Gel Electrophoresis: Nucleic Acids  book cover
1st Edition

Gel Electrophoresis: Nucleic Acids

ISBN 9781872748283
Published December 31, 1995 by Garland Science
192 Pages

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Book Description

Through its clear presentation of the basic concepts, Gel Electrophoresis: Nucleic Acids breaks new ground by describing the principles of the technique without resorting to complicated protocols and recipes.

Table of Contents

Abbreviations -- Preface -- PART 1: BASIC PRINCIPLES AND METHODS -- 1. Introduction: the Variety and Forms of Nucleic Acids -- Overview -- Electrophoresis and the properties of nucleic acids -- The variety and forms of nucleic acid -- References -- 2. The Theory of Nucleic Acid Electrophoresis -- The movement of nucleic acids in liquids and in gels -- Electric currents and buffer solutions -- Nucleic acids in solution -- Nucleic acids in gels -- The movement of nucleic acids through gels in constant electric fields -- Ogston sieving -- Reptation -- Migration as rigid rods -- The movement of nucleic acids through gels in pulsed electric fields -- References -- 3. The Electrophoresis of Native and Denatured Nucleic Acids -- The control of base pairing -- Physical and chemical denaturants for nucleic acid gel electrophoresis -- Temperature -- Alkaline conditions -- Methyl mercuric hydroxide, glyoxal and formaldehyde: denaturants for RNA in agarose gels -- Urea and formamide -- The binding of proteins to nucleic acids during gel electrophoresis -- References -- 4. The Choice of Format: Horizontal or Vertical, Agarose or Polyacrylamide? -- Apparatus for horizontal and vertical gels -- Apparatus for horizontal agarose gels -- Apparatus for vertical polyacrylamide gels -- Power supplies for nucleic acid gel electrophoresis -- Agarose gel media for nucleic acid electrophoresis -- Modified agarose preparations -- Low melting point agarose -- Higher strength agarose -- High-sieving agarose -- Visigel"' -- Ready-made agarose gels -- Polyacrylamide gel media for nucleic acid gel electrophoresis -- Modified polyacrylamide formulations -- Ready-made polyacrylamide gels -- Wedge shaped gels -- Toxicity of the components of polyacrylamide gels -- References -- 5. The Detection of Nucleic Acids Following Electrophoretic Separation -- Overview -- The principles of nucleic acid detection -- Binding with fluorescent dyes -- Labeling with radioactive nucleotides -- Labeling with fluorescent nucleotides -- Labeling nucleic acids with specific proteins -- Indirect DNA-protein coupling -- Direct DNA-protein coupling -- Conventional staining -- Nucleic acid detection in situ and after transfer to membranes -- Summary of nucleic acid detection methodology -- References -- PART 2: TECHNIQUES AND APPLCATIONS -- 6. Guide to Techniques and Applications -- Matching the molecule to the technique -- Strategic considerations: avoiding elementary mistakes -- 7. Nondenaturing Agarose Gel Electrophoresis -- Buffers for nondenaturing agarose gels -- The resolving power of agarose: altering the gel concentration; altering the field strength -- Estimating the length of unknown fragments using semi-log plots -- Solving a separation problem: resolving the 5148 and 4973 hp bands in the HindIII-EcoRI restriction digest of phage A DNA -- Alternative methods for staining with ethidium bromide -- Passive diffusion; can agarose gels be left safely overnight before photography? -- Amounts of DNA: estimating unkown quantities and before photography? -- Estimating unknown quantities -- Overloading and underloading a gel -- DNA conformation and the mobility of molecules in agarose gels -- Heating A DNA markers -- A note on taking pictures -- Recording the position of marker bands when DNA is transferred to a membrane -- Research applications: overview -- Research application: Nondenaturing agarose gel electrophoresis: Southern blotting -- Background -- Procedure -- Interpretation -- References -- 8. Denaturing Agarose Gel Electrophoresis -- Research application. Denaturing gel electrophoresis of single-stranded RNA molecules: Northern blotting -- Background -- Procedure -- Interpretation -- References -- 9. Pulsed Field Agarose Gel Electrophoresis -- The principles of pulsed field technology -- Pulsed field electrode geometry -- Sample preparations for pulsed field gels -- Research application. Pulsed field agarose gels for genome mapping -- Background -- Procedure -- Interpretation -- References -- 10. Nondenaturing Polyacrylamide Gel Electrophoresis -- Overview -- Research application. High-resolution separation of double-stranded DNA fragments -- Background -- Procedure -- Interpretation -- Single- and double-strand conformational polymorphisms: SSCP and DSCP -- The principle of using gel electrophoretic conformational polymorphisms to detect mutations -- Research application. Nondenaturing polyacrylamide gel electrophoresis: using SSCP analysis to detect mutations -- Bandshift or gel retardation assays -- The principles of the bandshift assay -- Research application. Bandshifting assays -- References -- 11. Denaturing Polyacrylamide Gel Electrophoresis -- Overview -- DNA sequencing -- The principles of DNA sequencing -- Research application. The use of a dGTP base analog to overcome a sequencing gel compression artifact -- DNA footprinting -- The principle of DNA footprinting -- Research application. DNA footprinting -- RNase protection assays -- The principle of the RNase protection assay -- Research application. RN ase protection assay -- Nuclease S1 protection assays -- The principle of nuclease S1 protection assays -- Research application. Nuclease S1 protection assay -- Primer extension assays -- The principle of the primer extension assay -- Research application. Primer extension assays -- References -- Appendices -- Appendix A: Glossary -- Appendix B: Suppliers -- Index.

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