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Recombinant DNA and Genetic Engineering Chapter 8 Purpose: Co-opt bacteria in order to grow large quantities of a particular DNA sequence (e.g., a gene). Why is this important? Ex: Human genome = 3 x 109 bp One gene = 103 to 104 bp, or ~0.0003% of total genomic DNA Clone = Genetically identical individuals. Examples: Bacteria cells in a single colony. Many cells Colony = pile of bacteria cells in one spot on petri dish, all derived from one cell following many rounds of cell division -- Genetically identical. DNA clone = many identical copies of a single DNA molecule. Example: A DNA molecule present in the bacteria cells in a single colony. Many cells o o o = plasmid DNA o o o o o o o o o o o o o o By extracting plasmid DNA from colony of bacteria cells, one can obtain useable quantities of a DNA with identical sequence. Restriction Enzymes Enzymes produced by bacteria. Purpose: primitive immune system to fight infection by bacteriophage. Bind to specific DNA sequences & make cuts in DNA backbone. Example: EcoRI - Binds to & cuts GAATTC CTTAAG Modification Enzymes: Protect bacteria cell’s own DNA from its own restriction enzymes. Example: EcoRI Methylase - Adds methyl groups to middle A’s of EcoRI recognition sequence m GAATTC CTTAAG m Restriction Enzymes 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ Many restriction enzymes’ recognition sites are palindromic. Those that make staggered nicks, produce restriction fragments with “sticky ends”. Sticky ends = short complementary single strands. Making Recombinant DNA Molecules 1. Cut DNA you want to clone & vector DNA with restriction enzyme to create complementary sticky ends. 2. Mix DNAs together & add DNA ligase. 3. Complementary sticky ends base pair, but DNA ligase must seal nick to permanently join the DNAs together. Forms phosphodiester bonds