Generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification

We have developed a technique called the generation of longer cDNA fragment s from serial analysis of gene expression (SAGE) tags for gene identificati on (GLGI), to convert SAGE tags of 10 bases into their corresponding 3' cDN A fragments covering hundred bases, A primer containing the 10-base SAGE ta g is used as the sense primer, and a single base anchored oligo(dT) primer is used as an antisense primer in PCR, together with Pfu DNA polymerase. By using this approach, a cDNA fragment extending from the SAGE tag toward th e 3' end of the corresponding sequence can be generated. Application of the GLGI technique can solve two critical issues in applying the SAGE techniqu e: one is that a longer fragment corresponding to a SAGE tag, which has no match in databases, can be generated for further studies; the other is that the specific fragment corresponding to a SAGE tag can be identified from m ultiple sequences that match the same SAGE tag. The development of the GLGI method provides several potential applications. First, it provides a strat egy for even wider application of the SAGE technique for quantitative analy sis of global gene expression. Second, a combined application of SAGE/GLGI can be used to complete the catalogue of the expressed genes in human and i n other eukaryotic species. Third, it can be used to identify the 3' cDNA s equence from any exon within a gene. It can also be used to confirm the rea lity of exons predicted by bioinformatic tools in genomic sequences. Fourth , a combined application of SAGE/GLGI can be applied to define the 3' bound ary of expressed genes in the genomic sequences in human and in other eukar yotic genomes.