Putting differences aside

Eisenstein, Michael

doi:10.1038/nmeth0406-324

Classic Protocol
Published: April 2006

Putting differences aside

Michael Eisenstein

Nature Methods volume 3, page 324 (2006)Cite this article

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Scientists have performed nucleic acid hybridization experiments since the early 1960s, but it was not until the late 1970s that an enterprising Stanford University graduate student named Fred Alt would demonstrate the potential of comparative hybridization for directly measuring changes in gene expression between two cell types. He and his colleagues were studying AT-3000, a murine sarcoma cell line known to greatly overexpress the enzyme dehydrofolate reductase (DHFR) relative to the parental line from which the cells had been derived. They generated radiolabeled cDNA from AT-3000 mRNA, which they hybridized to excessive quantities of parental cell line mRNA and purified on hydroxyapatite resin, separating out all double-stranded nucleic acid hybrids. This process readily yielded DHFR cDNA, left behind after 'subtraction' of transcripts common to both cell types owing to considerably reduced DHFR expression levels in parental lines. These findings would prove important as the first demonstration of both the phenomenon of gene multiplication and of the principle behind the method later known as 'subtractive hybridization'¹.

Shortly afterward, William Timberlake of Wayne State University presented a modified version of this procedure, which he dubbed 'cascade hybridization', for the isolation of genes differentially expressed at various developmental stages in the fungus Aspergillus nidulans^2,3. His results would demonstrate the potential for performing broader comparative studies, yielding hundreds of previously unidentified life stage–specific transcripts, and the combined impact of his work with that of Alt and others in the field would make subtractive hybridization an immensely popular means for comparing gene expression states.

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