Oligonucleotide hybridization analysis

As the density of information derived from efforts to sequence, map and identify human genes increased, so did the demand for analytical tools capable of exploiting this information. DNA microarrays were developed in response to this demand. Southern(69) was the first to describe parallel, in situ ohgonucleotide synthesis as a means of generating oligonucleotide probe arrays on solid supports for highly parallel hybridization analysis. Southern s method uses standard nucleotide synthetic reactions to synthesize the oligonucleotides. The reactions are carried out in a movable chamber, which provides a physical barrier between the reaction chamber and the intended synthesis area. 

With one common mutation, a very successful and efficient hybridization analysis is the PCR allele-specific oligonucleotide (ASO) assay. This requires that the sequence of the common mutation is known. 

Hruban RH, van Mansfeld AD, Offerhaus GJ, et al. K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization. Am J Pathol. 1993 143 545-554. 

Fig. 1. In situ hybridization analysis of alternatively spliced transcripts from the FMRFamide neuropeptide locus in the pulmonate mollusc Lymnaea stagnalis, using a battery of oligonucleotides specific for each of the exons see also ref. d)- (A) The locus comprises five exons (1-V) and gives rise to two mRNAs, both sharing exon I, but containing either exon II or alternatively exons III-V (3). (B) The set of consecutive serial sections from the CNS shown here has been probed with an oligonucleotide specific for exon II (left), exon III (middle), and exon I (right). It is clear that expression of exon II and III is mutually exclusive. In addition, because the two transcripts that con-...

RT-PCR, reverse transcription-polymerase chain reaction ASO, allele-specific oligonucleotide hybridization SSCP, single-stranded conformational polymorphism analysis. 

The principle behind DNA chips was bom from the basic Southern blot technique, and it is now a fast-moving field at the cutting edge of genome research. The two main scientific elements of DNA chips are the manufacture of microarrays with single-stranded oligonucleotides attached to pixels on the surface and hybridization analysis with spatial resolution. 

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

Daigo Y, Chin S-F, Gorringe KL, et al. Degenerate oligonucleotide primed-polymerase chain reaction-based array comparative genomic hybridization for extensive Amplicon profiling of breast cancers. A new approach for the molecular analysis of paraffin-embedded cancer tissue. Am. J. Pathol. 2001 158 1623-1631. 

---