Molecular Genetics

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Investigators often are interested in genes that are expressed in particular cell types or under particular circumstances. Subtractive cDNA hybridization (left) and differential display (right) provide two means for isolating such genes without knowing their function. Also useful is a technique, the two hybrid system, for isolating genes for an unknown protein that interacts with a target protein.

Illustrated is the basic approach to subtractive hybridization. Many variations on the approach (eg., which molecules are hybridized, how the separation of hybridized and non-hybridized molecules is obtained) exist. The enrichment for treatment-specific sequences is not 100% since hybridization of the common sequences is seldom driven to absolute completion. Typically, several cycles of hybridization-selection are used.

For polyA-containing mRNAs, the cDNA synthesis primer can be an oligo dT terminated by one of the three other nucleotides. Differential display is more rapidly done than subtractive hybridization and provides a greater selectivity. Differential display is subject to PCR artefacts. A variety of primers must be used to find all treatment-specific mRNAs. Differential display has almost supplanted subtractive hybridization as the method of choice to isolate treatment-specific transcribed sequences.

A technique gaining in popularity is comparative hybridization to EST microarrays.

• By random or biased random cloning techniques, a population of cDNA sequences is generated. These are called expressed sequence tags or ESTs. Alternatively, from completely sequenced genomes, representative sections representing actual genes can be used.
• The EST sequences are spotted at high density in microarrays.
• One array is hybridized with fluorescently labeled cDNA obtained from the source of interest. Another array is hybridized with cDNA from the reference source and labeled with a different fluorophore.
• Relative intensities of hybridization at each of the spots of the microarray are compared by precise scanning for the fluorescence emissions.
• Increases and decreases in gene expression relative to the reference can be detected.
• Standards for such procedures have been proposed.

In a variation on differential display, double-stranded cDNA products generated by PCR are digested with restriction endonucleases before display by gel electrophoresis.