LECTURE3ABdunn

LECTURE3ABdunn - Lecture 4 Gene cloning : the tools needed...

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Lecture 4 Gene cloning : the tools needed and how to use them Griffiths et al: Chapter 20. Pages 715-735; 739-741; 745-753
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An overview: Fig 11-1 (8 th edition)
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A vector is a molecule that can contain a piece of foreign DNA. Ideally: - It should be self-replicating , and thus being able to self- propagation (contains an origin of replication). - It should be able to accept foreign molecules (presence of restriction sites = or cloning site). - It should contain selectable markers (Amp and Tet). Fig 11-7 (8 th edition) Fig. 20-5
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CLONING: Generating recombinant (hybrid) DNA molecules Fig 20-2 vector (Fragment of ) Gene of interest
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In more detail: Fig 20-3
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Cloning: how amplification works… Fig. 20-4
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… the bacteria amplifies the DNA Fig. 20-4
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Fig. 20.5 Or BamH1 LacZ Lac insert Z X-Gal Blue White (Up to 10 kb) Selecting plasmids with DNA insertion With a multiple cloning site
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Clones containing DNA fragment will form colonies Fig. 5-3
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In practice . .. But 10 kb is a relatively small DNA insert as compared to the human genome And we still do not know which WHITE colony contains the target gene
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Figure 20-6 step 2 Cloning in phage λ Phage λ : A bacteriophage (48,502 bp) that can infect E.coli . One third of its genome (15 kb) is not required for lytic growth, and can be replaced by exogenous DNA. (required vector DNA is 30 kb). Because only 45-50 kb molecules will fit in the virus head, insert DNA is restricted to 15-20 kb size. Partial digestion with restriction enzyme Sau3A and size select DNA fragments 15-20 kb long. Ends of these fragments from Sau3A digestion hybridize to BamH1 ends on the λ vector
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Figure 20-6 step 3 Cloning in phage λ
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Figure 20-6 step 4 Cloning in phage λ One third of its genome (15 kb) is not required for lytic growth, and can be replaced by exogenous DNA. (required vector DNA is 30 kb)
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Figure 20-6 step 5 Cloning in phage λ
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Figure 20-6 step 6 Cloning in phage λ
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Figure 20-6 step 7 Cloning in phage λ On a lawn of bacteria
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In practice . .. Fig 5-26
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are plasmid vectors that can program assembly into λ phage heads. Cosmids: plasmids containing cos sites -DNA enclosed between two cos sites will be packaged into virus heads as long as the DNA is between 45 and 50 kb in length, plasmid is 5 kb, thus inserts between 40-45 kb. - After infection, the phage DNA will be replicated as
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This note was uploaded on 12/21/2010 for the course BIOL BIOL 202 taught by Professor D.schoen during the Summer '08 term at McGill.