73 Bioanalytical chemistry 4. Gel Electrophoresis Required reading: Sections 9.1, 9.2.3, 9.2.4, 9.5.1, 10.1 to 10.7, 11.1 to 11.5, and 15.5 of Mikkelsen and Cortón, Bioanalytical Chemistry Some objectives for this section ⇒ You will know what DNA agarose gel electrophoresis is ⇒ You will know what the difference between normal and pulsed field electrophoresis of DNA ⇒ You will know what SDS-PAGE is. ⇒ You will understand how the basis for molecular basis for size-based separation of proteins by SDS- PAGE. ⇒ You will know what IEF is. ⇒ You will know how SDS-PAGE and IEF can be combined in 2-dimensional gel electroophoresis ⇒ You will know what a Western Blot is. ⇒ You will appreciate how these techniques can be used in the analysis of DNA and proteins. Primary Source Material • Chapter 4 and 6 of Biochemistry: Berg, Jeremy M.; Tymoczko, John L.; and Stryer, Lubert (NCBI bookshelf). • Chapter 3 and 7 of Molecular Cell Biology 4th ed. (Ch. 9, 5th ed.): Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. (NCBI bookshelf). • Chapter 12 of Introduction to Genetic Analysis Anthony: J.F. Griffiths, Jeffrey H. Miller, David T. Suzuki, Richard C. Lewontin, William M. Gelbart (NCBI bookshelf). • Some animations are from http://www.wiley-vch.de/books/info/3-527-30300-6/. • http://www.piercenet.com/ Electrophoresis 74 The velocity of migration (v) of a molecule in an electric field depends on the electric field strength (E), the net charge on the protein (z), and the frictional coefficient (f). Ez v = f The frictional coefficient f depends on both the mass and shape of the migrating molecule and the viscosity (η) of the medium. For a sphere of radius r, f = 6πηr € The speed of migration is therefore proportional to the charge:mass ratio. z v ∝ r € • Electrophoresis is a technique for separating, or resolving, molecules in a mixture under the influence of an applied electric field. Dissolved molecules in an electric field move, or migrate, at a speed determined by their charge:mass ratio. • For example, if two molecules have the same mass and shape, the one with the greater net charge will move faster toward an electrode. € Agarose gel electrophoresis is typically used 75 for the separation of DNA molecules • Agarose is a polysaccharide that is obtained from the cell wall of some seaweed species. • The basic repeating unit of agarose is 1,3-linked β-D-galactopyranose and 1,4-linked 3,6-anhydro-α-L- galactopyranose. Two units of this repeating structure, known as agarobiose, are shown on the slide. • The average molecular mass of the agarose polysaccharide is ~120,000 daltons, which corresponds to ~400 agarobiose units. Some of the sugar groups in the polysaccharide are sulfated. • Agarose is essentially insoluble at room temperature but can be dissolved in water at high temperature. By heating and cooling a solution of agarose, a firm gel can be formed. The pores within this gel depend on the concentration of agarose used, but can be in the range of 100 to 300 nm. This pore size is sufficient to allow DNA molecules to pass through in an applied electric field. However, there is very little passive diffusion of DNA through the gel. • The mechanism for gelation of agarose upon cooling is thought to involves the formation of double helix structures in the initial stages of gelation. These double helixes then pack together into bundles to give the final gel structure. • Some information and the gelation mechanism figure from www.Lonza.com (MB_SB_appendixB_207-209.pdf) • What is agar and agarose? See http://www.lsbu.ac.uk/water/hyagar.html DNA is typically fragmented with restriction 76 enzymes prior to electrophoresis image credit: U.S. Department of Energy Human Genome Program, http://www.ornl.gov/hgmis. • Gel electrophoresis is used to separate DNA and RNA molecules by size and to estimate the size of nucleic acid molecules of unknown length by comparison with the migration of molecules of known length. • DNA and RNA molecules are highly charged near neutral pH because the phosphate group in each nucleotide contributes one negative charge. As a result, DNA and RNA molecules move toward the positive electrode during gel electrophoresis. Smaller molecules move through the gel matrix more readily than larger molecules, so that molecules of different length, such as restriction fragments, separate. Because the gel matrix restricts random diffusion of the molecules, molecules of different length separate into “bands” whose width equals that of the well into which the original DNA mixture was placed. Pulsed field agarose gel electrophoresis can 77 separate very large DNA molecules Normal mode Pulsed field • Agarose gel electrophoresis with a constant electric field can resolve DNA fragments up to ≈20 kb in length. Larger DNAs, ranging from 20 kb to 10 megabases (Mb) in length, can be separated by size with pulsed-field gel electrophoresis. • This technique depends on the unique behaviour of large DNAs in an electric field that is turned on and off (pulsed) at short intervals. When an electric field is applied to large DNA molecules in a gel, the molecules migrate in the direction of the field and also stretch out lengthwise. If the current then is stopped, the molecules begin to “relax” into random coils. The time required for relaxation is directly proportional to the length of a molecule. • The electric field then is reapplied at 90° or 180° to the first direction. Longer molecules relax less than shorter ones during the time the current is turned off. Since the molecules must relax into a random coil before moving off in a new direction, longer molecules start moving in the direction imposed by the new field more slowly than shorter ones. Repeated alternation of the field direction gradually forces large DNA molecules of different size farther and farther apart. • Pulsed-field gel electrophoresis is very important for purifying long DNA molecules up to ≈107 base pairs in length. Very large chromosomes must be digested into fragments of 107 base pairs or less before they can be analyzed. Such large restriction fragments can be generated with restriction enzymes that cut at rarely occurring 8-bp restriction sites. DNA fragments can be detected by 78 autoradiography or fluorescent dyes Detection of DNA fragments in gels: 1. Radioactive label + photographic film or a phosphorimager 2. Fluorescent dye a. Ethidium bromide Ethidium b. SYBR or monomeric cyanine dyes Yo-pro-1 c. Dimeric cyanine dyes "" Yoyo-1 http://www.probes.com/handbook/sections/0804.html • Whereas agarose gel electrophoresis is typically used to resolve DNA molecules that differ by at least ~100 base paris or so, the resolving power of polyacrylamide gel electrophoresis is so great that single-stranded DNA molecules up to about 500 nucleotides long can be separated if they differ in length by only 1 nucleotide. This high resolution is critical to the DNA-sequencing procedures described later. • DNA molecules composed of up to ≈2000 nucleotides can be separated electrophoretically on polyacrylamide gels, and molecules from 500 nucleotides to 20 kb on agarose gels. • 3 gels labeled A to C: (A) a polyacrylamide gel is used to fractionate single-stranded DNA. In the size range 50 to 200 nucleotides, DNA molecules that differ in size by only a single nucleotide can be separated from each other. (B) an agarose gel with medium-sized pores is used to separate double-stranded DNA molecules. This method is most useful in the size range 300 to 10,000 nucleotide pairs. (C) the technique of pulsed-field agarose gel electrophoresis has been used to separate 16 different chromosomes that range in size from 220,000 to 2.5 million nucleotide pairs. DNA molecules as large as 107 nucleotide pairs can be separated in this way. • Radioactively labeled DNA can be visualized by autoradiography of the gel. In this case, the gel is laid against a sheet of photographic film in the dark, exposing the film at the positions where labeled DNA is present. When the film is developed, a photographic image of the DNA is observed. Radiolabeled DNA bands also can be detected by laying the gel against a phosphorimager screen, which counts β-particles released by labeled molecules in the gel. • If the DNA is not radiolabeled, the gel is incubated in a solution containing the fluorescent dye ethidium: This planar molecule binds to DNA by intercalating between the base pairs. Binding concentrates ethidium in the DNA and also increases its intrinsic fluorescence. As a result, when the gel is illuminated with ultraviolet light, the regions of the gel containing DNA fluoresce much more brightly than the regions of the gel without DNA. 79 Analyzing very complex mixtures of genomic DNA: the Southern blot • "Gels used for electrophoresis of nucleic acids and proteins are permeable. This obvious fact didn't dawn on me until I tried to dissolve some agarose by floating it on a solution of sodium perchlorate and noticed a bead of liquid form on the top. I reasoned that if DNA molecules were carried through with the flow it would be possible to capture them on a nitrocellulose membrane, using the setup shown in the sketch. The big thrill came when, at the first attempt, I saw genes lit up as bands after hybridizing with radiolabeled probes. The Journal of Molecular Biology [initially] rejected the first manuscript1 as a 'methods paper' and the sketch is what I sent to people who had heard of the method and wanted to get on and use it." -- Ed Southern, Oxford University • Restriction-enzyme digestion of genomic DNA can result in so many fragments that a stained electrophoretic gel shows a smear of DNA.