Semi-nested PCR analysis of unknown tags on serial analysis of gene expression.

Serial analysis of gene expression (SAGE) is a powerful technique for studying gene expression at the genome level. However, short SAGE tags limit the further study of related data. In this study, in order to identify a gene, we developed a semi-nested PCR-based method called the two-step analysis of unknown SAGE tags (TSAT-PCR) to generate longer 3'-end cDNA fragments from unknown SAGE tags. In the procedure, a modified lock-docking oligo(dT) with two degenerate nucleotide positions at the 3'-end was used as a reverse primer to synthesize cDNAs. Afterwards, the full-length cDNAs were amplified by PCR based on 5'-RACE and 3'-RACE. The amplified cDNAs were then used for the subsequent two-step PCR of the TSAT-PCR process. The first-step PCR was carried out at an appropriately low annealing temperature; a SAGE tag-specific primer was used as the sense primer, and an 18 bp sequence (universal primer I) located at the 5'-reverse primer end was used as the antisense primer. After 15-20 PCR cycles, the 3'-end cDNA fragments containing the tag could be enriched, and the PCR products could be used as templates for the second-step PCR to obtain the specific products. The second-step PCR was performed with a SAGE tag-specific primer and a 22-bp sequence (universal primer II) upstream of universal primer I at the 5'-reverse primer with a high annealing temperature. With our innovative TSAT-PCR method, we could easily obtain specific PCR products covering SAGE from those transcripts, especially low-abundance transcripts. It can be used as a method to identify genes expressed in different cell types.