Abstract
Quantitation of the copy number of a specific gene in a given sample on the genomic as well as on the steady state RNA transcriptional level is of great interest in both basic and clinical research. Traditionally, the copy number of a gene in a given sample is estimated by Southern blot or dot blot hybridization techniques. A certain amount of total genomic DNA of a sample is digested with restriction enzymes, size fractionated by nondenaturing agarose gel electrophoresis, and transferred and permanently bound to a nitrocellulose or nylon membrane (Southern blot); alternatively, DNA can be transferred and bound to a membrane without previous manipulations (dot blot). The membrane is then incubated with a radioactive, labeled molecular probe, which specifically binds to the gene of interest. After washing steps to remove unbound probe, the membrane is exposed to film and a signal is detected. The intensity of the signal, which can be quantitated (e.g., by densitometry), correlates to the amount of specific DNA (gene of interest) per amount of total genomic DNA and gives an estimate of the copy number of this gene.
Similarly, the degree of expression of a gene can be estimated after transferring a certain amount of RNA to a membrane (Northern blot) and hybridization to a specific probe. In each experiment, control samples with a known degree of gene expression have to be analyzed in parallel.
However, there are several limitations of the above techniques.
First, the sensitivity of these assays allows only for detection of gross differences in copy number and expression of a certain gene. Usually, only differences between samples of more than 3- to 4-fold can be detected. Moreover, very low levels of expression of a gene of interest may not be detectable at all.