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SAGE (Serial analysis of Gene Expression)

SAGE (Serial Analysis of Gene Expression) is a technique that allows for the rapid and comprehensive analysis of gene expression patterns in a given cell population. It works by isolating mRNA, synthesizing cDNA, ligating short sequence tags to the cDNA, and then counting the number of times each tag is observed to quantify gene expression levels. The tags are concatenated and sequenced to generate vast amounts of data that must be analyzed computationally to identify which genes particular tags correspond to and to compare expression profiles between cell types. SAGE provides an overview of a cell's complete transcriptional activity and has been applied to study differences in cancer vs normal cells and to identify targets of oncogenes and tumor suppressor genes.

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Mohammed TalhaKhatkhatay1SAGE(Serial Analysis of Gene Expression)SAGE(Serial Analysis of Gene Expression)
WHAT IS GENE EXPRESSION?OUTLINESAGE AND ITS PRINCIPLE…STEPS IN SAGE, ITS APPLICATIONS AND PROBLEMS.REFERENCES.2
What is Gene Expression?A process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins or functional RNA. DNA RNA Protein3
SAGE:Serial analysis of gene expression (SAGE) is an approach that allows rapid and detailed analysis of overall gene expression patterns.SAGE provides quantitative and comprehensive expression profiling in a given cell population.An overview of a cell’s complete gene activity.4
SAGE invented at Johns Hopkins University in USA (Oncology Center) by Dr. Victor Velculescu in 1995.5
Principle Underlining SAGE methodology:A short sequence tag (10-14bp) contains sufficient information to uniquely identify a transcript provided that tag is obtained from a unique position within each transcript.Sequence tag can be linked together to form long serial molecules that can be cloned and sequenced.Quantitation of the number of times a particular tag is observed provides the expression level of the corresponding transcript.6
Steps In Brief…7
8
SAGE Flowchart…1. Isolate mRNA.B2. (a) Add biotin-labeled dT primer: (b) Synthesize ds cDNA.B3.(a) Bind to streptavidin-coated beads. (b) Cleave with “anchoring enzyme”.9B
(c) Discard loose fragments.4. (a) Divide into two pools and add linker sequences (b) Ligate.10B
5. Cleave with “tagging enzyme”11B6. Combine pools and ligate.7. Amplify ditags, then cleave with anchoring enzyme.
8. Ligate ditags. 9. Sequence and record the tags and frequencies.12
SAGE In Details…Trapping of RNA with beadsmRNA’s end with a long string of “A” (Adenine)
Molecules that consist of 20 or so dT’s acts like a attractant to capture mRNAs.
Coating of microscopic magnetic beads with “TTTTT” tails is done.
A magnet is used to withdraw the bead and the mRNA is isolated.13
14mRNAmRNAmRNAmRNAmRNAMicroscopic bead coated with TTTT’smRNAmRNAmRNAmRNAmRNA
15mRNAmRNAmRNAmRNAmRNAMicroscopic bead coated with TTTT’smRNAmRNAmRNAmRNAmRNA15
cDNA synthesisds cDNA is synthesized from the extracted mRNA by means of biotinylated oligo(dT) primer.
cDNA synthesis is immobilized to streptavidin beads.16
17BBBiotinylated oligo dT (primers) BBBBmRNABStreptavidin beadsBcDNABB
Enzymatic cleavage of cDNAThe cDNA molecule is cleaved with a restriction enzyme.
Type II restriction enzyme used (E.g. NlaIII.)
Average length of cDNA – 256bp with sticky ends created.18
19Nla III (Restriction enzyme)BBBB
Ligation of Linkers to bound cDNACaptured cDNA are then ligated to linkers at their ends.
Linkers must contain:
NlaIII 4-nucleotide cohesive overhang.
Type IIs recognition sequence.
PCR primer sequence.20
21LinkersBBBBPool APool B
Cleaving with tagging enzymeTagging enzyme, (usually BsmF1)cleave DNA, releasing the linker-adapted SAGE tag from each cDNA.
Repair of ends to make blunt ended tags using DNA polymerase (Klenow fragments) and dNTPs.22
23Bsm FI(tagging Enzyme)BLinker adapted SAGE tagB
Formation of DitagsThe left thing is the collection of short tags taken from each molecule.
Two groups of cDNAs are ligated to each other, to create a “ditag” with linkers on either end.
Two tags are linked together using T4 DNA ligase.24
25Add DNA ligase
PCR amplification of DitagsThe linker-ditag-linker constructs are amplified by PCR using primers specific to the linkers.26
27PCR Amplification
Isolation of DitagsThe cDNA is again digested by the Anchoring enzyme (AE)
Breaking the linker off right where it was added in beginning.
This leaves a “sticky” end with the sequence GTAC (or CAGT on the other strand) at each end of the ditag.28
Nla III(Anchoring enzyme)2929292929292929