Quantitation of gene copy number and mRNA using the polymerase chain reaction.

Abstract

Listen

Translate article

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...