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

摘要

We have developed a technique called the generation of longer cDNA fragments from serial analysis of gene expression (SAGE) tags for gene identification (GLGI), to convert SAGE tags of 10 bases into their corresponding 3' cDNA fragments covering hun- dred bases. A primer containing the 10-base SAGE tag 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 the 3' end of the corresponding sequence can be generated. Application of the GLGl technique can solve two critical issues in applying the SAGE technique: 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 identi- fied from multiple sequences that match the same SAGE tag. The development of the GLGl method provides several potential ap- plications. First, it provides a strategy for even wider application of the SAGE technique for quantitative analysis of global gene ex- pression. Second, a combined application of SAGE/GLGl can be used to complete the catalogue of the expressed genes in human and in other eukaryotic species. Third, it can be used to identify the 3' cDNA sequence from any exon within a gene. It can also be used to confirm the reality of exons predicted by bioinformatic tools in genomic sequences. Fourth. a combined application of SAGE/GLGl can be applied to define the 3' boundary of expressed genes in the genomic sequences in human and in other eukaryotic genomes.