1.Biotinylated primers are used to reverse-transcribe mRNA. The resultant cDNA is then digested with a frequent-cutting enzyme (anchoring enzyme AE). Using streptavidin-coated beads, the 3' end of the cDNA is bound and then split into two pools, A and B. A primer (A' and B') containing a recognition site for a type IIS restriction enzyme is ligated to the nucleotides in each of these pools.
2.Following digestion with a relevant type IIS tagging enzyme, eluted DNA fragments are ligated and amplified using the A' and B' primers. Following PCR, the anchoring enzyme removes the priming sites A and B. Concatamers are then formed and cloned into a vector. A ditag containing the sequence information of two independent cDNA tags is punctuated by the anchoring site.*
3.Cloned inserts are verified by agarose gel and sequenced. SAGE analysis tools then analyze these sequences. Finally, a comparison of the sequence data determines variations in expression patterns.
Applied Biosystems Capillary Electrophoresis Systems and SAGE Technology
Most SAGE investigations require analysis of more than 50,000 tags. Obtaining sequencing information quickly and accurately is vital. Applied Biosystems capillary electrophoresis systems and BigDye terminator chemistries offer an easy, hands-free approach to sequencing. ABI PRISM 3700 and 3100 DNA sequencers accurately and reliably determine the transcript information necessary to build a profile. ABI PRISM BigDye terminators provide signal uniformity and sensitivity. Together they offer a cost-effective solution to largescale transcript analysis using SAGE technology.
Accurate basecalling at long readlengths is important for ditag analysis. BigDye terminators provide the hig