Physiology and Molecular Biology of Stress ... - KHAM PHA MOI

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A.K. Tyagi, S. Vij and N. Saini
2.2. Serial Analysis of Gene Expression
SAGE is a powerful technique that can be used for studying global gene expression
profiles. This method relies upon the generation of unique short sequences, 9-17 base
pairs (Velculescu et al., 1995; Saha et al., 2002) which contain sufficient information to
identify a transcript (Fig. 1). This technique generates a large number of tags, can
potentially identify >4 9 (>2,62,144) which is far more than the estimated number of genes
in Arabidopsis (25,498, The Arabidopsis Genome Initiative, 2000) and rice (~45,000,
Rice Genome Program, http://rgp.dna.affrc.go.jp/). The number of times a particular tag
appears in a population of SAGE tags would provide direct information of cellular level
of gene expression. This technique is much suited in those organisms whose genome
has been sequenced. SAGE analysis has been extensively applied in yeast (Velculescu
et al., 1997), humans (Zhang et al., 1997; Chen et al., 2002) and mouse (Gunnersen et al.,
2002). Only a few studies have been conducted for analysis of global genes expression
in plants by SAGE. Matsumura et al. (1999) and Gibbings et al. (2003) used SAGE for
cDNA generated with
biotin-oligo (dT) primer
Restriction digest double-stranded cDNA
with a 4-base cutter "anchoring enzyme"
(Nla III); bind to streptavidin coated beads
Divide the population and ligate different
linkers A and B, both of which have a
restriction site for the "tagging enzyme"
(Bsm FI)
Tagging enzyme recognizes the linker
sequences and cuts downstream in a sequence
independent fashion; fill-in 5' overhang to
generate blunt ends
Blant end ligate pool A to pool B, and PCR
amplify with primers specific to linker
sequences A and B
Digestion with Nla III, ligation, cloning
and sequencing of SAGE tags
Figure 1. Schematic diagram of SAGE (modified from Song, 2003)