Direct Sequencing of Differential Display PCR Products

The polymerase chain reaction (PCR)-based mRNA differential display (1 ) has become an increasingly popular alternative technique for isolating genes of interest in a variety of in vitro and in vivo systems (for review, see ref. 2 ), as compared to such conventional techniques as differential screening and subtractive hybridization. The method of mRNA differential display consists of two basic steps: (1) reverse transcription (RT) using a set of 3′-anchored primers, T12 MN (M = G, A or C; N = G, A, T or C); and (2) PCR amplification of cDNA fragments using arbitrary 10-mer primers (upstream) and anchored downstream primers. One critical feature of this technique is to display most of the mRNA population on a sequencing gel after PCR.