Serial analysis of gene expression (SAGE)
SAGE is based on the principle that every gene can be identified by a unique tag located near the 3′-end of its mRNA. As there are 4 nitrogen bases A, T, G and C, a 10 bp tag gives us 410 possible combinations. So a 10 bp tag is most commonly employed in SAGE. Perhaps the biggest advantage of SAGE is that it does not require prior knowledge of the sequence of a gene, in other words, it is a useful technique to identify unknown genes. It is especially useful in those organisms whose genome has not been well characterized. However, there still remains the problem of identifying the function of a newly discovered gene, for which biological assays need to be designed.
The technique of SAGE requires RNA as the starting material, since we are looking for unique tag in the mRNA. The mRNA (present in a sample of total RNA) is first immobilized onto poly T beads. Thereafter cDNA is synthesized on them by the process of reverse transcription using universal oligo dT primers. The cDNA is then treated with a restriction enzyme, usually Nla III that cuts at a 4bp recognition site (i.e. it cuts once every 256 bp). The cDNA attached to the oligo dT beads (via the mRNA) is cut at several places, including the region nearest to the 3′ end of the cDNA. The region 3′ to the site of restriction enzyme cutting constitutes the tag (minus the poly T region). This is oligo dT bead bound cDNA is then separated from the rest of the RNA/cDNA/DNA contaminants.
The 5’ends oligo dT bound cDNA are filled in with Klenow DNA polymerase, following which the total cDNA is divided into roughly two equal portions. Each portion is now ligated to a special oligonucleotide (called linker) at its 5’end. There are two linkers A and B. The special feature of these linkers is that their 3′ end has a recognition site for the second restriction enzyme that will be used in SAGE, called the “Tagging enzyme”. The tagging enzyme is a type II S restriction endonuclease that cuts 10bp downstream from its recognition site. In most instances BsmF1 is used as the tagging enzyme.
The cDNAs ligated to the linkers (A or B) are now digested with the “tagging enzyme” which cuts the cDNA at a point 10bp downstream of its recognition site, thereby releasing it from the oligo dT support. Now, the cDNA tagged with the two linker oligos A and B are mixed and ligated together giving rise to “ditags”. A ditag comprises two cDNAs one having linker A at its 5’end and the other having linker B at its 5’end.
These ditags are then amplified by PCR using primers that are complementary to the linker sequences A and B. After PCR, the ditags are released from the linkers by digestion with the anchoring enzyme NlaIII (as we might remember, the NlaIII sites were filled in by Klenow DNA polymerase after the first digestion. So these sites are intact in the 5′ end of each ditag).
The ditags so released are separated on a gel. The bands formed are cut out and serially ligated. The resultant serial tags are cloned into an appropriate vector and sequenced. Usually a tag is considered to be significantly upregulated if it is amplified more than 5 fold at p Circ. Res. 91(7), 565 (2002).
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