nucleic acid hibridization (nash)

8/14/2019 Nucleic Acid Hibridization (NASH)

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TECHNIQUES IN PLANT VIROLOGYCIP Training Manual2.5 DETECTION/Molecular Methods

Section 2.5.1Nucleic Acid

Hybridization (NASH)

Viruses are infective protein nucleic acid complexes (nucleoproteins)capable of directing their own replication when infecting a specific hostcell. Viroids behave similarly, but are made of only circular nucleic acidwithout a protein coat.

The chemical structure and molecular conformation of nucleic acids havemade possible the recent development of highly sensitive and specifichybridization techniques for detecting viruses and viroids (see Figure 1).

Nucleic acids are molecules formed by nucleotides. Typically, everynucleotide includes three characteristic components:

a) Nitrogen base. This may be derived from a purine (adenine orguanine) or from a pyrimidine (cytosine, uracil, and thymine)(Figure 2).


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P.V. Sec 2.5.1 998 Page 2 - INTERNATIONAL POTATO CENTER

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 ofthe 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 ofphosphodiester linkage

Figure 6. Linkage of nitrogen bases through hydrogen bonds


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

It is possible to obtain double-stranded DNA chains, double-strandedRNA chains, and double-stranded DNA-RNA chains (Figure 7). Thesedouble-stranded chains present a double-helix tridimensionalconformation. The two strands can be easily separated when thehydrogen bonds between the paired bases are broken.

Denaturation of the double strands can be obtained by heating the DNAsolution or by adding an alkali or acid to ionize the bases. If the solution iscooled slowly or if the pH of the solution is neutralized, the DNA strandswill pair again and the renaturation of the double helix occurs (Figure 8).The decoiling or melting of DNA causes an increase in the absorbancevalue at 260 nm. This is called a hyperchromic effect.

Melting temperature (Tm) is the temperature at which half the helicalstructure 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 ofits bases: one strand is complementary to the other. If a sample ofdenatured nucleic acid is fixed to a nitrocellulose membrane and if, undercertain conditions of temperature and ionic strength, it comes into contactwith another denatured sample, hybridization will occur if the strands arecomplementary (Figure 10).


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Figure 10. Hybridization between two nucleic acids of complementarynucleotide sequence.

Recommended LiteratureOld, R.W., S.B., Primrose. 1985. Principios de manipulacin gentica.

Editorial Acribia, S.A., Espaa. 375 p.

Hames, B.D., and S.J. Higgins (eds.). 1988. Nucleic acid hybridization, apractical approach. IRL Press, England. 245 p.

nucleic acid hibridization (nash)

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