Chapter5Notes 11.45.23 PM

Chapter5Notes 11.45.23 PM - Page 1 of 25 Chapter 5 Notes...

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Page 1 of 25 Chapter 5 Notes Biochemistry 461 Fall 20 10 CHAPTER 5 , EXPLORING GENES: LECTURE TOPICS 1) RESTRICTION ENZYMES 2) GEL ELECTROPHORESIS OF DNA 3) DNA SEQUENCING, RNA SEQUENCING, DNA SYNTHESIS 4) POLYMERASE CHAIN REACTION (PCR) 5) RECOMBINANT DNA CONSTRUCTION AND GENE CLONING 6) DNA CLONING VECTORS 7) GENE LIBRARIES: MAKING AND SCREENING THEM 8) CHROMOSOME MAPPING 9) EXPRESSION OF CLONED GENES 10) ENGINEERING NOVEL PROTEINS
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Page 2 of 25 Recombinant DNA technology (started in late 1970's) ! An incredibly powerful set of tool for gene manipulation. ! Methods associated with this "technology" make genetic engineering a reality. ! DNA (genes), RNA, and protein structure and function can be altered by design for beneficial (or detrimental -biological warfare/terrorism ?) results. KEY TOOLS and METHODS OF GENE EXPLORATION ! ENZYMES to cut, join and replicate DNA in test tubes ( in vitro ) a) restriction enzymes are DNA cutters b) DNA ligases are DNA joiners c) DNA replication requires DNA polymerases ! GEL ELECTROPHORESIS to separate and isolate specific DNA s ! BLOTTING METHODS based on hybridization (BASE-PAIRING) of complementary DNA and/or RNA ! SOLID PHASE methods to sequence and synthesize DNA ! POLYMERASE CHAIN REACTION for gene detection and amplification.
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Page 3 of 25 RESTRICTION ENZYMES (ENDONUCLEASES) ! DNA scissors - hundreds of restriction enzymes are known ! Recognition sequences - different lengths (often 4-8bp), palindromic (2 -fold rotational axis of symmetry), specific cleavage sites (Fig. 6.1) ! They can leave overhanging ends or blunt ends ! Named (ex: Hin dIII ) for source bacterial strain: H = Haemophilus in = influenzae d = strain d III = third one identified ! Number of cuts in a specific DNA ranges from few (if long recognition site) to many (short or ambiguous recognition site) ! patterns of fragments are diagnostic of a given DNA species and physical maps of whole chromosomes can be made. (Fig.6.-2)
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Page 4 of 25 GEL ELECTROPHORESIS OF DNA ! Agarose gels separate DNA restriction fragments ! Visualize DNA by staining or autoradiography ! even differences of one base pair can be detected on gels. ! Hybridization - Base-pairing of DNA-DNA, DNA-RNA: Complementary single- stranded DNA and RNA molecules form base-paired structures even if only 2 or 3 bases can pair - like at ends of restriction fragments ! [ IMPORTANT: Hybridization (DNA-DNA, DNA-RNA) is almost always used in one or more ways to to detect particular DNA or RNA sequences and to construct new combinations of DNA fragments.] NUCLEIC ACID BLOTTING AND HYBRIDIZATION: DNA bands (and patterns) on gels
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Page 5 of 25 can be transferred to nitrocellulose filters (Southern blotting) and identified by hybridization with a specific gene probe. (Fig.6.3) ! Southern (DNA), Northern (RNA), and Western (protein) blotting methods are all powerful probes of gene function. ! Rest
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Chapter5Notes 11.45.23 PM - Page 1 of 25 Chapter 5 Notes...

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