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Molecular RNA Probe
The document discusses molecular RNA probes, detailing their design, labeling methods, and applications in biotechnology. It differentiates between radioactive and non-radioactive labeling techniques, highlighting their advantages and safety considerations. The document also covers RNA probe applications such as northern blotting, fluorescence in situ hybridization, and RNA protection assays.
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Molecular RNA Probe
- 1. Molecular RNA Probe BharatBhushan Negi (174106012) Manoj Kumar S. (174106010) Umesh Kushwah (174106034) Medisetti Rajmohan Naidu (174106006) Vikkurthi Rajesh (174106009) Presented By : M.Tech Biotechnology Students IIT Guwahati
- 2. Contents: 1. Introduction 2. ProbeDesign - Gene Probe - Oligonucleotide Probe 3. Types of Labeling - Radioactive - Non-Radioactive labeling 4. Methods - Primer extension - RNA Polymerases - End Labeling Nucleic Acid 5. Applications of RNA Probe
- 3. What is RNAprobe ? • A stretch of RNA that can detect a target sequence in the genome • Probe and target base sequences must be complementary to each other
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- 8. Labelling Characteristics of starting material Origin Type HybridizationProbes DNA Cell-Based Cloning or PCR Normally ds;0.1kb to 1000kb for conventional DNA clones; 0.1kb to >20kb for PCR DNA Polymerase based DNA strand synthesis RNA Transcription from insert DNA cloned into vector Ss usually upto a thousand nucleotides ‘Run-off’ transcription from cloned DNA
- 9. RNA Probes fromplasmids Bacteriophage promoter
- 12. Types of Labeling Radioactive labels Non- radioactive labels Radioactive labels • 32P •35S • 3H • 125I Non- radioactive labels • Biotin • Enzyme labels • Fluorescence chemicals • Antibodies • Digoxigenin (DIG) System.
- 13. End- Labelling of NucleicAcids One may proceed straight to the labeling reactions. For either 5’ -, or 3’ - labeling, the RNA should be spin column- or PAGE-purified prior to experiments.
- 14. Radioactive labels Detection byautoradiography or Geiger muller counter. Safety considerations High cost Disposal of radioactive waste products. Radio labeled probes used to be the most common type but are less popular today because- Radiolabel probes • Radio labeled probes are the most sensitive. • provide the highest degree of resolution. Half life time • 32P- Relative short half life14.3 days • 35S- longer half-life (87.4 days) • 3H- longest half-life (12.3 year). • 125I- longer half-life (60 days)
- 15. Non-Radioactive Labels PROBE TARGET Compared toradioactive labels, the use of nonradioactive labels have several advantages. Safety Higher stability of probe Detection in situ Less time taken to detect the signal Efficiency of the labeling reaction
- 16. Methods Based onRNA Polymerases RNA polymerases catalyzes the synthesis of RNA from nucleoside triphosphates using a DNA template. Thus they can incorporate labeled ribonucleotides into RNA during transcription if such labeled nucleotides are provided to it. eg: RNA Polymerase obtained from E.coli,T7 etc.,
- 17. Biotin Labeled Probe Avidinor Streptavidin High affinities for biotin Detected with Streptavidin couple to a fluoroscent marker.
- 18. Enzyme Labeled • Enzymesattached to probe. • Attached with the help of Glutaraldehyde. Enzyme • Detected by reaction with a substrate that changes color called “reporter group. • Substrate for HRP, forms a purple insoluble product. • HRP catalyzes the oxidation of luminol, a chemiluminogenic substrate for HRP. Substrate • Alkaline phosphatase. • Horseradish peroxidase (HRP). • β- galactosidase. • Xanthine oxidase Examples
- 19. Antibody Labeled Probe Antigenicgroup or Antibody is coupled to the probe Detected by using specific antibodies or secondary Ab Conjugated with HRP/AP. The signal is then detected by when catalyzed with oxidation of Luminol with HRP and give color reaction.
- 20. Chemiluminescence Labeled Chemiluminescent chemicals attached tothe probe. Detected by their Light Emission using a Luminometer. Fluorescence chemicals Labeled Fluorescence chemicals attached to probe . Using Ultraviolet (UV) light, This type of label is especially useful for direct examination of microbiological or cytological specimens under the microscope-a technique known as fluorescent in situ hybridization (FISH).
- 21. Digoxigenin (DIG) Labeled DIGSYSTEM • Most sensitive • Comprehensive • Convenient • Effectivesystem for labeling and detection of DNA, RNA, and oligonucleotides. ANTIBODY • Anti-digoxigenin antibody–alkaline phosphatase conjugate SIGNAL DETECTION • The signal is then detected with colorimetric or chemiluminescent alkaline phosphatase substrates.
- 22. Applications of RNAProbe RNA Protection Assay Biomedical Northern Blotting In-situ Hybridization • RNA separation by electrophoresis • Detection with complementary hybridized probe • To reveal the location of specific nucleic acid sequences on chromosomes or in tissues • Detection of pathogenic microorganism Example : Actinomyces, Bacteriodes, Borrelia • Detection of food spoilage • Highly sensitive and sequence specific
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- 24. Fluorescence In SituHybridization (FISH) • Assay Speed and Dynamic • Safety & Cost • Multi Probe Targeting • Genetic Disorders • Bacterial infections • Different stages of cancer development Resolution is diffraction limited Advantages Applications Limitation
- 25. RNA Protection Assay(RPA)
- 26. References: 1. Keller,G. H. and Manak, M. M. (1989) DNA Probes, Stockton, New York. 2. Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 3. Karcher, S. J. (1995) Molecular Biology: A Project Approach, Academic, San Diego, CA. 4. Hugenholtz, P., Tyson, G. W., and Blackall, L. L. (2002) Design and evaluation of 16S rRNAtargetedoligonucleotide probes for fluorescence in situ hybridization, in Gene Probes: Principles and Protocols (Aquino de Muro, M. and Rapley, R., eds.), Humana, Totowa, NJ, pp. 29– 42. 5. Boehringer Mannheim GmbH (1995) The DIG System User’s Guide for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany. 6. Boehringer Mannheim GmbH (1996) Nonradioactive In Situ Hybridisation Manual: Application Manual, 2nd ed. Boehringher Mannheim GmbH, Mannheim, Germany. 7. Alphey, L. and Parry, H. D. (1995) Making nucleic acid probes, in DNA cloning 1: Core Techniques (Glover, D. M. and Hames, B. D., eds.), IRL, Oxford, pp. 121–141. 8. Feinberg, A. P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Analy. Biochem. 132, 6–13. 9. Feinberg, A. P. and Vogelstein, B. (1984) Addendum. Analy. Biochem. 137, 266–267. 10. Aquino de Muro, M. and Priest, F. G. (1994) A colony hybridization procedure for the identification of mosquitocidal strains of Bacillus sphaericus on isolation plates. J. Invertebr. Pathol. 63, 310–313. 11.CHAPTER FOURTEEN RNA Radiolabeling Rishi Porecha, Daniel Herschlag1 Department of Biochemistry, Stanford University, Stanford, CA 12. Rio, D. C. (2011) RNA: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press 13. Cooper, Geoffery M. (2007) The Cell: A Molecular Approach. 4th ed. Washington D.C.: ASM Press
- 28. Questions Q.1 Why thereis the need of RNA probe ? Q.2 Why RNase can’t cleave the double stranded RNA ? Q.3 Which is the best method for labeling of probe ?