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DIGESTION OF DNA WITH RESTRICTION ENZYMES

DIGESTION OF DNA WITH RESTRICTION ENZYMES. What are restriction enzymes?. are enzymes that have the ability to cleave the sugar phosphate backbones in double stranded DNA at specific restriction sites . Found naturally in a wide variety of prokaryotes. (Molecular scissors).

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DIGESTION OF DNA WITH RESTRICTION ENZYMES

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  1. DIGESTION OF DNA WITH RESTRICTION ENZYMES

  2. What are restriction enzymes? • are enzymes that have the ability to cleave the sugar phosphate backbones in double stranded DNA at specific restriction sites. • Found naturally in a wide variety of prokaryotes (Molecular scissors)

  3. Biological Role In bacteria • Most bacteria use Restriction Enzymes as a defense against bacteriophages. • Restriction enzymes prevent the replication of the phage by cleaving its DNA at specific sites. • They do not cleave the host DNA as the restriction sites are methylated

  4. How Restriction Enzyme cut the DNA?Restriction fragments can be blunt ended or sticky ended 5’ G A A T T C 3’ 5’ G A T A T C 3’ 3’ C T T A A G 5’ 3’ C T A T A G 5’ Sticky Ends Blunt Ends • Sticky ends or blunt ends can be used to join DNA fragments. • Sticky ends are more cohesive compared to blunt ends.

  5. Example of Restriction Enzymes Over 300 RE have been isolated and the nomenclature depends on the organism from which they are derived e.g. Bam HI from Bacillus amyloliquefaciens H

  6. 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.

  7. Uses of Restriction Enzymes Restriction Enzymes can be isolated from bacteria and then used to generate a restriction map. This can provide useful information in characterizing a DNA molecule.

  8. Uses…. Restriction enzymes are most widely used in recombinant DNA technology.(gene cloning)

  9. Uses…. Restriction Fragment Length Polymorphism is a tool to study variations among individuals & among species

  10. Restriction Fragment Length Polymorphism (RFLP)-Example technique in molecular biology to differentiate minor nucleotide sequence variations in homologous fragments of DNA.  The technique relies on the specificity of restriction endonucleases, which are highly sequence-specific and cut the double-stranded DNA only at their recognition sites. PCR for the region that you want to do the study on DNA Extraction Agarose gel to visualized your results Incubation of the DNA with RE

  11. Restriction Fragment Length Polymorphism (RFLP)-Example

  12. Restriction Fragment Length Polymorphism (RFLP)-Example • Agarose gel electrophoresis • what do you think these samples (normal or abnormal)

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