Serial Analysis of Gene Expression (SAGE)
Schematic representation of the SAGE protocol
Adapted from Velculescu V.E, Zhang L., Vogelstein B. and Kinzler K. W. (Science 270:484-487, 1995)
Legend
| Step 1 | The restriction enzyme Nla III, "anchoring enzyme" (AE), cleaves at the sequence 5'-CATG, leaving a four nucleotide 3' overhang. Biotinylated fragments are isolated using streptavidin beads. |
| Step 2 | Divide in half and ligate to linkers A or B. The linkers contain unique primer binding sites, the recognition sequence (5'-GGGAC) for a tagging enzyme (TE), in this case BsmFI, and an Nla III compatible sticky end. |
| Step 3 | Cleave with BsmFI tagging enzyme (TE) and blunt-end-fill in. The enzyme BsmFI cuts 10 and 14 bases in the 3' direction from its recognition site, thus adding the "Tag" sequence to the linkers. |
| Step 4 | Ligation and amplification using primers A and B. |
| Step 5 | Restriction with anchoring enzyme, isolation of ditags, concatenate and clone. X and O represent nucleotides from different transcripts. |
Tags and Ditags from Sequencing Gel
SAGE software searches GenBank for matches to each tag
This allows assignment to 3 categories of tags:
- mRNAs derived from known genes
- anonymous mRNAs, also known as expressed sequence tags (ESTs)
- mRNAs derived from currently unidentified genes
Advantages of SAGE Technology
- Analyzes all transcripts (Transcriptome) without prior selection of known genes
- Provides quantitative data on both known and unknown genes
- Ideally suited for determining changes on gene expression as consequence of an experimental treatment (e.g. carcinogen, hormone)