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Lecture Molecular biology (Fifth Edition): Chapter 5 - Robert F. Weaver

Chapter 5 - Molecular tools for studying genes and gene activity. This chapter describe the most popular techniques that molecular biologists use to investigate the structure and function of genes. Most of these start with cloned genes. Many use gel electrophoresis. Many also use labeled tracers, and many rely on nucleic acid hybridization. We have already examined gene cloning techniques. | Molecular Biology Fifth Edition Chapter 5 Molecular Tools for Studying Genes and Gene Activity Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5.1 Molecular Separations • Often mixtures of proteins or nucleic acids are generated during the course of molecular biological procedures A protein may need to be purified from a crude cellular extract A particular nucleic acid molecule made in a reaction needs to be purified Gel electrophoresis is used to separate different species of: Nucleic acid Protein 5- DNA Gel Electrophoresis Melted agarose is poured into a form equipped with removable comb Comb “teeth” form slots in the solidified agarose DNA samples are placed in the slots An electric current is run through the gel at a neutral pH to allow the sample to travel through the gel matrix 5- DNA Separation by Agarose Gel Electrophoresis DNA is negatively charged due to the phosphates in . | Molecular Biology Fifth Edition Chapter 5 Molecular Tools for Studying Genes and Gene Activity Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5.1 Molecular Separations • Often mixtures of proteins or nucleic acids are generated during the course of molecular biological procedures A protein may need to be purified from a crude cellular extract A particular nucleic acid molecule made in a reaction needs to be purified Gel electrophoresis is used to separate different species of: Nucleic acid Protein 5- DNA Gel Electrophoresis Melted agarose is poured into a form equipped with removable comb Comb “teeth” form slots in the solidified agarose DNA samples are placed in the slots An electric current is run through the gel at a neutral pH to allow the sample to travel through the gel matrix 5- DNA Separation by Agarose Gel Electrophoresis DNA is negatively charged due to the phosphates in its backbone and moves toward the positive pole Small DNA pieces have little frictional drag so they move rapidly Large DNAs have more frictional drag so their mobility is slower Distributes DNA according to size Largest near the top Smallest near the bottom DNA is stained with fluorescent dye that intercalates between the bases 5- DNA Size Estimation Mobility of fragments are plotted v. log of molecular weight (or number of base pairs) Electrophoresis of unknown DNA in parallel with standard fragments permits size estimation upon comparison Same principles apply to RNA separation 5- Electrophoresis of Large DNA Special techniques are required for DNA fragments larger than about 1 kilobases Instead of constant current, alternate long pulses of current in forward direction with shorter pulses in either opposite or sideways direction Technique is called pulsed-field gel electrophoresis (PFGE) 5- Protein Gel Electrophoresis Separation of proteins is done using polyacrylamide gel