Blotting and Hybridization Techniques PDF

9. Blotting & Hybridization Techniques Hybridization Probe Probe: It is a fragment of DNA or RNA of variable length (100 – 10,000 bp) which can be radioactively or fluorescently labelled. Hybridization: A phenomenon in which single-stranded DNA or RNA molecules bind to...

9. Blotting & Hybridization Techniques Hybridization Probe Probe: It is a fragment of DNA or RNA of variable length (100 – 10,000 bp) which can be radioactively or fluorescently labelled. Hybridization: A phenomenon in which single-stranded DNA or RNA molecules bind to complementary sequence on sample DNA or RNA. It can be used to detect presence of DNA or RNA sequence, which is complementary to the sequence of the probe. Blotting Blotting is a technique for transferring DNA, RNA, and proteins onto a carrier so they can be separated. Often include the use of gel electrophoresis. Types of Blotting Southern Blot Northern Blot Western Blot It is used to It is used to It is used to detect DNA. detect RNA. detect protein. Basic Blotting Techniques Southern, northern and western blotting share basic blotting techniques. It begins with electrophoretic separation of DNA/RNA/protein on gel. Bands on the gel are transferred to a membrane (nitrocellulose, polyvinylidene difluoride (PVDF), and etc) where they are immobilized. Radiolabeled or enzymatically labelled antibody or DNA probes bind to immobilized target. molecule of interest is visualized with detected methods. Molecular probe Specific DNA/RNA can be detected by hybridization single-stranded nucleic acid tagged with fluorophore with samples. 1. Southern Blotting This method was developed by Sir Edwin Southern in 1975. Used to detect a specific DNA sequence in DNA samples. The technique involves four key steps: 1. DNA digestion by restriction enzyme. 2. Separation of DNA fragments. 3. Transfer to membrane. 4. Hybridization of probe to DNA fragments. https://youtu.be/GPVf_AWMYZ4 Procedure of Southern Blotting 1. DNA digestion with restriction enzyme. 2. DNA fragments are separated by gel electrophoresis. 3. DNA denaturation. 4. Transfer to nitrocellulose paper (blotting). 5. Hybridization with specific radiolabeled DNA probe. 6. Washing step. 7. Autoradiograph. Applications of Southern Blots Southern blots are used in: To detect a particular DNA fragment. To isolate a particular DNA fragment. To identify mutations. To identify genetic diseases. To identify infectious agents. For forensic and paternity testing. Application of Southern Blotting in Diagnostics. Unaffected Affected Affected 2. Northern Blotting It is a technique to determine the identity, size and abundance of specific RNA sequences. Developed by James Alwine and George Stark. This method immoblizes the molecule of interest on a membrane (nitrocellulose or nylon). It uses hybridization techniques to identify specific nucleic acids and genes.  Procedure of Northern Blotting 1. RNA is isolated from samples. 2. RNA samples are loaded onto agarose/polyacramide gel and will be separated according to size. 3. Gel is transferred to a membrane, nylon or nitrocellulose paper, by creating the sandwich arrangement. 4. Membrane is placed in a dish containing labelled probe that corresponds to the sequence of interest. 5. Membrane is washed to remove unbound probes. 6. Labelled probe is detected by autoradiography. Advantages and disadvantages of Northern Blot. Advantage: Can detect RNA size. Relatively cheap. Disadvantages: Less sensitive compared to RT-qPCR. Sensitive to even slight degradation of RNA samples. Require large amount of target RNA fragments. Applications of Northern Blot Gene expression studies. Diagnosis of diseases such as Crohn’s disease. Detection of viral microRNAs that play key roles in viral infection. Screen recombinants by detecting the mRNA produced by transgene. Application of Northern Blot in diagnostic With this procedure, a 1065 bp PCR product associated with the inflammation that occurs in Crohn’s disease was identified, cloned and sequenced. North- ern blot hybridisations showed that this novel sequence originates from a unique RNA species of 3.1 kb. 3) Western blotting https://youtu.be/AnjBg587mGg Western blot procedure In order to detect the expression of protein, sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel is used. Steps: i. Denaturation: sample is mixed with SDS (with β- mercaptoethanol) and bromophenol blue are mixed with protein sample. The SDS binds to the protein and form a negatively charged micelle around the protein. ii. Polyacrylamide gel Electrophoresis There are 2 types of vertical gel: continuous gel electrophoresis; ii) discontinuous gel electrophoresis. Discontinuous gel typically consists of two sections with different densities: (i) a stacking gel, and (ii) a separating gel. Stacking gel: i) Continuous ii) Discontinuous To concentrate all proteins in one band, so they will migrate in gel at the same time. Separating gel/running gel/resolving gel: To separate the proteins according to their molecular weight. An electric field is applied across the loaded gel with the positive pole positioned on the opposite side of the samples. iii. Blotting to membrane Proteins are transferred to a solid support membrane; e.g. nitrocellulose, polyvinylidene difluoride (PVDF), and nylon. In the transfer process voltage is applied to transfer the proteins from the gel to the membrane. The setup includes sponges, filter papers, the gel, and the membrane, which is placed between the gel and the positive electrode. Transfer Approaches iv. Detection of proteins on membrane. The membrane is then incubated with the primary antibody to the target protein. Primary antibody is detected by a tagged secondary antibody. The tagged secondary antibody catalyzes an enzymatic reaction with the substrate, which can be detected by film or a digital imager. Use of western blotting in clinical diagnosis A Western blot test is typically used to confirm a positive HIV diagnosis. Summary of western blot procedure Comparison between Northern Blot, Southern Blot and Western Blot. Extra: In situ hybridization is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue. It was invented by Joseph Gall 01 FlSH Fluorescence in situ If the probes are Types hybridization (FISH) fluorescently-labelled. CISH If the probe is labelled with a chromogen. Chromogenic in situ hybridization (CISH) 02 Characteristics of in situ hybridization methods Technique Visualization method Advantage Application CISH Bright-field microscopy Able to view CISH signal Molecular pathology and tissue morphology at diagnostics. the same time. DNA-FISH Fluorescence microscopy Multiplexible: visualize Gene presence, gene several targets in the location, mutation same sample. analysis. RNA-FISH Fluorescence microscopy Multiplexible: visualize Gene expression, RNA and flow cytometry several targets in the location. same sample. Appearance of FISH and CISH staining FISH CISH Procedures of FISH  Application of ISH Prenatal testing: e.g. testing for chromosomal abnormalities such as Down Syndrome. Pharmacogenomics: Predicting how quickly a drug is metabolize. Example: enzyme CYP2C19 metabolizes several drugs, such as anti-clotting agent Clopidogrel, into their active forms. Some patients are polymorphic for CYP2C19 gene that make poor metabolisers of those drugs. Pathogenomics: Molecular diagnostic are used to identify infectious diseases such as chlamydia, influenza virus and tuberculosis.  Case study Case A: Susan is a 23-year-old whose father, age 55, and paternal aunt, age 61, have been diagnosed with Huntington’s chorea. A paternal uncle, age 66, appears to be unaffected by the disease. Susan wants to know if she inherited the mutated gene from her father so that she can prepare for that future if necessary. She arranges to undergo DNA testing for Huntington’s disease. Her 17-year old brother, John, also decides to be tested after talking with Susan. DNA samples: ANSWER Susan (patient) Father (affected) This amplification can be detected by Southern blot analysis, since the Aunt (affected) size of the fragment bound by the probe is increased as a result of the amplification of the triplet repeat. Alternatively, PCR can be used to Uncle (unaffected) isolate the region containing the triplet repeats; the relative size of the John (brother) repeat region can be determined by running the PCR products on a gel. Control DNA with HD mutation Control DNA, normal (without HD mutation) For Southern blot: Digest the DNA samples with restriction enzyme, and then perform a Southern blot with the Huntington’s probe. By Which diagnostic technique can be used comparing the sizes of the fragments bound by the probe, by Susan to check if she has Huntington’s determine the Huntington’s gene status of Susan and her brother. disease? For PCR: Use the HD primers to perform PCR on the DNA samples. Run the PCR products on a gel References Southern blotting: https://youtu.be/CSrUm-EgTK4 Western blotting: : https://youtu.be/OkH8u84t84M Western blot simulation: https://www.labxchange.org/library/items/lb:LabXchange:227cccb5:lx_ simulation:1 https://www.labxchange.org/library/items/lb:LabXchange:ee936a17:lx _simulation:1 THANK YOU