COLD SPRING HARBOR, N.Y. (Wed., Sept. 1, 2010) -- A cell devotes a significant amount of effort to maintaining the stability of its genome, preventing the sorts of chromosomal rearrangements characteristic of many cancers. Assays that measure the rate of gross chromosomal rearrangements (GCRs) are needed in order to understand the individual genes and the different pathways that suppress genomic instability. In the September issue of Cold Spring Harbor Protocols, Richard Kolodner and colleagues from the University of California, San Diego's Ludwig Institute for Cancer Research present "Determination of Gross Chromosomal Rearrangement Rates," a genetic assay to quantitatively measure the rate at which GCRs occur in yeast cells. The assay measures the rate of simultaneous inactivation of two markers placed on a nonessential end of a yeast chromosome. This simple protocol for determining GCR mutation rates in a variety of genetic backgrounds coupled with a diversity of modified GCR assays has provided tremendous insight into the large numbers of pathways that suppress genomic instability in yeast and appear to be relevant to cancer suppression pathways in humans. This featured protocol is freely available on the journal's website.
Large segments of DNA can vary in copy number between individuals. Such copy number variations (CNVs) contribute greatly to genetic diversity and are also thought to be associated with susceptibility or resistance to some diseases, including cancer. "Simple Copy Number Determination with Reference Query Pyrosequencing (RQPS)," featured in the September issue of Cold Spring Harbor Protocols, provides an assay for determining the copy number of any allele in the genome. The method, from Raphael Kopan and colleagues at Washington University, takes advantage of the fact that pyrosequencing can accurately measure the ratio of DNA fragments in a mixture that differ by a single nucleotide. A reference allele with a known copy number and a query allele with an unknown copy number are engineered with single nucleotide variations, and the ratio seen between these probes and genomic DNA reflects the copy number. RQPS can be used to measure copy number of any transgene, differentiate homozygotes from heterozygotes, detect the CNV of endogenous genes, and screen embryonic stem cells targeted with bacterial artificial chromosome (BAC) vectors. RQPS is rapid, inexpensive, sensitive, and adaptable to high-throughput approaches. The article is freely available on the journal's website.
|Contact: David Crotty|
Cold Spring Harbor Laboratory