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Restriction Enzymes

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Restriction Enzymes Presentation

Restriction Enzymes Presentation

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  • 1. Restriction EndonucleaseMCB5706:BIOMOLECULAR ENGINEERING Prof Tan Wen Siang
  • 2. History and Definition Type I Type IIRestriction Types enzymes Type III Artificial RE Recombinant DNA genotype DNA Application DNA sequencing DNA storage – libraries
  • 3. DNA Palindromes The palindrome in which the same forward and backwards are on a single strand of DNA strand, as in GTAATG The palindrome is also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (GTATAC being complementary to CATATG) Inverted repeat palindromes are more common and have greater biological importance than mirror- like palindromes.
  • 4. Back
  • 5. HindIII EcoRI AluI HaeIII
  • 6. Back
  • 7. Example: EcoR1 Genus: Escherichia Species: coli Strain: R Order discovered: 1Back
  • 8.  Large enzymes Combination restriction-and-modification Cleave outside of their recognition sequences Require two recognition sequences in opposite orientations within the same DNA molecule
  • 9.  Cleave only normal and modified DNA (methylated, hydroxymethylated and glucosyl-hydroxymethylated bases). Recognition sequences have not been well defined Cleavage takes place ~30 bp away from one of the sites
  • 10.  generated by fusing a natural or engineered DNA binding domain to a nuclease domain  can target large DNA sites (up to 36 bp)  can be engineered to bind to desired DNA sequencesBack
  • 11.  Application in cut and paste DNA make genetic engineering possible with coordination of Ligase Enzyme Rrestriction site could be added to primers (forward&Reverse) that will be used for amplifying gene Both vector and amplified gene will be cut by RE to produce sticky ends In ligation mixture the target gene will be inserted to vector with the aid of sticky ends and DNA ligase
  • 12. i. Designing of primer with restriction siteii. Amplifying target gene by PCR and designed primeriii. Double digestion of vector and amplified gene by same REsiv. Ligation of gene and vectorv. Transformationvi. Expression Back
  • 13. ( ) ingle ucleotide olymorphisms distinguish gene alleles by specifically recognizing single base changes in DNA By use of SNP the restriction enzyme can be used to without the need for expensive gene sequencing
  • 14. DNA molecule 1 differs from DNAmolecule 2 at a single base-pair Backlocation (a C/T polymorphism)
  • 15.  (RFLP) Restriction Fragment Length Polymorphism Molecular biologists have identified regions of the human genome where restriction fragment lengths are highly variable between individuals Electrophoresis of these RFLP’s produce different patterns of DNA bands
  • 16. Restriction Enzymes for RFLP _ DNA is negatively charged from the phosphate backbone + Visualize DNA with ethidium bromide – fluoresces ONLY when bound to DNA