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

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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.

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

Lecture PowerPoint to accompany Molecular Biology Fifth Edition Robert F. Weaver Chapter 5 Molecular Tools for Studying Genes and Gene Activity 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 5-2
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 5-3
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 5-4
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 5-5
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) 5-6
Protein Gel Electrophoresis • Separation of proteins is done using polyacrylamide gel electrophoresis (PAGE) – Treat proteins to denature subunits with detergent such as sodium dodecyl sulfate (SDS) • SDS coats polypeptides with negative charges so all move to anode • Masks natural charges of protein subunits so all move relative to mass not charge – As with DNA smaller proteins move faster toward the anode 5-7
Summary • DNAs, RNAs, and proteins of various masses can be separated by gel electrophoresis • Most common gel used in nucleic acid electrophoresis is agarose but polyacrylamide is typically used in protein electrophoresis • SDS-PAGE is used to separate polypeptides according to their masses 5-8
Two-Dimensional Gel Electrophoresis • While SDS-PAGE gives good resolution of polypeptides, some mixtures are so complex that additional resolution is needed • Two-dimensional gel electrophoresis: – Nondenaturing gel electrophoresis (no SDS) uses 2 consecutive gels each in a different dimension – Sequential gels with distinct pH separation and polyacrylamide gel concentration 5-9
A Simple 2-D Method • Samples are run in 2 gels – First dimension separates using one concentration of polyacrylamide at one pH – Second dimension uses different concentration of polyacrylamide and pH – Proteins move differently at different pH values without SDS and at different acrylamide concentrations 5-10
A More Powerful Two-Dimensional Gel Electrophoresis Technique A two process method: • Isoelectric focusing gel: mixture of proteins electrophoresed through gel in a narrow tube containing a pH gradient – Negatively charged protein moves to its isoelectric point at which it is no longer charged – Tube gel is removed and used as the sample in the second process 5-11
A More Powerful Two-Dimensional Gel Electrophoresis Technique continued • Standard SDS-PAGE: – Tube gel is removed and used as the sample at the top of a standard polyacrylamide gel – Proteins partially resolved by isoelectric focusing are further resolved according to size • When used to a compare complex mixtures of proteins prepared under two different conditions, even subtle differences are visible 5-12
Ion-Exchange Chromatography • Chromatography originally referred to the pattern seen after separating colored substances on paper • Ion-exchange chromatography uses a resin to separate substances by charge • This is especially useful for proteins • Resin is placed in a column and the sample is loaded onto the column material 5-13
Separation by Ion-Exchange Chromatography • Once the sample is loaded buffer is passed over the resin + sample • As ionic strength of elution buffer increases, samples of solution flowing through the column are collected • Samples are tested for the presence of the protein of interest 5-14
Gel Filtration Chromatography • Protein size is a valuable property that can be used as a basis of physical separation • Gel filtration uses columns filled with porous resins that let in smaller substances and exclude larger substances • As a result larger substances travel faster through the column 5-15
Affinity Chromatography • The resin contains a substance to which the molecule of interest has a strong and specific affinity • The molecule binds to a column resin coupled to the affinity reagent – Molecule of interest is retained – Most other molecules flow through without binding – Last, the molecule of interest is eluted from the column using a specific solution that disrupts their specific binding 5-16
Summary • High-resolution separation of proteins can be achieved by two-dimensional gel electrophoresis • Ion-exchange chromatography can be used to separate substances according to their sizes • Gel filtration chromatography uses columns filled with porous resins that let in smaller substances but exclude larger ones • Affinity chromatography is a powerful purification technique that exploits an affinity reagent with strong and specific affinity for a molecule of interest 5-17
5.2 Labeled Tracers • For many years “labeled” has been synonymous with “radioactive” • Radioactive tracers allow vanishingly small quantities of substances to be detected • Molecular biology experiments typically require detection of extremely small amounts of a particular substance 5-18
Autoradiography Autoradiography is a means of detecting radioactive compounds with a photographic emulsion – Preferred emulsion is x-ray film – DNA is separated on a gel and radiolabeled – Gel is placed in contact with x- ray film for hours or days – Radioactive emissions from the labeled DNA expose the film – Developed film shows dark bands 5-19
Autoradiography Analysis • Relative quantity of radioactivity can be assessed looking at the developed film • More precise measurements are made using a densitometer – Area under peaks on a tracing by a scanner – Proportional to darkness of the bands on autoradiogram 5-20