TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual

2.5 DETECTION/Molecular Methods

Section 2.5.1
Nucleic Acid
Hybridization (NASH)

Viruses are infective protein nucleic acid complexes (nucleoproteins)

capable of directing their own replication when infecting a specific host
cell. Viroids behave similarly, but are made of only circular nucleic acid
without a protein coat.

The chemical structure and molecular conformation of nucleic acids have

made possible the recent development of highly sensitive and specific
hybridization techniques for detecting viruses and viroids (see Figure 1).

Nucleic acids are molecules formed by nucleotides. Typically, every

nucleotide includes three characteristic components:

a) Nitrogen base. This may be derived from a purine (adenine or

guanine) or from a pyrimidine (cytosine, uracil, and thymine)
(Figure 2).
 Figure 1. Nucleotide chain of RNA

Figure 2. Nitrogen bases of nucleic acids

Figure 3. Pentose sugar present in nucleic acids

b) Pentose sugar. Deoxyribose for DNA and ribose for RNA (Figure 3).

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 c) Phosphoric acid. Supplies the necessary energy for the formation of
the phosphodiester linkages in the nucleotide chain (Figures 4 and 5).

Figure 4. Triphosphate group component of nucleotides

Figure 5. Liberation of phosphoric acid energy for the formation of

phosphodiester linkage

Figure 6. Linkage of nitrogen bases through hydrogen bonds

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 The nitrogen bases form pairs of bases through hydrogen bonds. Adenine
is linked to thymine or uracil through two hydrogen bonds, while
cytosine is linked to guanine through three hydrogen bonds
(Figure 6).

It is possible to obtain double-stranded DNA chains, double-stranded

RNA chains, and double-stranded DNA-RNA chains (Figure 7). These
double-stranded chains present a double-helix tridimensional
conformation. The two strands can be easily separated when the
hydrogen bonds between the paired bases are broken.

Denaturation of the double strands can be obtained by heating the DNA

solution or by adding an alkali or acid to ionize the bases. If the solution is
cooled slowly or if the pH of the solution is neutralized, the DNA strands
will pair again and the renaturation of the double helix occurs (Figure 8).
The decoiling or melting of DNA causes an increase in the absorbance
value at 260 nm. This is called a hyperchromic effect.

Melting temperature (Tm) is the temperature at which half the helical

structure is dissociated (Figure 9).

Figure 7. Double stranded DNA, RNA, and DNA-RNA chains.

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 Figure 8. Denaturation and renaturation of nucleic acids.

Figure 9. Melting temperature of double-stranded nucleic acids.

The most important characteristic of the double helix is the specificity of

its bases: one strand is complementary to the other. If a sample of
denatured nucleic acid is fixed to a nitrocellulose membrane and if, under
certain conditions of temperature and ionic strength, it comes into contact
with another denatured sample, hybridization will occur if the strands are
complementary (Figure 10).

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 Figure 10. Hybridization between two nucleic acids of complementary
nucleotide sequence.

Recommended Literature
Old, R.W., S.B., Primrose. 1985. Principios de manipulación genética.
Editorial Acribia, S.A., España. 375 p.
Hames, B.D., and S.J. Higgins (eds.). 1988. Nucleic acid hybridization, a
practical approach. IRL Press, England. 245 p.

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