Gel Electrophoresis in Medical Virology PDF

Gel electrophoresis - It is a method for separation and analysis of macromolecules (nucleic acid and proteins) and their fragments based on their molecular weight. - Why should we perform electrophoresis of virus components? Because molecular masses of capsid proteins and viral nucleic acid are...

Gel electrophoresis - It is a method for separation and analysis of macromolecules (nucleic acid and proteins) and their fragments based on their molecular weight. - Why should we perform electrophoresis of virus components? Because molecular masses of capsid proteins and viral nucleic acid are distinctive features of viruses. Gel types Agarose Polyacrylamide Composition - Polysaccharide extracted - Cross-linked polymer from sea weed. of acrylamide Gel casted - horizontally - vertically Toxicity - Non-toxic - Potent neuro-toxic Separation - large molecules - small molecules Usage - nucleic acid separation - Nucleic acid and protein separation Staining - Before or after running - After running the gel the gel (Ethidium bromide or RedSafe is used to visualize DNA under UV as it binds to DNA and causes fluorescence). Determination of the Molecular Mass of Coat Proteins by Polyacrylamide Gel Electrophoresis (PAGE) Step - Components - purpose - Purified virus - Denature the viral capsid + into the polypeptide Denaturation subunits denaturation buffer 1  Denaturation buffer may contain: - Sodium dodecyl sulfate (SDS): A detergent that eliminates the influence of the structure and charge by binding to the protein backbone giving it a net negative charge. - 2-Mercaptoethanol (2-ME): a reducing agent that cleaves disulfide bonds so that the proteins unfold into linear chains. 1- Upper stacking - Proteins are concentrated gel (~4% while migrating through Electrophoresis acrylamide) the stacking gel before entering the separating gel. The stacking occurs due to the difference in the rate of migration of glycine ions, chloride ions and proteins. 2- Lower - Separation of protein fragments resolving gel (~12% acrylamide) - After pouring the separating gel, overlay with water or isopropanol to level the gel and to prevent contact with oxygen which prevents polymerization. - Proteins migrate at different rates based on the concentration of the separating gel. High acrylamide concentration is suitable for separation of smaller proteins as it forms gel with smaller mesh size. 2 - After denaturation, proteins become negatively charged and can be separated in the gel on the basis of chain length. Smaller proteins migrate faster. - The distance migrated is inversely proportional to the molecular mass of the protein. - During the polyacrylamide gel electrophoresis, acrylamide and bisacrylamide are added to form a gel matrix. Acrylamide forms polymers in which bisacrylamide creates a cross-linking. - Ammonium persulfate (APS) is the one that initiates polymerization of the acrylamide and bisacrylamide. - TEMED reacts with the APS and causes splitting of the persulfate ions into sulfate free radicals. Formed free radicals initiate the polymerization (free radical reaction) of the acrylamide and cross-linking with bisacrylamide. - A protein-specific, - To make proteins visible dye binding or Staining color-producing chemical. - General steps include: - Water wash: to remove electrophoresis buffers. - Fix: an acid or alcohol wash to limit diffusion of protein bands from the matrix. - Water wash: to remove the acid or alcohol used in fixation. - Stain: using a dye or chemical to diffuse into the gel and bind to (or react with) the proteins. - Destain: to remove excess dye from the gel background. - Examples of the stains: - Coomassie dye stain: In acidic conditions, Coomassie dye binds to basic and hydrophobic residues of proteins, changing the color to intense blue. - Silver stain: the most sensitive in detecting protein. It involves the deposition of metallic silver onto the surface of a gel at the locations of protein bands (by binding to certain protein functional groups) then gets reduced to metallic silver, resulting in a brown-black color. 3  Microarray -Microarray is a collection of thousands to millions microscopic spots (contain known nucleic acid fragments called probes or reporters) attached to a solid surface (silicon, plastic or glass slide), referred to as a “chip”. The chip is then bathed with DNA or RNA isolated from a study sample. Complementary base pairing between the sample and the chip-immobilized fragments produces fluorescence that can be detected using a microarray scanner. - Hybridization is the process of forming a double stranded DNA molecule through base pairing between two complementary single- stranded DNA probe and a single-stranded DNA target. -Antibody microarrays: These are protein-specific microarrays that contain a collection of capture antibodies spotted and fixed on a solid surface and the interaction between the antibody and its target antigen is detected. -Microarray is a laboratory tool used to detect: Presence of virus in a sample. Gene expression Comparative gene hybridization (CGH) CGH Normal cell Infected cell Gene expression in normal cells is equal to that in infected cells. Gene expression in normal cells is higher than infected cells. Gene expression in infected cells is higher than normal cells. then wash 4  Blotting is a technique by which a macromolecule such as DNA, RNA, or protein is transferred to a solid support (such as: nitrocellulose, polyvinylidene difluoride or nylon membrane) after a gel electrophoresis and detected with a specific probe. ❖ Southern blotting (DNA) Types: ❖ Northern blotting (RNA) ❖ Western blotting (protein) -Steps involved in western blotting: 1-Sample preparation: western blotting -for protein extraction, chemical lysis buffers such as Tris-HCl are used to disrupt the cellular membranes and solubilize the target proteins. -Protease inhibitor cocktail should be used to avoid loss of protein. -DNase is used to reduce the viscosity of the released DNA. Southern and Northern blotting -for DNA or RNA extraction, detergent lyses the entire cell. - Protease is used for protein lysis. -DNA or RNA is purified and precipitated by alcohol. 2-Gel electrophoresis: Western blotting Polyacrylamide gel electrophoresis (PAGE) is used for separation of protein fragments. Southern and Northern blotting PAGE or agarose gel electrophoresis is used for separation of nucleic acid molecules. (-if DNA is double strand, it must be denatured into single strand by soaking the gel into denaturation buffer -RNA always exists in secondary structure so formaldehyde is added in the gel to denature RNA) 5  3- Blotting (transfer): -the separated fragments are transferred from the gel to a solid support membrane, usually made of chemically inert substances, such as nitrocellulose or polyvinylidene difluoride (PVDF). electro-blotting (electric current) nitrocellulose polyvinylidene difluoride Low background due to lower High background due to its higher protein binding capacity protein binding capacity Brittle, so it is not suitable for Higher mechanical strength, so it is stripping and reprobing suitable for stripping and reprobing cheap expensive -Two methods of transfer: Wet transfer Semi-dry transfer Transfer sandwich is placed Transfer sandwich is placed vertically horizontally large amount of buffer Small amount of buffer slow fast High efficient (80-100%) Less efficient (60-80%) -Ponceau red is a reversible protein stain, used for visualization of proteins in membrane. 4- Immobilization The fragments on the membrane can be immobilized by exposing the membrane to UV radiation or placing the membrane in oven at 80°C for 2-3 hours or 6 5- Hybridization Western blotting blocking buffer is used - a solution of primary antibody is incubated with the membrane to block unoccupied overnight at 4°C. binding surface on the - Following incubation, the membrane is washed several times to membrane, preventing remove unbound primary antibody. non-specific binding -add the secondary antibody, which is labeled with radioactively, of antibodies. fluorescent dye,or enzyme that can generate a signal that is used to indicate the location of protein such as horseradish peroxidase (HRP). Southern and Northern blotting - add a labelled nucleic acid with a homologous sequence to the target sequence. 5- washing: -wash the blot to remove excess probe. 6- Detection with substrate: -the bound, labelled probe is detected using the method required for the particular label used. -signal is detected when HRP is exposed to colorimetric or chmiluminescent substrate. Tetramethylbenzidine (TMB) Luminol Colorimetric substrate (purple to chmiluminescent substrate black) Detection limit: nanogram Pecogram to femtogram it is not suitable for stripping and it is suitable for stripping and reprobing reprobing Dot blot: a technique for detecting, and identifying proteins DNA, or RNA. This technique is similar to western, southern and northern blot, but protein, DNA, or RNA samples are not separated using electrophoresis; instead, samples are spotted through circular templates directly onto the membrane. 7  PCR (Polymerase chain reaction) A method used to make millions to billions of copies of a specific DNA sample rapidly. This amplification occurs through cycles of repeated heating and cooling using thermal cycler instrument. 1- DNA template contains the DNA target region to amplify. 2- DNA polymerase (Taq polymerase) Heat resistant enzyme, isolated from Thermus aquaticus polymerizes new DNA strands 3- Forward and reverse primers specific primers that are complementary to the DNA target region. 4- Deoxynucleoside triphosphates (dNTPs) The building blocks from which the DNA polymerase synthesizes a new DNA strand 5- Buffer solution Provides a suitable chemical environment for optimum activity and stability of DNA polymerase 6- Cations Mg2+ is the most common. 8  Steps 1- Initial Denaturation (94°C for 1min.) This causes DNA melting, or denaturation, of the double-stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single-stranded DNA molecules. 2- Denaturation -This causes DNA melting, or denaturation, of the double-stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single-stranded DNA molecules. -Higher temp is associated with high G≡C content. cycle 3- Annealing -annealing of the primers to each of the single-stranded DNA templates. Annealing temp is about 3–5 °C below the Tm of the primers used. - Melting temp (Tm) is the temperature at which one half of the DNA double stranded will dissociate to become single stranded, it depends on the length of DNA molecule and its specifc nucleotide sequence (GC content). 4- Extension/elongation - The temp at this step depends on the DNA polymerase used, synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture in the 5' to 3' direction -The time required for elongation depends on DNA polymerase used and the length of DNA region to amplify. -Multiple cycles are required to amplify the DNA target to millions of copies. 5- Final elongation (optional) -temp 70-74℃ for 5-15 min. is suitable for optimal activity of polymerase -ensures that any remaining ssDNA is fully elongated. 6- Final hold Cools the reaction chamber to 4-15℃ for an indefinite time and may be employed for short-term storage of PCR products. 9 RT-PCR (Reverse transcription polymerase chain reaction) is a laboratory technique combining reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA target. Reaction components Reverse transcriptase (RNA cDNA) + other PCR components Multiplex PCR is the simultaneous detection of multiple targets in a single reaction, with a different pair of primers for each target. -the primer design should ensure all primer pairs are highly specific. -all primers pairs should work at the same annealing temperature. -amplicon size shouldn’t differ too much. 10  QPCR (Real-time polymerase chain reaction) is a PCR-based technique that couples amplification of a target DNA with quantification of the concentration of that DNA. qPCR system 1-Thermal cycler 2-Optical module to detect fluorescence in the tubes during the run 3-Computer to translate the fluorescence data in results. Reaction components Real-time chemistries + other conventional PCR components Threshold line: is the level of detection or the point at which a reaction reaches a fluorescent intensity above background. Cycle threshold (Ct): is the cycle number at which the fluorescence of a PCR product can be detected above the background signal. Base line fluorescence -qPCR detection chemistries: (1) Non-specific (binding dye) Tm SYBR green -is a dye that binds to the minor groove of dsDNA. -when SYBR green binds to the dsDNA, the intensity of the fluorescent emission increases. Temp℃ 11 - after PCR cycle, add melt curve step 40 to 95℃ (slow ramp), thus to overcome the non-specificity of SYBR green. (2) Specific (hydrolysis probe/dual labeled probe) TaqMan probe -consists of a fluorophore covalently attached to the 5' end of the oligonucleotide probe and a quencher at 3' end. - Reporter (fluorophore): a molecule that emits light of certain wavelength after having absorbed light of a specific but different wavelength, such as FAM, Joe and Vic. - Quencher: a molecule that accepts energy from a fluorophore in the form of light and dissipates this energy in the form of light or heat (quenching), such as TAMRA, BQ, MGB. - 5'-3' exonuclease activity of Taq polymerase cleaves a dual labeled probe during hybridization to the complementary target sequence, thus relieving the quenching effect and allowing fluorescence of the fluorophore. 12