Target Discovery and Validation Reviews and Protocols

Gene Target Discovery 123
digestion, ligation, and amplification of cDNA. Ligated concatemers of up to
50 tags are cloned into plasmid vectors and sequenced. Compared with conventional
EST sequencing, SAGE is 30–50 times more effective per plasmid. The
abundance of a particular tag relates directly to the expression level of the gene
from which it is derived, resulting in a statistical representation of the transcripts
within the investigated cell type. The technology enables high throughput and has
the ability to compare data from numerous sources. Since the development of
SAGE in 1995, it has been applied in a large number of studies involving different
cell types and organisms (reviewed in ref. 44). The principle of SAGE is as follows:
dscDNA synthesized using biotinylated oligo(dT) primers, is cleaved with a
so-called “anchoring enzyme,” a four-base restriction enzyme. The resulting 3′
ends are recovered on beads via biotin-streptavidin binding, by using biotinylated
polyT primer. These cDNA fragments are then ligated to linkers containing the
recognition site for a restriction enzyme that cuts approx 14 bp downstream of the
recognition site. The small fragments, each with a 9- to 17-bp gene-specific tag are
concatenated into long sequences and cloned into plasmids that are sequenced.
The greatest advantage with the methodology is the high throughput it
allows. It also provides an absolute quantitation of gene expression, enabling
the combination of results from different laboratories deposited in the public
SAGEmap database (http://www.ncbi.nlm.nih.gov/SAGE) and the omnibus
database (GEO) (http://www.ncbi.nlm.gov/projects/geo/). In addition, SAGE
has the power to measure very rare transcripts and to discover novel transcript
not detectable with EST sequencing (see Chapter 4, Volume 1).
The main concern with the method is the short length of the specific tags,
~10 bp. This length may be too short to be unique for a specific transcript; especially
in cases were the tag recognizes conserved domains and gene families.
Thus, multiple genes may share the same tag. In addition, these short tags are
very sensitive to sequencing errors and single nucleotide polymorphisms (45).
These effects may be compensated for by introducing alternative restriction
enzymes, which generate tags with up to 17 bp (46). For a transcript to be
included in the analysis, its gene must have a restriction site for the chosen
restriction enzyme. More transcripts would be present in the tag library by generating
libraries by using several enzymes (47). Whereas a limitation with the
original SAGE was the need of large amounts of starting material (2.5–5 µg of
mRNA), modified strategies such as MicroSAGE (requiring < 10 cells) (48),
SAGE-lite (100 ng of total RNA) (49), and miniSAGE (1 µg of total RNA without
PCR amplification (50) have been developed.
A more recently developed method based on a similar principle to SAGE is
massively parallel signature sequencing (51). In this method, millions of transcripts
are captured and identified by bead-based EST signature sequencing. The
level of expression is analyzed by counting the number of mRNA molecules that