Molecular RNA Probe

What are molecular probes ? What is RNA probe? Applications of Probe ?
Presented By: M.Tech Biotechnology Students (IIT Guwahati). Bharat Bhushan Negi
Manoj Kumar S.
Umesh Kushwah
Medisetti Rajmohan Naidu
Vikkurthi Rajesh

1. Molecular RNA Probe Bharat Bhushan 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: Introduction Probe Design - Gene Probe - Oligonucleotide Probe Types of Labeling - Radioactive - Non-Radioactive labeling Methods - Primer extension - RNA Polymerases - End Labeling Nucleic Acid 5. Applications of RNA Probe

3. What is RNA probe ? A stretch of RNA that can detect a target sequence in the genome Probe and target base sequences must be complementary to each other

4. RNA Probes from plasmids

5. End- Labelling of Nucleic Acids O ne 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.

6. Radioactive labels Detection by autoradiography or Geiger muller counter. Radio labeled probes used to be the most common type but are less popular today because-

7. Methods Based on RNA 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.,

8. Biotin Labeled Probe

9. Enzyme Labeled

10. Antibody Labeled Probe

11. .

12. Digoxigenin (DIG) Labeled

13. Applications of RNA Probe 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

14. Northern Blotting

15. Fluorescence In Situ Hybridization (FISH) Assay Speed and Dynamic Safety & Cost Multi Probe Targeting Genetic Disorders Bacterial infections Different stages of cancer development Resolution is diffraction limited A dvantages Application s Limitation

16. RNA Protection Assay (RPA)

17. 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

18. Questions Q.1 Why there is 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 ?