1. Restriction enzymes are used in genetic engineering
A. How restriction endonucleases cut DNA
1. Endonucleases recognize a variety of 4-6 nucleotide sequences.
2. Enzyme binds at recognition site and cleaves both strands of DNA at same time.
3. For a sticky end cutter, the site where DNA is cut has offset ends.
B. Why restriction endonucleases are so useful
1. Each enzyme always cuts at the same sequence.
2. Sticky ends can pair with each other.
a. Two fragments can be joined together by DNA ligase.
b. Any two fragments cleaved by the same endonuclease can be joined together-fragments can be from entirely different organisms.
2. Sample genetic engineering experiment-cut segments of DNA are inserted into bacteria via plasmids
A. DNA from human cell containing insulin gene is cut with restriction enzymes that recognize specific nucleotide sequences at the ends of the gene.
B. Production of recombinant DNA-inserting the insulin gene into bacterial DNA
1. Cut bacterial plasmid DNA with same restriction enzyme(s) as the insert was cut.
2. Place plasmid and insert together with DNA ligase-sticky ends will pair easily.
C. How does the plasmid get into a bacterial cell so the inserted DNA can be reproduced?-Transformation
1. A plasmid carrying the desired insert could enter the cell by temporary pores in the cell membrane created by chemicals (calcium chloride) and heat shock (today's lab).
2. A plasmid could enter the cell by temporary pores in the cell membrane created by delivering an electrical charge to the bacteria.
3. These treatments make the cells competent (capable of accepting a plasmid).
D. How do we determine which bacterial cells got a plasmid?-Screening the clones
1. Not all the bacterial cells will have taken in a plasmid.
2. Use a commercial plasmid (cloning vector) that contains a gene for antibiotic resistance (ours has ampicillin resistance).
3. Spread transformed bacteria on a nutrient plate with antibiotic in it (selection medium).
4. Only bacterial cells with the resistance gene carried on the plasmid will survive and reproduce (ampicillin resistant bacteria).
5. Bacterial cells without the plasmid will still be ampicillin sensitive and will die.
E. How do they get insulin from the bacteria?
1. Bacteria now carrying the desired gene can be multiplied.
2. Gene product (insulin, etc.) can be extracted from the bacteria and purified.