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Sunday, January 31, 2010 @ 5:43 PM
Entry03: Methods of Studying Viruses The indirect methods fall into 3 categories: 1. Multiplication in a suitable culture system of the virus by the effects that it causes. 2. Serology which makes use of the interaction between a virus and antibody directed specifically against it. 3. Detection of viral nucleic acid. Different techniques used to study viruses: * Agarose gel electrophrosis * Polyacrylamide gel electrophrosis (PAGE) - Formamide or urea added to denature nucleic acic - Sodium dodecyl sulfate added to denature proteins (SDS- PAGE) * ELISA * Western Blot * Northern Blot * Southern Blot * Column Chromatography - Molecular Sieve - Ion- Exchange - Affinity * Centrifugation * Ultracentrifugation - Density Gradient Based (Buoyant Density) - Rate Zonal (Isokenetic) Northern Blot 1. RNA is separated by gel electrophoresis, usually an agarose gel. Because there are so many different RNA molecules on the gel, it usually appears as a smear. 2. The RNA is transferred to a sheet of special blotting paper called nitrocellulose. The RNA molecules retain the same pattern of separation they had on the gel. 3. The blot is incubated with a probe which is single-stranded DNA. This probe will form base pairs with its complementary RNA sequence and bind to form a double-stranded RNA-DNA molecule. The probe cannot be seen but it is either radioactive or has an enzyme bound to it. 4. The location of the probe is revealed by incubating it with a colourless substrate that the attached enzyme converts to a coloured product that can be seen or gives off light which will expose X-ray film. If the probe was labelled with radioactivity, it can expose X-ray film directly. Southern Blot 1. DNA (genomic or other source) is digested with a restriction enzyme and separated by gel electrophoresis, usually an agarose gel. The DNA is denatured into single strands by incubation with NaOH. 2. The DNA is transferred to a membrane which is a sheet of special blotting paper. The DNA fragments retain the same pattern of separation they had on the gel. 3. The blot is incubated with many copies of a probe which is single-stranded DNA. This probe will form base pairs with its complementary DNA sequence and bind to form a double-stranded DNA molecule. The probe cannot be seen but it is either radioactive or has an enzyme bound to it. 4. The location of the probe is revealed by incubating it with a colourless substrate that the attached enzyme converts to a coloured product that can be seen or gives off light which will expose X-ray film. If the probe was labelled with radioactivity, it can expose X-ray film directly. Western Blot 1. Denatured or native proteins, by length of the polypeptide or by 3-D structure of the protein, are separated by the means of gel electrophoresis. 2. After the proteins are transferred to a nitrocellulose membrane, the proteins are then detected using antibodies. 3. Each protein is attached to an antibody, and an antibody is used to detect antigen. A sensitive indicator is used to label an antibody, and there will be a colour reaction with streptavidin.  http://google.com ELISA The antigen is detected by antibody and an indicator, such as horse radish peroxidise, is used to label the antibody. A colour reaction is shown. In this method, one molecule must be binded to a solid surface.  http://google.com Agarose Gel Electrophoresis of DNA Agarose gels, the most commonly-used means of isolating and purifying fragments of DNA, which is a prerequisite for building any type of recombinant DNA molecule. Equipments: •An electrophoresis chamber and power supply •Gel casting trays, The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis. •Sample combs, around which molten agarose is poured to form sample wells in the gel. •Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE). •Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded. •Ethidium bromide, a fluorescent dye used for staining nucleic acids. Transilluminator (an ultraviolet lightbox), which is used to visualize ethidium bromide-stained DNA in gels. NOTE: always wear protective eyewear when observing DNA on a transilluminator to prevent damage to the eyes from UV light. Agarose Concentration: By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilite separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs. Electrophoresis Buffer: Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel! Conversely, if you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it. SDS-Polyacrylamide Gel Electrophoresis (PAGE) In their native form, proteins fold into a variety of shapes, some compact, some elongated. The rate of migration of native proteins through a sieving medium is therefore more a reflection of their relative compactness, and less an accurate measure of molecular weight. Denaturing the proteins nullifies structural effects on mobility, allowing separation on a true charge/mass ratio basis. It also separates subunits in multimeric proteins, allowing analysis of large, complex aggregates. The most commonly used denaturant is sodium dodecyl sulfate (SDS). SDS is an amphipathic surfactant. It denatures proteins by binding to the protein chain with its hydrocarbon ‘tail’, exposing normally buried regions and ‘coating’ the protein chain with surfactant molecules. The polar ‘head’ group of SDS adds an additional benefit to the use of this denaturant. Proteins solubilized in SDS bind the detergent uniformly along their length to a level of 1.4 g SDS/g protein. This creates a charge/mass ratio which is consistent between proteins. For this reason, separation on a polyacrylamide gel in the presence of SDS occurs by mass alone SDS is the most commonly used detergent in protein electrophoresis. Treatment with SDS creates a uniform charge to mass ratio between different proteins. PAGE + Urea or Formamide Acid urea PAGE allows the electrophoretic separation of different forms of a tRNA, discriminated by changes in bulk, charge, and/or conformation that are brought about by aminoacylation, formylation, or modification of a tRNA. Preparation of acid urea polyacrylamide gel - Gel dimension: • 0.4 mm × 20 cm × 45 cm - Gel composition: • 6.5% polyacrylamide (19:1 acrylamide/bisacrylamide) • 0.1 M sodium acetate pH 5.0 • 8 M urea • Dissolve the above ingredients under stirring (without heating the solution); adjust • Volume to 50 ml and degas for 5−10 min • Add TEMED (0.15% v/v) and ammonium persulfate (0.7% w/v) • Cast gel and allow to polymerize for approximately 2 hours - Acid urea sample buffer: • 0.1 M sodium acetate pH 5.0 • 8 M urea • 0.05% bromophenol blue • 0.05% xylene cyanol FF Gel electrophoresis •It is recommended that a short pre-electrophoresis (~30 minutes) be performed prior to loading the samples and that the sample wells be cleaned carefully. The electrophoresis buffer is 0.1 M sodium acetate pH 5.0. •Typically, 0.01−0.5 OD of tRNA (<>SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a method to resolve proteins in a mixture based on their molecular size. Negatively charged sodium dodecyl sulfate (SDS) in the sample buffer binds to heat denaturated proteins. The proteins migrate toward the positive pole in an electrical gradient, impeded by the polymerized and cross-linked polyacrylamide. (To convert all proteins to the same shape - we use SDS)  Labels: #entry |