Transcript Gene Expression Analysis by SAGE and MPSS

Gene
Expression
Analysis by
SAGE
Gene Expression
Some challenges:
– Large number of genes
• How do you keep samples and
equipment small and affordable?
• How do you analyze that amount of
data?
– Need to know sequence of some
or all genes
Methods
• SAGE (Serial Analysis of
Gene Expression)
– Victor E. Velculescu, Lin Zhang,
Bert Vogelstein, Kenneth W.
Kinzler
–October 1995
SAGE – introduction
• SAGE uses short tags of cDNA
made from all mRNAs in a cell
– The assumption: a tag of only 9 or 10
bases, from a specific position in the
mRNA, is enough to identify the transcript
• These tags are linked together so
that they can be cloned in large
groups
• Sequencing identifies genes, and
they can be counted to determine
relative expression
SAGE – the method
The 3’ end of
biotinylated, double
stranded cDNA
adheres to streptavidin
beads
This and all other pictures of the SAGE method are from
http://sciencepark.mdanderson.org/ggeg/SAGE_technique.htm
SAGE – the method
•Anchoring enzyme (AE) usually
recognizes a 4-bp site
•Restriction may occur before
binding to beads
•cDNA is divided in half, and each
half is ligated to a different linker (A
or B)
•Linkers have a IIS restriction
enzyme site and are
complementary to a PCR primer
SAGE – the method
•Tagging enzyme is a type IIS
restriction enzyme
•Unique in that they cleave at a
defined distance away from the
site that they recognize
•Tagging enzyme recognizes site in
the linker, and cuts off a short “tag” of
cDNA, leaving a blunt end.
•Two groups of cDNAs are ligated to
each other, to create a “ditag” with
linkers on either end
SAGE – the method
•PCR is used to amplify the ditags, using a primer that is
complementary to the linker.
•The cDNA is again digested by the AE, breaking the linker off right
where it was added in the beginning. This leaves a “sticky” end with
the sequence GTAC (or CATG on the other strand) at each end of the
ditag.
SAGE – the method
•Ditags are ligated together to form long concatemers. Between each
ditag is the AE site, allowing the scientist and the computer to
recognize where one ends and the next begins.
•The concatemers are sequenced, and the tags are matched up with
the gene that they uniquely represent. By counting the number of
times each tag appears, the relative expression levels can be
determined.
SAGE – advantages
• No hybridizing, so no crosshybridizing can occur
• Knowledge of genes to be
detected can wait until after
experiment
• Can help identify new genes by
using tag as a PCR primer
SAGE – disadvantages
• Cost and time required to perform
so many PCR and sequencing
reactions
• Type IIS restriction enzyme can
yield fragments of the wrong length
depending on temperature.
• Multiple genes could have the same
tag
• As with microarrays, mRNA levels
may not represent protein levels in
a cell
MOUSE SAGE SITE
http://mouse.biomed.cas.cz/sage/compare.cgi
Brain vs. Diseased Brain >2x fold
change
Retina Mutant/Wt
Crx+/+ vs Crx-/-
SAGE DATA