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:
1. Introduction
2. Probe Design
- 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 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. Characteristics
Single
Stranded
Higher
Specificity
Improved
Signal
Lack of
Probe/Probe
Hybridization

5. Nucleic Acid
Hybridization
Probe Target

6. Probe
Design
Gene Probes
Oligonucleotide
Probes

7. Gene
Probes
>500
bases
Cloned
Probes
PCR
Greater
Specificity
Oligonucleotide
Probes
18-30 bases
40-60%
GC
No
Complimentary
Regions

8. Labelling
Characteristics of
starting material
Origin
Type
Hybridization Probes
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 from plasmids
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
Nucleic Acids
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 by autoradiography 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 to radioactive 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 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.,

17. Biotin Labeled Probe
Avidin or
Streptavidin
High affinities for
biotin
Detected with
Streptavidin couple to
a fluoroscent marker.

18. Enzyme Labeled
• Enzymes attached 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
Antigenic group 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 to the 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
DIG SYSTEM
• 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 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

23. Northern Blotting

24. 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
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 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 ?