Hybridization, nucleic acids

Double-stranded DNA denatures into single strands as the temperature rises but renatures into a double-stranded structure as the temperature falls. Any two single-stranded nucleic acid molecules can form double-stranded structures (hybridize) provided that they have sufficient complementary nucleotide sequence to make the resulting hybrid stable under the reaction conditions. 

The concentration of a specific nucleic acid sequence in a sample can be measured by hybridization with a suitable labeled DNA probe. After hybridization, nuclease is used to destroy unhybridized probe and the probe remaining is a measure of the concentration of the target sequence. The hybridization conditions can be altered to ensure that only identical sequences (high stringency conditions) or identical plus related sequences (low stringency conditions) will hybridize with the probe and hence be detected. 

Southern blotting involves electrophoresis of DNA molecules in an agarose gel and then blotting the separated DNA bands on to a nitrocellulose filter. The filter is then incubated with a labeled DNA probe to detect those separated DNA bands that contain sequences complementary to the probe. 

Northern blotting is analogous to Southern blotting except that the sample nucleic acid that is separated by gel electrophoresis is RNA rather than DNA. 

For in situ hybridization, a tissue sample is incubated with a labeled nucleic acid probe, excess probe is washed away and the location of hybridized probe is examined. The technique enables the spatial localization of gene expression to be determined as well as the location of individual genes on chromosomes. 

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O. G. PNA-related oligonucleotide mimics and their evaluation for nucleic acid hybridization studies and analysis. Nucleosides, Nucleotides Nucleic Acids 2001 20 419-428. 

Nucleic acid hybridization can be detected by means of the piezoelectric QCM (= quartz crystal microbalance) (a) Y Okahata, Y Matsunobu, K Ijiro, M Mukae, A Murakami, K Makino. J. Am. Chem. Soc. 114 8299-8300, 1992 (b) S Yamaguchi, T Shimomura. Anal. Chem. 65 1925-1927,1993 (c) KIto, KHashimoto, Y Ishimori. Anal. Chim. Acta 327 29-35,1996 ... 

The difficulty with protein arrays is that proteins do not behave as uniformly as nucleic acid. Protein function is dependent on a precise, and fragile, three-dimensional structure that may be difficult to maintain in an array format. In addition, the strength and stability of interactions between proteins are not nearly as standardized as nucleic acid hybridization. Each protein-protein interaction is unique and could assume a wide range of affinities. Currently, protein expression mapping is performed almost exclusively by two-dimensional electrophoresis and mass spectrometry. The development of protein arrays, however, could provide another powerful... 

Nucleic acid hybridization Fluorescent in situ hybridization (FISH)... 

Two principal types of nucleic acid-based methods, nucleic acid hybridization and polymerase chain reaction (PCR), are commonly used for the rapid identification of bacteria. A few other nucleic acid-based methods will also be mentioned. 

Nucleic acid hybridization methods use oligonucleotide DNA probes with sequences complementary to a portion of the nucleic acid of the target bacterium38,60 and designed to hybridize with immobilized DNA or RNA on a membrane. After any unbound probe has been washed off, the hybridized probe can be detected.64 66... 

Masuko M, Ohtani H, Ebatal K, Shimadzu A (1998) Optimization of excimer-forming two-probe nucleic acid hybridization method with pyrene as a fluorophore. Nucleic Acids Res 26 5409-5416... 

Montone KT, Brigati DJ, Budgeon LR. Anatomic viral detection is automated the application of a robotic molecular pathology system for the detection of DNA viruses in anatomic pathology substrates, using immunocytochemical and nucleic acid hybridization techniques. Yale J. Biol. Med. 1989 62 141-158. 

Similar techniques can be used to devise (strept)avidin-biotin assay systems for detection of nucleic acid hybridization. DNA probes labeled with biotin can be detected after they bind... 

Diamandis, E.P. (1993) Time-resolved fluorometry in nucleic acid hybridization and Western blotting techniques (Review). Electrophoresis 14, 866-875. 

Ghosh, S.S., Kao, P.M., and Kwoh, D.Y. (1989) Synthesis of 5 -oligonucleotide hydrazide derivatives and their use in preparation of enzyme-nucleic acid hybridization probes. Anal. Biochem. 178, 43-51. 

Nucleic acid structures and sequences primary and secondary structure of DNA fragments, translocation of genes between two chromosomes, detection of nucleic acid hybridization, formation of hairpin structures (see Box 9.4), interaction with drugs, DNA triple helix, DNA-protein interaction, automated DNA sequencing, etc. 

D2. Dattagupta, N., Rae, P. M. M., Huguenel, E. D., Carlson, E., Lyga, A. el al.. Rapid identification of microorganisms by nucleic acid hybridization after labeling the test sample. Anal. Biochem. 177, 85-89 (1989). 

LI. Landry, M. L., Nucleic acid hybridization in viral diagnosis. Clin. Biochem. 23, 267-277 (1990). 

W2. Wetmur, J. G., DNA probes Applications of the principles of nucleic acid hybridization. Crit. Rev. Biochem. Mol. Biol. 26, 227-259 (1991). 

Wetmur JG. 1991. DNA probes applications ofthe principles of nucleic acid hybridization. Grit Rev Biochem Mol Biol 26 227. 

Anderson, M.L.M. and Young, B.D., Quantitative filter hybridization, in Nucleic Acid Hybridization A Practical Approach, Hames, B.S. and Higgis, S.J., Eds., IRL Press, Oxford, 1985, chap. 4. 

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