Principles of SAGE Technology

The serial analysis of gene expression - SAGE - is a sequencing-based method of generating expression profiles from any given cell type or tissue. As an open system, SAGE characterizes a short segment of DNA from a defined location in each expressed gene, as a unique identifier for that gene. The ability to count many thousands of short DNA segments, called SAGE tags, allows the detection of genes that are expressed at very low levels in a high-throughput manner. From a historical point of view, SAGE is a logical progression ofapproaches that simply count gene transcripts, like counting plaques in a cDNA library or counting expressed sequence tags (ESTs) for a single sequence. However, SAGE has revolutionized the generation of countable, gene-specific tags and has introduced a way of rapid counting. But the basic principle of counting still plays a fundamental role in the statistics of SAGE. It is one of the major differences to technologies like microarrays, which are based on analogue results relying on measurement ofsignal intensities resulting from nucleic acid hybridization.

SAGE is a patented technology developed in the laboratory of Bert Vogelstein and Ken Kinzler [1], which has been used since then in many laboratories and has led to about 300 publications. SAGE has been selected by the US NCI as a method of choice for the Cancer Genome Anatomy Project (CGAP) [2].

Four principle steps underlie the SAGE technology (Figure 2.1):

1. Isolation of tags: a short DNA fragment (SAGE tag), which is unique for each mRNA species, is isolated.

2. Concatenation: tags are ligated to form large DNA molecules (concatemers).

3. Sequencing: the concatemers are sequenced.

4. Expression profiling: tags are identified, annotated, and counted. The expression profile is deduced by comparing the nature and frequency of tags within two or more libraries.

Fig. 2.1 Outline of SAGE technology.
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