Recombinant DNA rDNA “rDNA” contains DNA from 2 or more different sources the DNA has been “spliced” together! See page 501 (Mader) Making rDNA: animation of "sticky ends" Need 2 Enzymes: 1. Restriction Enzymes (“molecular scissors”) -a specific restriction enzyme will cut DNA at a specific cleavage site (a sequence of bases) animation of section of DNA inserted into a plasmid Restriction enzymes recognize a specific short nucleotide sequence This is known as a Restriction Site 2. DNA ligase - seals any breaks in the DNA molecule. Need a “VECTOR” (something to carry the DNA into a host cell) -we often use bacterial PLASMIDS (small, circular pieces of bact. DNA) The Procedure: 1. A bacterial plasmid is isolated. 2. The desired human gene is “cut” out of the human DNA using restriction enzymes (example of a restriction enzyme is “EcoR1”.) 3. A bacterial plasmid is also “cut” with the SAME restriction enzyme. (this leaves the human DNA and the plasmid DNA with the same “sticky ends”) 4. The two kinds of DNA are mixed together with some DNA ligase. 5. Bacteria are mixed with the newly engineered plasmids. (they readily take up plasmids especially if you treat them with CaCl2, which makes them more porous) 6. Bacteria are allowed to replicate. 7. Bacteria are tested for the presence of the human gene. NB: The gene that is inserted into the plasmid will only work if it DOESN’T have any introns. One way to do this is to synthesize the gene in a machine. Another method is to isolate the mRNA for the gene and use “REVERSE TRANSCRIPTASE” to make a DNA copy of it. (= complementary DNA (cDNA)) v. detailed animation of the whole process Uses for rDNA: Mass production of hormones ,proteins, and chemicals ex. Insulin 1. 2. Producing safer vaccines. Eg. Hepatitis B vaccine 3. Producing Transgenic plants and animals : pest -resistant crop species : larger cows (they produce more growth -hormone) Farms in the Future? 4. Gene Therapy (See page 508, Mader) - Ex vivo (outside the body) Cells from the patient are given normal genes to replace defective ones, then the cells are returned to the patient to replicate and grow. In vivo (inside the body) eg. A virus carrying a normal gene is inhaled by the patient. The virus is able to provide the patient with the normal gene product that the patient was missing due to a genetic disorder.