Nucleic acid hybridization

Overview

Hybridization is the process, discovered by Alexander Rich, of combining complementary, single-stranded nucleic acids into a single molecule. Nucleotides will bind to their complement under normal conditions, so two perfectly complementary strands will bind to each other readily. This is called annealing. However, due to the different molecular geometries of the nucleotides, a single inconsistency between the two strands will make binding between them more energetically unfavorable. Measuring the effects of base incompatibility by quantifying the rate at which two strands anneal can provide information as to the similarity in base sequence between the two strands being annealed. Annealing may be reversed by heating the double stranded molecule of DNA (or RNA or DNA:RNA) to break the hydrogen bonds between bases and separate the two strands. This is called melting or denaturation.

Experimental Procedure

1. The double-stranded DNA helix is heated in solution, usually buffered for pH.
2. Due to external conditions, the hydrogen-bonded base pairing becomes thermodynamically unfavorable and the complementary strands separate.
3. One set of denatured DNA is then mixed with another set of denatured DNA.
4. The combined sets are then cooled slowly to allow the DNA to reanneal and form a new "hybridized" DNA molecule.

By analyzing the rate at which different sets of denatured DNA reannealed, the relative differences and similarities between species or between individuals within a species can be mapped and compared based on a DNA Difference Score.

Southern and Northern blotting

There exist two kinds of nucleic acid hybridization. Both are methods in which radioactive labeled single DNA strands of known base sequences are used as a probe to detect the nucleotide sequence of another single stranded DNA or RNA molecule.

The first one is widely used for detection of specific genes in cellular DNA. It was developed by E.M. Southern and is called Southern Blotting. The DNA is digested with a restriction enzyme and the fragments are separated by electrophoresis. The gel is then overlaid with a nitrocellulose filter, to which the DNA fragments are transferred (blotting) to yield a replica of the gel. Following, the filter is incubated with a radiolabeled probe, which hybridizes to the complementary strand. This fragment is then visualized by exposure of the filter to X-ray film.

The second method is called northern blotting, it is used to identify the base pair sequence of RNA. RNA is extracted and fractionated according to size by electrophoresis. Then the procedure is the same as in Southern Blotting. Northern blotting is frequently used in studies of gene expression (e.g. whether specific mRNA is present in different types of cells).

References

• "Glossary of Biotechnology Terms". Anatomy of a Comparative Gene Expression Study.
• Cooper, Geoffrey M.; Hausman, Robert E. (2004), The Cell: A Molecular Approach (4 ed.), Sinauer Associates, Inc.

External links

• "Discovery of the Hybrid Helix and the First DNA-RNA Hybridization" by Alexander Rich
• Immunohistochemistry - In Situ Hybridization

See also

• base pairing
• PCR
• melt curve
• complementary DNA
• homology
• Western blotting