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.     

Enhanced concatemer cloning-a modification to the SAGE (Serial Analysis of Gene Expression) technique.

The Serial Analysis of Gene Expression (SAGE) method, described in 1995 by Velculescu et al ., represents a powerful means to compare gene expression between two mRNA populations. An improvement to SAGE that removes contaminating linker molecules, which compromise the efficiency of the method, has been developed. This modification utilises biotinylated PCR primers, which generate biotinylated linkers at an early stage in the SAGE protocol, thus allowing removal of the unwanted linkers by binding to streptavidin-coated magnetic beads at a later stage. The application of this modification resulted in the rapid generation of high ditag yields and clones with large average insert sizes.     

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.     

Serial analysis of gene expression in the silkworm, Bombyx mori

The silkworm Bombyx mori is one of the most economically important insects and serves as a model for Lepidoptera insects. We used serial analysis of gene expression (SAGE) to derive profiles of expressed genes during the developmental life cycle of the silkworm and to create a reference for understanding silkworm metamorphosis. We generated four SAGE libraries, one from each of the four developmental stages of the silkworm. In total we obtained 257,964 SAGE tags, of which 39,485 were unique tags. Sorted by copy number, 14.1% of the unique tags were detected at a median to high level (five or more copies), 24.2% at lower levels (two to four copies), and 61.7% as single copies. Using a basic local alignment search tool on the EST database, 35% of the tags matched known silkworm expressed sequence tags. SAGE demonstrated that a number of the genes were up- or down-regulated during the four developmental phases of the egg, larva, pupa, and adult. Furthermore, we found that the generation of longer cDNA fragments from SAGE tags constituted the most efficient method of gene identification, which facilitated the analysis of a large number of unknown genes.     

Tandem arrayed ligation of expressed sequence tags (TALEST): a new method for generating global gene expression profiles.

We have developed a new and simple method for quantitatively analyzing global gene expression profiles from cells or tissues. The process, called TALEST, or tandem arrayed ligation of expressed sequence tags, employs an oligonucleotide adapter containing a type IIs restriction enzyme site to facilitate the generation of short (16 bp) ESTs of fixed position in the mRNA. These ESTs are flanked by GC-clamped punctuation sequences which render them resistant to thermal denaturation, allowing their concatenation into long arrays and subsequent recognition and analysis by high-throughput DNA sequencing. A major advantage of the TALEST technique is the avoidance of PCR in all stages of the process and hence the attendant sequence-specific amplification biases that are inherent in other gene expression profiling methods such as SAGE, Differential Display, AFLP, etc. which rely on PCR.