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  Section: Biotechnology Methods » Molecular Biology
 
 
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In Situ Hybridization

 
     
 
Content
Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Chromosomes
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Transformation
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method

A modern approach to the specific location of genes on chromosomes is a technique for the hybridization of DNA and RNA “in situ.” With this procedure, specific radioactive RNA or DNA (known as probes) can be isolated (or
synthesized “in vitro”) and then annealed to chromosomes that have been treated in such a manner that their basic double-stranded DNA has been “melted” or dissociated.

In theory, and fortunately in practice, when the DNA is allowed to reanneal, the probe competes for the binding, but only where it mirrors a complementary sequence. Thus, RNA will attach to the location on the chromosome where the code for its production is to be found. DNA will anneal to either RNA that is still attached to a chromosome, or to the complementary sequence DNA strand within the chromosome. Since the probe is radioactive, it can be localized via autoradiographic techniques.

Finally, it is possible to produce an RNA probe that is synthesized directly from repetitive sequences of DNA, such as that found within the nucleolar organizer region of the genome. This RNA is known as cRNA (for copied RNA)
and is a convenient source of a probe for localizing the nucleolar organizer gene within the nucleus, or on a specific chromosome.

The use of in situ hybridization begins with good cytological preparations of the cells to be studied, and thepreparation of pure radioactive probes for the analysis. The details depend upon whether the hybridization is between DNA (probe) and DNA (chromosome), DNA (probe) and RNA (chromosome), or between RNA (probe) and DNA (chromosome).


Preparation of the Probe

Produce radioactive RNA by incubating the cells to be measured in the presence of 3H-uracil, a specific precursor to RNA. Subsequent to this incubation, extract rRNA from the sample and purify through differential centrifugation, column chromatography or electrophoresis. Dissolve the radioactive RNA probe in 4X saline-citrate containing 50% formamide to yield a sample that has 50000 to 100000 counts per minute, per 30 microliter sample, as determined with a scintillation counter. Add the formamide to prevent the aggregation of RNA.


Preparation of the Slides

Fix the materials to be studied in either 95% ethanol or in 3:1 methanol:water, attach to presubbed slides (as squashes for chromosomes) and air dry.


Hybridization
Place the air-dried slides into a moist chamber, usually a disposable petri dish containing filter paper, and carefully place 30 microliters of RNA probe in 4X SSC-50% formamide onto the sample. Carefully add a cover slip (as in the preparation of a wet mount), place the top on the container and place in an incubator at 37°C for 6–12 hours.


Washing
  1. Pick up the slides and dip into 2X SSC so that the coverglass falls off.
  2. Place the slides in a coplin jar containing 2X SSC for 15 minutes at room temperature.
  3. Transfer the slides to a treatment with RNase (50 microgram/mL RNase A, 100 units/mL RNase T1 in 2X SSC) at 37°C for 1 hour.
  4. Wash twice in 2X SSC, 15 minutes each.
  5. Wash twice in 70% ethanol, twice in 95% ethanol, and air dry.

Autoradiography

Add photographic emulsions to the slides and after a suitable exposure period, develop the slides, counterstain, and add cover slips.
Analyze the slides by determining the location of the radioactive probe on the chromosomes or within the nuclei.

 
     
 
 
     




     
 
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