Generation of high-quantity and quality tag/ditag cDNAs for SAGE analysis

The serial analysis of gene expression (SAGE) technique is an important tool for genome-wide gene expression analysis. However, the requirement of a large amount of mRNA for the analysis and the difficulties in generating high-quality tag and ditag fragments for the construction of a SAGE library often interfere with the successful performance of the SAGE technique. We developed two procedures to solve these issues: (i) introducing low-cycle PCR amplification of the 3' cDNA before the BsmFI digestion of the 3' cDNAs and (ii) gel purifying the BsmFI-released tag fragments before ditag formation. These modifications provide a large quantity of initial 3' cDNAs and high-quality tags and ditags for the construction of SAGE libraries.     

Correct identification of genes from serial analysis of gene expression tag sequences

SAGE (serial analysis of gene expression) is a remarkable technique for genome-wide analysis of gene expression. It is crucial to understand the extent to which SAGE can accurately indicate a gene or expressed sequence tag (EST) with a single tag. We analyzed the effect of the size of SAGE tag on gene identification. Our observation indicates that SAGE tags are in general not long enough to achieve the degree of uniqueness of identification originally envisaged. Our observations also indicate that the limitation of using SAGE tag to identify a gene can be overcome by converting SAGE tags into longer 3' EST sequences with the generation of longer cDNA fragments from SAGE tages for gene identification (GLGI) method.