f5 - DNA Cloning by Recombinant DNA Technology Ch 9 pp...

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Ch. 9, pp. 361-380 DNA Cloning by Recombinant DNA Technology
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Restriction Map • Restriction digestion generates a reproducible set of DNA fragments. • The order of restriction sites and the distance between them is called a restriction map . • DNA molecules with different sequences will have different restriction maps. • Is useful in gene mapping (also used in forensics!)
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Tube 1 DNA+ EcoR I Tube 2 DNA+ EcoR I+ Sal I Tube 3 DNA+ Sal I Determination of a Restriction Map 1) Independently digest DNA sample with restriction enzyme A, restriction enzyme B, and both restriction enzymes A and B together 2) Separate the restriction fragments by gel electrophoresis 3) From the size of fragments on gel, you can determine original restriction map (order of restriction sites and distance between them).
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Restriction Mapping
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DNA ligase catalyzes the formation of phosphodiester bond between 2 DNA strands Only the restriction fragment with compatible sticky ends can be ligated by DNA ligase. Any two blunt-ended DNA can be joined by ligase DNA ligase: Connects the restriction fragments of DNA
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Gel Electrophoresis
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DNA Gel Electrophoresis: Analysis of Restriction Digestions
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DNA Gel Electrophoresis: Analysis of Restriction Digestions • The digested DNA are analyzed using gel electrophoresis – Agarose gels: -for analyzing longer DNA fragments, ~ 500 - 20,000 base pairs –Acrylamide gels (similar to protein gels): -for analyzing short DNA fragments, ~1 - 2000 base pairs - has the power to resolve differences between 1 nt EtBr • The restriction fragments are separated in the gel, containing EtBr, fluorescent dye that intercalates with DNA. • DNA bands are visualized under UV light
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1. Ethidium bromide intercalates between the base pairs; can be visualized by UV; Fluorescent dyes are also used 2. Radioactively labeled DNA can be visualized by autoradiography of the gel Ethidium Bromide (EtBr) Visualizing DNA bands on a gel
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Cloning a gene Ch. 9, pp. 361-380
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Vectors: Plasmids • Plasmids are circular double stranded DNA…separate from the host chromosomal DNA -These occur naturally in bacteria, yeast, and lower eukaryotic systems - Can be parasitic or symbiotic with the host cell • Replicate separately from the chromosomal DNA of the cell due to presence of origin of replication • Are usually ~3 Kb and are used to clone relatively small DNA fragments (e.g. < 5 kb)
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(1) a replicon : origin of replication (2) a selectable marker : allows for selection; - is often a bacterial gene that confers antibiotic resistance - some of the often used antibiotic resistance are amp r , kan r , tet r , etc.
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