DNA

Deoxyribonucleic acid (DNA) is the molecule that carries genetic information for the
development and functioning of an organism

DNA is made of two linked strands that wind around each other to resemble a
twisted ladder
— a shape known as a double helix

Each strand has a backbone made of alternating sugar (deoxyribose) and phosphate groups.
Attached to each sugar is one of four bases: adenine (A), cytosine (C), guanine (G) or thymine (T).
The two strands are connected by chemical bonds between the bases: adenine bonds with thymine,
and cytosine bonds with guanine.
 RNA
• Ribonucleic acid (abbreviated RNA) is a nucleic acid present in all living cells that has structural
similarities to DNA.
• Unlike DNA, however, RNA is most often single-stranded.
• An RNA molecule has a backbone made of alternating phosphate groups and the sugar ribose, rather
than the deoxyribose found in DNA.
• Attached to each sugar is one of four bases: adenine (A), uracil (U), cytosine (C) or guanine (G).
• Different types of RNA exist in cells: messenger RNA (mRNA), ribosomal RNA (rRNA) and
transfer RNA (tRNA).
• In addition, some RNAs are involved in regulating gene expression. Certain viruses use RNA as their
genomic material.
Histochemical Demonstration

Hydrolysis of the nucleic acids will yield

(1) phosphate groups
(2) (2) sugars and
(3) (3) nitrogenous bases.

The demonstration of nucleic acid depends either upon the reaction of dyes with the
phosphate groups or the production of aldehydes from sugar deoxyribose.

No histochemical methods are available to demonstrate nitrogenous base

 Demonstration of nucleic acids

Method

Feulogen reaction DNA

Methylene green pyronin DNA and RNA

Fluorscent Method DNA and RNA

Gallocynin Chrome DNA and RNA

 Feulogen reaction

Mild acid hydrolysis ,employing 1 M HCl at 600C is used to break purine deoxyribose bond, the resulting
exposed aldehydes are then demonstrated by the used of schiff's reagent.
Elements containing DNA are staining a red-purple colour.
The ribose bond is unaffected by the hydrolysis and RNA is not demonstrated
The hydrolysis is a critical part of the method, an increasingly stronger reaction is obtained as the hydrolysis
time is increase until the optimum is reached. beyond the reaction becomes weaker, and if the hydrolysis is
continued the reaction may fail completely.
An important consideration is selecting the correct hydrolysis time is the fixative used to fix the tissue.
Bouin's fixative is not suitable as it causes overhydrolyis of the nucleic acid during fixation.
 Feulogen-naphthoic acid-hydrazide (NAH)method

This is used as control method for standard Feulogen reaction

The sections are hydrolysed in 1 M HCl as in Feulogen reaction

The aldeydes produced by this hyrdolysis is coupled by 2-hydroxy-3-naphthoic acid, which in turn is
coupled to diazonium salt fast blue B.
The results with this technique are identical to true Feulgen reaction
 Demonstration of RNA

The method of choice for demonstrating RNA is the methyle green-pyronin technique

RNA can also be demonstrated, along with DNA, by :

• Acridine orange
•Gallocyanin-chrome alum technique
Suitable extraction procedure to be applied
 Methyle green-pyronin techigue

Principle
Both dyes are cationic, when used in combination methyl green binds preferentially and
specifically to DNA leaving pyronin to bind to RNA .

The methyle green-specific reactivity is attributed to the spatial aligment of the NH4 groups of
the dye to phosphate radicals on the DNA double helix.

Pyronin stain does not show specific affinity and any negatively charged tissue constitutes will stain
red
 Methyle green-pyronin techigue

DNA : Green
RNA : Red
 Gallocyanin-Chrome alum method for DNA and RAN

This technique relies on the combination of phosphoric acid residue of the nucleic acids with
Gallocynin at an acid pH.
 Fluorescent Method

This method demonstrate both nucleic acids the most popular one is acridine orange technique
where DNA is stained yellow and RNA red
 Digestion methods for nucleic acids

Method Target
Enzymatic DNAase DNA
RNAase RNA
Chemical Perchloric Acid Both RNA & DAN
Trichloroacitic Both RNA & DAN
Acid
The Feulgen reaction has a limited diagnostic role, but is commonly used in conjunction with
microdensitometry to study cancer cell ploidy.
Use to study inverse relationship between DNA content of certain lymphoma and the tumor
prognosis.
These histochemical methods that are used for the demonstration of nucleic acids is
replace by other new methods

• In situ hybridization
• Immunohistochemistry
• Real time PCR

Feulgen reaction is now used in combination with image cytometry to assess the quality of the
DNA in single cell
Factors influence demonstration of nucleic acids

-Fixation
-Decalcification
-Basophilia
 Fixation

The nucleic acids are best preserved in alcoholic and acidic fixatives .e.g. Carnoy's fluid which
contains both alcohol and glacial acetic acid

Formalin has only a limited reaction with DNA and RNA, but for routine work gives acceptable
results.

Low (40C )temperature fixation in neutral buffered formalin has been shown to prevent DNA
degradation by cell nucleases, which is of some importance when carrying out molecular biology
studies
 Decalcification

EDTA decalcification gives the best results

Treatment of tissue with strong inorganic acids such as nitric acid or

hydrochloric acid is to be avoided as nucleic acids are progressively
extracted

Using an organic acid e.g. formic acid for short periods of time
(8hours) to decalcify tissue will normally permit acceptable
results with the standard special stains like methyle green-pyronin
staining

Over decalcification using an acid will result in pyroninophilic with the

methyle-green pyronin stain. The denatured DNA stains red rather
than the usual green coloration.
 Basophllia

Both DNA and RNA stain strongly with most cationic dyes.
A distinction has been drown between the link formed by simple cataionic dyes such as neutral red and
methylene blue and nucleic acid and that formed by metal complex dyes such as alum haematoxylin.
Staining with simple cationic dyes can be markedly reduced by prior treatment with acids.
Alum heamatoxylin staining of cell nucleus is much less affected by prior acid treatment.