Direct sequencing of DNA isolated from mRNA differential display.

A recently developed mRNA differential display technique has a potential power for identifying genes that are differentially expressed in a variety of in vitro and in vivo systems. One critical feature of this technique is to display most of the mRNA population on a sequencing gel after polymerase chain reaction using a 5' decamer and a 3' T12MN anchored primer. However, these primers are too small to be successfully used as a sequencing primer using the classical sequencing protocol. In the present report we described the application of an extended primer set for the re-amplification after mRNA differential display, which renders the amplified DNA suitable for direct sequencing using either of these extended oligonucleotides as a primer without further subcloning. This improved technique will greatly save the time, cost and labor-intensive work for the discovery of genes using mRNA differential display.