dec 4 Restriction, Modification, and Cloning

dec 4 Restriction, Modification, and Cloning - 2 17.1...

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Restriction, Modification, and Cloning 12/4/06 Major ideas and questions: 1. Restriction enzymes limit the incorporation of foreign DNA into bacteria. 2. Modification systems protect the host against its own restriction enzymes. 3. How can restriction enzymes be used to make recombinant DNA molecules? 4. Given the DNA sequence or restriction pattern for a gene, you should be able to design a scheme to clone that gene into a plasmid or phage vector. Problems in Klug: 1, 6, 7, 9, 11, 12, 20
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1 Restriction-Modification Systems How do bacteria protect themselves against invading DNA (e.g. viral infection)? 17.1 Figure 17.1 Restriction endonuclease 17.4 Figure 17.4 figure 17-04.jpg Type II restriction endonuclease • Binds to a specific 4 - 8 bp sequence • Sequence is usually a palindrome • Endonuclease cuts at or near the recognition site
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Unformatted text preview: 2 17.1 Figure 17.1 Restriction endonuclease 17.4 Figure 17.4 * * * * Restriction-Modication Systems Bacteria have a modifying enzyme (DNA methylase) that alters the recognition sequence and prevents cutting by the restriction enzyme. Using restriction enzymes for cloning Cutting is specic - sequence recognition DNA strands can be separated - melt Complementary strands will form a duplex Breaks can be repaired by DNA ligase 17.4 Figure 17.4 figure 17-04.jpg 3 AatII GACGT^C AccI GT^NNAC AceIII CAGCTC AciI CCGC AclI AA^CGTT AII C^TTAAG AgeI A^CCGGT AhaIII TTT^AAA AlfI GCANNNNNNTGC AluI AG^CT AlwNI CAGNNN^CTG ApaI GGGCC^C ApaBI GCANNNNN^TGC ApaLI G^TGCAC ApoI R^AATTY AscI GG^CGCGCC AspCNI GCCGC Fig. 9.2 Fig. 9.5a2 4 ? How many possible recombinant molecules can be made ?...
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dec 4 Restriction, Modification, and Cloning - 2 17.1...

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