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Restriction Enzymes

Restriction Enzymes. What are restriction enzymes?. Restriction enzymes are proteins that cut DNA. Because they cut within the molecule, they are often referred to as restriction endo nucleases. Function. Found naturally in bacteria Protects the bacteria from invading viruses

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Restriction Enzymes

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  1. Restriction Enzymes

  2. What are restriction enzymes? Restriction enzymes are proteins that cut DNA. Because they cut within the molecule, they are often referred to as restriction endonucleases.

  3. Function • Found naturally in bacteria • Protects the bacteria from invading viruses • bacterium modifies its own restriction sites by methylation so enzyme cannot cut it • When virus injects its DNA the bacteria can cleave the viral DNA without affecting its own • Able to cut double stranded DNA molecules at a specific nucleotide pair sequence called the restriction site

  4. Nomenclature • Over 600 restriction enzymes are commercially available • Named for the bacteria from which they were isolated • 3 letter system • Based on Genus and species that the enzyme was isolated from • Additional letters added to signify particular strains and order of discovery • Bgl II - Bacillus globigi • EcoR I - E. coli Strain RY13 • Hind III - Haemophilus influenza

  5. Probability and Non-Random Sequences • In a DNA sequence with a random distribution of nucleotides, there is a clear relationship between the number of nucleotide pairs in the recognition site and the frequency of cutting • Enzymes which recognize 4 bases (4 base cutter) will cut more frequently than enzymes which have a 6 base recognition sequence (6 base cutter) and so on. • In random DNA, the probability of finding one nucleotide pair is independent of any other pair in the sequence. • In general the probability of a recognition site is (1/4)n where n is the number of nucleotides in the recognition site • 4 bases = (1/4)4 = 1/256 • 5 bases = (1/4)5 = 1/1024 • 6 bases = (1/4)6 = 1/4096 • 8 bases = (1/4)8 = 1/65,476 • DNA in organisms is not randomly distributed however • There may be an organism bias in G/C content • There may be a difference in G/C content between coding and non-coding DNA

  6. How were they discovered? In 1970 Hamilton Smith accidentally found that a DNase from the bacterium, Haemophilus influenzae, cut DNA at specific PALINDROME DNA SEQUENCES known as RESTRICTION SITES.

  7. Restriction enzymes bind DNAat specific sites

  8. In DNA, a PALINDROME SITE is a SEQUENCE OF BASE PAIRS in double stranded DNA that reads the same backwards and forwards across the double strand Example: The sequence of base pairs GAATTC is a palindrome because both sequences of the double strand READ THE SAME when read from either their respective "G" or "C" ends. 5’... G A A T T C …3’ 3’... C T T A A G …5’

  9. Based on the TYPES OF CUTS they make, there are two types of restriction enzymes. • BLUNT ENDS • STICKY ENDS 5’... G A A T T C …3’ 3’... C T T A A G …5’ 5’... G A A T T C …3’ 3’... C T T A A G …5’

  10. Blunt Ends

  11. Sticky Ends

  12. Sister’s Sequence

  13. Brother’s Sequence Perform a restriction digest on both siblings; how do they compare when you “run the results out on a gel”?

  14. Gel Electrophoresis(the separation of DNA based on size) agarose wells

  15. Loading a Gel

  16. DNA is negatively charged (click once to run animation) Electrical charge + -

  17. End Result:a DNA fingerprint

  18. Gel Analysis Ladder: DNA of known sizes (used as a standard) Large DNA pieces -these have a hard time migrating through the gel pores Small DNA pieces -these can migrate through the gel pores easily and migrate farther

  19. DNA sizes measured in Basepairs (bp) or KilobasepairsKb DNA samples

  20. Recombinant DNA (Chimeras) • making recombinant DNA from two different sources whether it be different species or different tissue sources is an important tool. • Using recombinant DNA technology can lead to important discoveries about the structure-function relationship of proteins, how mutations cause disease, how genes may be regulated, and to isolate a gene product for production (ex. Insulin)

  21. Virtual Restriction Digestions of Plasmids http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/virgel.html

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