFI, ABA AAGCTT TTCGAA </li><li> 9. How do I know what sequenceHow do I know what sequence each enzyme cut? Test by cutting DNA of known sequencey g q Commercial sources are tested already,y, and you find a catalog </li><li> 10. Some popular Biotechnology Companies Life Technologies (BRL/GIBCO) New England Biolabs Amersham Pharmacia Biotech Qiagen P Promega Clonetech InvitrogenInvitrogen Stratagene ... </li><li> 11. Nomenclature of restriction enzyme Eco R1: E coli Pst I: Providencia stuartii Hind III: Haemophilus influenza Not I: Norcardia otitidis-caviarum What do you name a restriction enzymeWhat do you name a restriction enzyme isolated from Xanthomonas graminis? </li><li> 12. How long is the recognition sequence 4 bp: e.g., Taq 1, HpaII, MspI 6 bp: e.g., EcoR1, HindIII, BamH1, PstI, salI 8 bp: Not I, Sfi I </li><li> 13. Recognition sequence may beRecognition sequence may be interrupted or ambiguous Acc I: GT(at/gc)AC( g ) Bgl I: GCCNNNNNGGCg Afl III: ACPuPyGTAfl III: ACPuPyGT </li><li> 14. Three types of ends producedThree types of ends produced by type II restriction enzymes 3-overhang (protruding) 5-overhang5 overhang Blunt end </li><li> 15. 5-overhang EcoR I 5-----------------------gaattc---------------------------35 -----------------------gaattc---------------------------3 3-----------------------cttaag--------------------------5 X EcoR1 5-----------------------g + aattc---------------------------3g 3-----------------------cttaa + aattc 3 g---------------------------5 </li><li> 16. 3-overhang Pst I: 5-----------------------ctgcag---------------------------35 -----------------------ctgcag---------------------------3 3-----------------------gacgtc--------------------------5 X PstI 5-----------------------ctgca-3 + 5-g---------------------------3g 3-----------------------g-5 + 5 g 3 3- actgc---------------------------5 </li><li> 17. Blunt end EcoR V 5-----------------------gatatc---------------------------35 -----------------------gatatc---------------------------3 3-----------------------ctatag--------------------------5 X EcoR V 5-----------------------gat + atc---------------------------3g 3-----------------------cta + atc 3 tag---------------------------5 </li><li> 18. Only Compatible, base-pairable ends li tcan ligate </li><li> 19. Odds of cutting at a segment of DNA 4 bp cutter: 44 = 256 bp 6 bp cutter: 46 = 4 kbp 8 bp cutter: 48 = 64 kb ??? How many do you predict Eco R1 to cut catfish genome of 8 x 109 bpg p </li><li> 20. What do you expect ifWhat do you expect if you digest with youryou digest with your plasmid DNA with 4-p bp cutters </li><li> 21. What do you expect ifWhat do you expect if you digest with youryou digest with your plasmid DNA with 6-p bp cutters </li><li> 22. What do you expect ifWhat do you expect if you digest with youryou digest with your plasmid DNA with 8-p bp cutters </li><li> 23. Restriction mapping EcoR1 EcoR1 EcoR1 PstI PstI E E E P P 10 kb 0.6 kb 2.4 kb 6.0 kb 1.0 kb 1.5 kb 0.8 kb 7.7 kb 10 kb What do you expect to see with double digest, if reaction is complete? 0.6 kb, 0.9 kb*, 1.5 kb, 6.0 kb, 0.2 kb, 0.8 kb. </li><li> 24. What do you expect ifWhat do you expect if you digest with youryou digest with your genomic DNA with 4-g bp cutters </li><li> 25. What do you expect ifWhat do you expect if you digest with youryou digest with your genomic DNA with 6-g bp cutters </li><li> 26. What do you expect ifWhat do you expect if you digest with youryou digest with your genomic DNA with 8-g bp cutters </li><li> 27. Selection of restrictionSelection of restriction enzymes Of Over 250 commercially available and over 2,000 total, how do I know what to use? Cutting frequency Easy to work with Economical </li></ol>

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