Serial Analysis of Gene Expression (SAGE) is a method that allows for the quantitative analysis of gene expression patterns in cells or tissues without prior knowledge of gene sequences. It works by extracting short sequence tags (10-14 base pairs) from the 3' end of transcripts and linking them together in long strings that can be cloned and sequenced simultaneously. This allows for many transcripts to be analyzed from a single sequencing reaction and provides quantitative data on gene expression levels based on tag frequencies. SAGE provides an accurate way to discover genes and analyze overall expression patterns without needing to know full mRNA sequences in advance.

1. S E R I A L A N A L Y S I S O F
G E N E E X P R E S S I O N
( S A G E )
PRESENTED BY:
A SHWINI.R
B.SC+M.SC -BIOTECH
A MITY INSTITUTE OF
BIOTECHNOLOGY

2. Serial Analysis of Gene Expression (SAGE)
• Serial analysis of gene expression (SAGE) is an approach that allows
rapid and detailed analysis of overall gene expression patterns.
• SAGE method allows for a quantitative and simultaneous analysis of
a large number transcripts in any particular cells or tissues, without
prior knowledge of the genes.
• This method takes advantage of the 3’-portion of mRNA as the gene
tag, but of much shorter form (9–10 bp).These tags can be serially
connected before cloning into a plasmid vector.
• Since the resulting plasmid clones contain multiple tags, sequences
of several dozens of mRNAs can be obtained by a single sequencing
reaction.

3. • A short sequence tag (10-14bp) contains sufficient
information to uniquely identify a transcript provided
that that the tag is obtained from a unique position
within each transcript;
• Sequence tags can be linked together to from long serial
molecules that can be cloned and sequenced; and
• Quantitation of the number of times a particular tag is
observed provides the expression level of the
corresponding transcript.
• Linking the tags together allows for rapid sequencing
analysis of multiple transcripts.
• By linking the tags together, only one sequencing event is
required to sequence every transcript within the cell.
• Makes the task of DNA expression profiling a much
easier
SAGE Principle

4. SAGE Flowchart
1. Isolate mRNA.
2. (a) Add biotin-labeled dT primer
(b) Synthesize ds cDNA.
3. (a) Bind to streptavidin-coated beads.

5. (b) Cleave with “anchoring enzyme”.
(c) Discard loose fragments.
4. (a) Divide into two pools and add linker
sequences
(b) Ligate.

6. 5. Cleave with “tagging enzyme”
6. Combine pools and ligate.
7. Amplify ditags, then cleave with
anchoring enzyme.

7. 8. Ligate ditags.
9. Sequence and record the tags and
frequencies.

9. Advantages:
• mRNA sequence does not need to be known
prior, so genes of variants which are not known
can be discovered.
• Its more accurate as it involves direct counting
of the number of transcripts.
Disadvantages:
• Length of gene tag is extremely short (13 or 14bp),
so if the tag is derived from an unknown gene, it is
difficult to analyze with such a short sequence.
• Type II restriction enzyme does not yield same
length fragments.
• mRNA levels and protein expression do not are
always correlate.