MBLAB-ELECTROPHORESIS

• ELECTROPHORESIS

  • The movement of molecules by an electric current.
  • This can occur in air or solution, or in a matrix to limit migration and contain the migrating material.
  • Electrophoresis is routinely applied to the analysis of proteins and nucleic acids. Each phosphate group on a nucleic acid polymer is ionized, making the molecule negatively charged.
• ELECTROPHORESIS

  • Under an electric current, DNA and RNA will migrate toward the positive pole (anode).
  • In a matrix of agarose or polyacrylamide (gel), migration under the pull of the current is impeded, depending on the size of the molecules and the spaces in the gel matrix.
  • Because each nucleotide has one negative charge, the charge-to-mass ratio of molecules of different sizes will remain constant. DNA and RNA will therefore migrate at speeds inversely related to the size or length of the polymer.
• GEL SYSTEMS

  • Electrophoresis is very much affected by the medium of where the materials are migrating. These mediums are called the Gel Systems.
  • Gel matrices provide resistance to the movement of molecules under the force of an electric current.
  • They prevent diffusion and reduce convection currents so that the separated molecules form a defined group, or “band.”
  • The gel can then serve as a support medium for analysis of the separated components.
• Agarose Gel

  • The most common and used in the microbiology.
  • Size of the DNA affects the concentration. Small pieces of DNA (50 to 500 base pairs) are resolved on more concentrated agarose gels, for example, 2% to 3%. It is inversely proportional.
  • Larger fragments of DNA (2,000 to 50,000 base pairs) are best resolved in lower agarose concentrations, for example, 0.5% to 1%.
• Agarose Gel

  • The gel strength of any concentration of agarose will also decrease over time and with exposure to chaotropic agents such as urea.
  • DRAWBACK: If it is too concentrated, it might impede the migration because the nucleic acid would not move anymore. If it is low concentration, that would also produce a weak gel that is easily destroyed. That’s why, identify a good or make a good solution of the agarose gel.
• Pulsed-Field Gel Electrophoresis (PFGE)

  • For very large DNA molecules (50,000 to 250,000+ bp)
  • Pulses of current applied to the gel in alternating dimensions enhance migration.
  • The current in this process is in various directions because of a large number of base pairs present.
• Pulsed-Field Gel Electrophoresis (PFGE)

  • The simplest approach to this method is field-inversion gel electrophoresis (FIGE).
  • FIGE works by alternating the positive and negative electrodes during electrophoresis. In this type of separation, the DNA goes periodically forward and backward. FIGE requires temperature control and a switching mechanism. (positive on one side and negative on the other side)
• Pulsed-Field Gel Electrophoresis (PFGE)

  • This method is used for bacterial typing for epidemiological purposes. Enzymatic digestion of genomic DNA will yield a set of fragments that will produce a band pattern specific to each type of organism.
  • By comparing band patterns, the similarity of organisms isolated from various sources can be assessed.
  • This is not used for clinical purposes but more for epidemiological purposes and even research purposes.
• Polyacrylamide Gels

  • Acrylamide + methylene bisacrylamide = polymerizes into a matrix that has consistent resolution characteristics.
  • It differs from agarose which is from a seaweed, polyacrylamide is therefore synthetic. By synthetic, this allows precise control of the polymer properties and higher resolution than can be achieved with agarose.
  • Requires a catalyst. This catalyst could be nucleating agents such as ammonium persulfate (APS) or tetramethylethylenediamine (TEMED), or light activation.
• Polyacrylamide Gels

  • APS produces free oxygen radicals in the presence of TEMED to drive the polymerization mechanism.
  • Catalyst is needed to control the properties of the polymer
  • In light activation, free radicals are generated by a photochemical process using riboflavin plus TEMED. Excess oxygen inhibits the polymerization process. Therefore, de-aeration, or the removal of air, of the gel solution is done before the addition of the nucleating agents.
• Polyacrylamide Gels

  • The main advantage of polyacrylamide over agarose is the higher resolution capability for small fragments. With a single-base resolution, polyacrylamide gel is used for nucleic acid sequencing, mutation analysis, nuclease protection assays and other applications requiring high resolution of nucleic acid. Especially that it can resolve even the small fragments.
• CAPILLARY ELECTROPHORESIS

  • Separates particles by size like the usual, and charge.
  • By size: small, fast migration; large, slow migration
  • By charge: negative, fast migration; positive, slow migration
  • Because the size and charge of DNA work counter to each other, a polymer (gel) in the capillary will resolve the DNA fragments mostly according to size.
• CAPILLARY ELECTROPHORESIS

  • Negatively charged molecules are completely ionized at high pH, whereas positively charged solutes are completely protonated in low-pH buffers. They will then migrate to the anode.
  • Nucleic acids do not separate well in solution. As the size or length of a nucleic acid increases (slowing migration), so does its negative charge (speeding migration), effectively confounding the charge/mass resolution.
• CAPILLARY ELECTROPHORESIS

  • Introducing a polymer inside the capillary establishes resolution by impeding nucleic acid migration according to size more than charge.
  • It is important that the nucleic acid be completely denatured (single stranded) so that it will be separated according to its size because the secondary structure will affect the migration speed.
  • ADVANTAGE: increased sensitivity and immediate detection
• BUFFERING SYSTEMS

  • The purpose of a buffer system is to carry the current and protect the samples during electrophoresis.
  • Tris buffers = the most commonly used in DNA --> Tris borate EDTA (TBE), Tris phosphate EDTA (TPE), Tris acetate EDTA (TAE)
  • TBE has a greater buffering capacity than TAE. Although the ion species in TAE are more easily exhausted during extended or high-voltage electrophoresis, DNA will migrate twice as fast in TAE than in TBE in a constant current.
• Buffer Additives

  • Buffer additives modify sample molecules in ways that affect their migration = formamide, urea, detergents
  • Denaturing agents (DNA) - Formamide and urea are denaturing agents. It breaks hydrogen bonds between complementary strands or within the same strand of DNA or RNA.
  • The point is to prevent them from reannealing or connecting from each other, especially that it is preferred that they must be single stranded.
  • Denaturing agents will block hydrogen bonding sites, hindering complementary sequences from reannealing.
• Buffer Additives

  • Urea and heat in the gel systems maintain this conformation such that intrachain hybridization (folding) of the nucleic acid molecules does not affect migration speeds, and separation occurs strictly according to the size or length of the molecule.
• Buffer Additives

  • Denaturing agents (RNA) - can use the same in RNA but there are some more specific denaturing agent which is the methylmercuric hydroxide (MMH).
  • Reacts with amino groups on the RNA to prevent base pairing between complementary nucleotides and with aldehydes (e.g., formaldehyde, glyoxal), which also disrupt base pairing. MMH is not used routinely because of its extreme toxicity.
• ELECTROPHORESIS EQUIPMENT

  Horizontal Submarine Gel System

  • the most commonly used. Horizontal gels are run in acrylic gel boxes or baths that are divided into two parts, with a platform in the middle on which the gel rests.
  • The electrodes (platinum wires) placed across the box at each end of the bath compartments are connected to a power supply through the walls of the container. The gel is submerged in an electrophoresis buffer that fills both compartments and makes a continuous system through which the current flows.
• ELECTROPHORESIS EQUIPMENT

  Horizontal Submarine Gel Systems

  • The thickness of the gel and the volume of the buffer affect the current, and therefore the migration of the sample, so these parameters are kept constant for consistent results.
  • Because the gel is submerged throughout the loading and electrophoresis process, horizontal gels are sometimes referred to as submarine gels.
• Gel

  • The volume of the gel solution will determine the thickness of the gel = which affects migration.
  • Agarose, supplied as a dry powder, is mixed at a certain percentage (w/v) with electrophoresis buffer and heated on a heat block or by microwave to dissolve and melt the agarose.
  • The molten agarose is cooled to between 55°C and 65°C.
• Combs

  • Inserted into the top of the gel to create holes, or wells.
  • The size of the teeth in the comb = the capacity of the well for the sample.
  • The number of teeth in the comb = the number of wells
  • Two types: the regular combs and the sharkstooth combs = Sharkstooth comb is used for vertical gel. The sample is loaded between the teeth of the comb.
• ELECTROPHORESIS EQUIPMENT

  Vertical Gel System

  • Vertical gels are cast between glass plates that are separated by spacers. The spacers determine the thickness of the gel, ranging from 0.05 to 4 mm.
  • The bottom of the gel is secured by tape or by a gasket in specially designed gel casting trays. After the addition of catalyst and nucleating agents, the liquid acrylamide is poured or forced between the glass plates with a pipet or a syringe.
• ELECTROPHORESIS EQUIPMENT

  Vertical Gel System

  • The comb (sharkstooth comb) is then placed on the top of the gel. For light-activated polymerization, the gel between the glass plates is exposed to a light source.
  • During this process, it is important to prevent air from getting into the gel or beneath the comb. Bubbles will form discontinuities in the gel, and oxygen will inhibit the polymerization of the acrylamide.