BIOB 375 1st Edition Lecture 32 Outline of Last Lecture II. DNA TechnologyIII. PCROutline of Current Lecture IV. Gene Amplification V. Cloning vectorCurrent LectureRestriction enzyme recognizes special DNA sequence, but some of them can produce the same endsTo connect two DNA molecules, they have to have the same matched endsIf DNA molecules are sheared by mechanical force, they have broken endsHow to fix the broken DNA ends?- By Klenow enxyme- Two functions of Klenow:o Filling in recessed 3’ ends ando Digesting away protruding 3’ overhangsGene amplification- Amplify the gene in a test tube via PCR- Insert the gene into a plasmid- Transform the plasmid into E. coliHowever in a living cell, three processes to ensure the accuracy of replication:1. Nucleotide selection2. Proofreading. Nucleotide selection3. Mismatch repairTo ensure the accuracy of replication, we use bacteria to amplify genes of our choice:Step 1. Amplify the full length of the gene and cut a plasmid with the enzyme that produces matched endsStep 2. Joining the gene sequence with the plasmid DNA by ligaseStep 3. Transform the plasmid to a E. coli strainStep 4. Pick a bacterial colony that carry “the gene”Three features of a cloning vector:1. Origin of replication2. Cloning sites3. Selectable markerTo insert a gene into a plasmid, they have to have matched
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