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DNA and Restriction Enzymes

DNA and Restriction Enzymes

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DNA and Restriction Enzymes

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  1. DNA and Restriction Enzymes

  2. Basic Structure of DNA • Built of nucleotides: • Pentose sugar • Phosphate • Nitrogen base • Purines – adenine, guanine • Pyrmidines – thymine, cytosine • N base attached to 1’ C of sugar

  3. Struct., cont. • 3’ C of 1 sugar bonds to 5’ phosphate to form phosphodiester bond

  4. Complementary Strands • DNA arranged in double helix (Rosalind Franklin’s work) • Chargaff’s Rule: A—T and G—C (purine to pyrimidine) • Antiparallel – run 5’3’ on 1 strand and 3’5’ on other • 2 strands are complementary; i.e. • 3’—AGTAC—5’ • 5’—TCATG—3’

  5. Properties of DNA • Negative charge • PO4- • Together, negative charge and polar sugar = soluble • Genes & non-coding sections • DNA is universal

  6. How can we manipulate DNA?

  7. Restriction Enzymes • Isolated from bacteria • Used as immune mechanism to protect from phages • Named after bacteria they are isolated from • Recognize specific sequences of DNA • Palindromes • Ex. EcoRI recognizes GAATTC

  8. “Cut” DNA in a specific pattern at the recognition site • May produce: • Blunt ends • Sticky ends

  9. Number & placement of recognition sites determine # and size of fragments • RFLPs • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

  10. Gel Electrophoresis • Separates DNA molecules based on size • Cut DNA using restriction enzyme(s) • Load DNA into gel • Run electrical current through buffer/gel • Stain DNA & compare unknowns to fragments of known sizes

  11. http://learn.genetics.utah.edu/content/labs/gel/