Direct Automated Sequencing of DDRT -PCR Fragments

Differential display is a powerful tool for comparing gene expression in different cell or tissue preparations (Liang and Pardee 1992). However, differential display does not give any indications regarding the identity of the differentially expressed genes. Thus, the next step is to identify the mRNAs that are represented in the differentially displayed bands and this is achieved by sequencing the DNA fragments. If the differential display is made from human cells, sequencing followed by searches in sequence databases will often identify the differentially expressed gene, since The Human Genome Project has resulted in the accumulation of a large number of human cDNA and gene sequences in sequence databases. In particular, the expressed sequence tag (EST) programs have produced a very large number of partial cDNA sequences consisting of “single read” sequences from the ends of randomly picked cDNAs (Adams et al. 1995). Since most differential display methods select and display fragments that originate from the 3’-ends of mRNAs, the sequences of the fragments will correspond to the 3’-EST sequences. Since the 3’-ESTs are linked to their corresponding 5’-ESTs which, in most cases, are derived from the coding region of the mRNAs, the 3’-ESTs will identify the gene.