Methods for marking molecules to identify gene alterations and metabolic shifts

May 2 2007

Physical alterations of DNA in chromosomes can cause serious diseases such as Down syndrome, Prader-Willi syndrome, or cancer.

Likewise, changes to the physiological environment of cells, with drugs or toxins, for example, can alter their metabolic output. To track these processes, scientists need ways to mark genes and their protein products. This month's release of Cold Spring Harbor Protocols (www.cshprotocols.org) features freely available methods for marking molecules to identify gene alterations and metabolic shifts.

The first method (http://www.cshprotocols.org/cgi/content/full/2007/10/pdb.prot4743) describes how to grow cells from bacteria, yeast, insects, or mammals in media containing the stable isotope nitrogen-15. As the cells grow, they produce nascent proteins that include nitrogen-15, a marker that distinguishes newly formed metabolic products from pre-existing ones. The differences between these cells and "normal" cells (grown in media without nitrogen-15) can then be quantified using a technique called mass spectrometry.

To identify structural features and abnormalities in chromosomes, scientists need to tag DNA probes with visual markers. The second freely available method (http://www.cshprotocols.org/cgi/content/full/2007/10/pdb.prot4730) describes how to label DNA probes with fluorescent markers of six different colors, which span the visible spectrum. These labeled DNA probes can be used simultaneously in FISH (fluorescent in situ hybridization) experiments, during which the probes bind to specific chromosomal regions of interest. The colorful chromosomes can then be viewed under a microscope.