[BIO 1306] Ch16_Lecture

[BIO 1306] Ch16_Lecture - 16 Recombinant DNA and...

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16 Recombinant DNA and Biotechnology
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16 Recombinant DNA and Biotechnology 16.1 How Are Large DNA Molecules Analyzed? 16.2 What Is Recombinant DNA? 16.3 How Are New Genes Inserted into Cells? 16.4 What Are the Sources of DNA Used in Cloning? 16.5 What Other Tools Are Used to Manipulate DNA? 16.6 What Is Biotechnology?
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16.1 How Are Large DNA Molecules Analyzed? Naturally occurring enzymes that cleave and repair DNA are used in the laboratory to manipulate and recombine DNA.
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16.1 How Are Large DNA Molecules Analyzed? Restriction enzymes ( restriction endonucleases ) cut double-stranded DNA into smaller pieces. Bacteria use these as defense against DNA from bacteriophage. DNA is cut between the 3′ hydroxyl group of one nucleotide and the 5′ phosphate group of the next— restriction digestion .
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Figure 16.1 Bacteria Fight Invading Viruses with Restriction Enzymes
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16.1 How Are Large DNA Molecules Analyzed? There are many restriction enzymes that cut DNA at specific base sequences— the recognition sequence , or restriction site .
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16.1 How Are Large DNA Molecules Analyzed? Restriction enzymes do not cut bacteria’s own DNA because the recognition sequences are modified. Methylases add methyl groups after replication; makes sequence unrecognizable by restriction enzyme.
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16.1 How Are Large DNA Molecules Analyzed? Bacterial restriction enzymes can be isolated from cells. DNA from any organism will be cut wherever the recognition site occurs. Eco RI (from E. coli ) cuts DNA at this sequence:
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16.1 How Are Large DNA Molecules Analyzed? The sequence is palindromic—it reads the same in both directions from the 5′ end. Eco RI occurs about once every four genes in prokaryotes. DNA can be chopped into small pieces containing a few genes.
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16.1 How Are Large DNA Molecules Analyzed? The Eco RI sequence does not occur anywhere in the genome of the phage T7. Thus it can survive in its host, E. coli .
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16.1 How Are Large DNA Molecules Analyzed? After DNA is cut, fragments of different sizes can be separated by gel electrophoresis . Mixture of fragments is place on a well in a porous gel. An electric field is applied across the gel. Negatively charged DNA fragments move towards positive end. Smaller fragments move faster than larger ones.
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Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)
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Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 2)
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Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 3)
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16.1 How Are Large DNA Molecules Analyzed? Electrophoresis provides information on: Size of fragments. Fragments of known size provide comparison. Presence of specific sequences. These can be determined using probes. DNA is denatured while in the gel, then transferred to a nylon filter to make a “blot.”
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Figure 16.3 Analyzing DNA Fragments by Southern Blotting
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16.1 How Are Large DNA Molecules Analyzed?
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