SAGE (Serial Analysis of Gene Expression)

SAGE is sequence sampling technique in which very short sequence tags are joined into long
concatamers. The size of the SAGE tag is optimal for high throughput analysis but genes can still be
identified unambiguously. A concatamer may contain more than 50 tags, and each SAGE sequence is
thus equivalent to more than 50 independent cDNA sequence experiments. SAGE is therefore
appropriate for the analysis of rare mRNAs.

SAGE (serial analysis of gene expression) is a sequence sampling technique that circumvents the
problems associated with random clone sequencing by sampling many different cDNAs in one
experiment. This is achieved by using very short ESTs and joining them into a long concatamer prior to
sequencing. The ESTs are just long enough to allow accurate and unambiguous gene identification.

The overall process of SAGE can be summarized as follows.

1. An mRNA population is reverse transcribe to generate cDNA, using a biotinylated oilgo dT-primer.

2. The cDNA is digested using restriction enzyme that cleaves very frequently, such as NlaIII, leaving 3’
fragments of few hundred base pairs labeled with biotin.

3. The 3’ end of cDNA is than captured and the other fragment is discarded.

4. The cDNA is divided into two pools, and each pool is ligated to a different linker (a ds oligonucleotide
with a specific overhang, in this case matching the overhang is generated by NlaIII). The linker carries an
internal recognition site for type II restriction enzymes such as FokI.

5. Cleavage with type II restriction enzyme generates a very short sequence tag attached to the linker,
and this known as a SAGE ditags.

6. The biotinylated fragments are discarded, and SAGE tags with different linkers are ligated tail to tail to
form dimmers called ditags.

7. The ditags are amplified by PCR using primer that anneals to the linkers.

8. The linker are removed using NlaIII and the ditags are randomly joined together in a long concatamer.

9. The concatamer is sequenced.

10. Sequence analysis identifies the tags and thus the associated mRNAs. The number of times each tag
appears in the concatamer is indicative of the mRNA abundance.