Chapter 5 - Chapter 5 Exploring Genes and Genomes...

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Chapter 5: Exploring Genes and GenomesRecombinant DNA technology depends first on having enzymes that can cut, join, and replicate DNA and reverse transcribe RNA.Base-pairing language allows complementary sequences to recognize and bind to each other, which helps to construct new combinations of DNA as well as to detect and amplify particular sequences.Novel proteins can be created by altering genes in specific ways to provide detailed views into proteins function.5.1The Exploration of Genes Relies on Key ToolsKey Techniques of Biotechnology:Restriction-enzyme analysis:precise, molecular scalpels.Blotting techniques:the Southern and Northern blots are used to separate and characterize DNA and RNA, respectively.DNA sequencing:the precise nucleotide sequence of a molecule of DNA can be determined.Solid-phase synthesis of nucleic acids:precise sequences of nucleic acids can be synthesized de novo and used to identify or amplify other acids.PCR:one molecules of DNA can be amplified to quantities that permit characterization and manipulation.Restriction Enzymes Split DNA into Specific FragmentsRestriction enzymes (restriction endonucleases) recognize specific base sequences in double-helical DNA and cleave both stands of that duplex.Restriction enzymes are found in a wide variety of prokaryotes to cleave foreign DNA molecules.The cell’s own DNA is not degraded since the sites recognized are methylated.The recognized sequence is palindromic (an inverted repeat) and the cleavage sites are symmetrically positioned.Restriction enzymes are used to cleave DNA molecules into specific fragments that are more readily analyzed and manipulated than the entire molecule.Restriction Fragments Can be Separated by Gel ElectrophoresisSmall differences between related DNA molecules can be readily detected because their restriction fragments can be separated and displayed by gel electrophoresis.The electrophoretic mobility of a DNA fragment is inversely proportional to the logarithm of the number of base pairs, up to a certain limit.A gel can be stained with ethidium bromide, which fluoresces an intense orange when bound to a double-helical DNA molecule.A restriction fragment containing a specific base sequence can be identified by
hybridizing it with a labeled complementary DNA strand.Southern BlottingA mixture of restriction fragments is separated by electrophoresis through an agarose gel, denatured to form single-stranded DNA, and transferred to a nitrocellulose sheet.The fragments are exposed to a 32P-labeled single-stranded DNA probe.The probe hybridizes with a restriction fragment having a complementary sequence, and autoradiography then reveals the position of the complex.

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