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Concept
Serial analysis of gene expression
Summary
Serial Analysis of Gene Expression (SAGE) is a transcriptomic technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. Several variants have been developed since, most notably a more robust version, LongSAGE, RL-SAGE and the most recent SuperSAGE. Many of these have improved the technique with the capture of longer tags, enabling more confident identification of a source gene.
Briefly, SAGE experiments proceed as follows:
The mRNA of an input sample (e.g. a tumour) is isolated and a reverse transcriptase and biotinylated primers are used to synthesize cDNA from mRNA.
The cDNA is bound to Streptavidin beads via interaction with the biotin attached to the primers, and is then cleaved using a restriction endonuclease called an anchoring enzyme (AE). The location of the cleavage site and thus the length of the remaining cDNA bound to the bead will vary for each individual cDNA (mRNA).
The cleaved cDNA downstream from the cleavage site is then discarded, and the remaining immobile cDNA fragments upstream from cleavage sites are divided in half and exposed to one of two adaptor oligonucleotides (A or B) containing several components in the following order upstream from the attachment site: 1) Sticky ends with the AE cut site to allow for attachment to cleaved cDNA; 2) A recognition site for a restriction endonuclease known as the tagging enzyme (TE), which cuts about 15 nucleotides downstream of its recognition site (within the original cDNA/mRNA sequence); 3) A short primer sequence unique to either adaptor A or B, which will later be used for further amplification via PCR.
After adaptor ligation, cDNA are cleaved using TE to remove them from the beads, leaving only a short "tag" of about 11 nucleotides of original cDNA (15 nucleotides minus the 4 corresponding to the AE recognition site).
The cleaved cDNA tags are then repaired with DNA polymerase to produce blunt end cDNA fragments.
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