Biology, 6th Edition

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Greg Steinberg AP Biology Chapter 20 DNA Technology and Genomics A. DNA Cloning a. DNA Technology i. Most DNA cloning processes are similar. ii. Plasmids are small circular strands of DNA. iii. Plasmid DNA is separated and then more DNA is added into it. iv. The plasmid is reinserted into the bacteria cell, as the cell divides the gene is reproduced. v. There are two separate goals for plasmid cloning. 1. Producing a protein 2. Prepare copies of the gene to study the nucleotide sequence. b. Restriction Enzymes i. Restriction Enzymes are used to cut DNA at a limited number of specific locations. ii. These enzymes are used by bacteria to cut up unwanted foreign DNA. iii. The restriction enzyme recognizes the restriction site of the DNA sequence. iv. When the restriction enzymes cut the DNA, the result is a double stranded DNA molecule with a single stranded tail called the “Sticky End” of the molecule. v. These ends form hydrogen bonds with their base pairs. vi. DNA Ligase permanently fuses the sticky ends together. c. A Closer Look i. The original plasmid of the system is known as the cloning vector. ii. The result is multiple copies of the cloned gene. d. Cloning Eukaryotes 1. Isolation of Vector and Gene-source DNA: DNA Plasmid and the cloning DNA are obtained. 2. Insertion of DNA into the Vector: The plasmid and DNA are digested by the same restriction enzyme. The enzyme mixes the DNA and joins them by base pairing. DNA Ligase then repairs the cut. 3. Introduction of the cloning vector into cells: The altered plasmid is put into the lacZ- bacteria. 4. Cloning of Cells: The bacteria are placed on a medium with ampicillin and X-gal. If the bacteria can grow in the ampicillin, it stays, if not, it is discarded and thrown away. 5. Identification of cell clones carrying gene in interest: The use of nucleic acid probes are then used. These RNA or
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DNA strands are complementary to the gene in interest. They form hydrogen bonds with their base pairs. But they are tagged, so now the gene in interest is tagged… You’re it. This process is known as nucleic acid hybridization. ii. Once the gene has been identified, it can be isolated and put into large tanks for mass use. iii. If a protein is wanted, the protein can be found by enzymatic and antibody use. e. Cloning and Expressing Eukaryotic Genes i. Gene expression is different in prokaryotes and eukaryotes. ii. And expression vector is a cloning vector that contains a prokaryote promoter upstream of the restriction site that allows a bacterium to produce a eukaryotic gene.
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