Serial Analysis of Gene Expression (SAGE)

SERIAL ANALYSIS OF GENE EXPRESSION (SAGE)
WHAT IS SAGE?
Serial analysis of gene expression (SAGE) is a powerful tool that allows digital analysis of overall gene expression patterns. Produces a snapshot of the mRNA population in the sample of interest.
SAGE provides quantitative and comprehensive expression profiling in a given cell population.
SAGE invented at Johns Hopkins University in USA (Oncology Center) by Dr. Victor Velculescu in 1995.
Addresses specific issues such as determination of normal gene structure and identification of abnormal genome changes.
Enables precise annotation of existing genes and discovery of new genes.
NEED FOR SAGE

Gene expression refers to the study of how specific genes are transcribed at a given point in time in a given cell. Examining which transcripts are present in a cell.
SAGE enables large scale studies of DNA expression; these can be used to create 'expression profiles.
Allows rapid, detailed analysis of thousands of transcripts in a cell. By comparing different types of cells, generate profiles that will help to understand healthy cells and what goes wrong during diseases.
THREE PRINCIPLES UNDERLIE THE SAGE METHODOLOGY:

a short sequence tag (10-14bp) contains sufficient information to uniquely identify a transcript provided 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 Quantitation of the number of times a particular tag is observed provides the expression level of the corresponding transcript.
STEPS IN BRIEF..
1. Isolate the mRNA of an input sample (e.g. a tumour). 2. Extract a small chunk of sequence from a defined position of each mRNA molecule. 3. Link these small pieces of sequence together to form a long chain (or concatamer).
4. Clone these chains into a vector which can be taken up by bacteria.

5. Sequence these chains using modern high-throughput DNA sequencers. 6. Process this data with a computer to count the small sequence tags.
SAGE TECHNIQUE (in detail)

Trap RNAs with beads
Messenger RNAs end with a long string of "As" (adenine)
Adenine forms very strong chemical bonds with another nucleotide, thymine (T)
Molecule that consists of 20 or so Ts acts like a chemical bait to capture RNAs Researchers coat microscopic, magnetic beads with chemical baits i.e. "TTTTT" tails hanging out When the contents of cells are washed past the beads, the RNA molecules will be trapped A magnet is used to withdraw the bead and the RNAs out of the "soup"
cDNA SYNTHESIS
Double stranded cDNA is synthesized from the extracted mRNA by means of biotinylated oligo (dT) primer.
cDNA synthesized is immobilised to streptavidin beads
ENZYMATIC CLEAVAGE OF cDNA.

The cDNA molecule is cleaved with a restriction enzyme. Type II restriction enzyme used. Also known as Anchoring enzyme. E.g. NlaIII. Any 4 base recognising enzyme used. Average length of cDNA 256bp with sticky ends created.
The biotinylated 3 cDNA are affinity purified using strepatavidin coated magnetic beads.
biotinylated 3 cDNA are affinity purified using strepatavidin coated magnetic beads.
LIGATION OF LINKERS TO BOUND cDNA

These captured cDNAs are divided into two halves, then ligated to linkers A and B, respectively at their ends. Linkers also known as docking modules. They are oligonucleotide duplexes. Linkers contain: NlaIII 4- nucleotide cohesive overhang Type IIS recognition sequence PCR primer sequence (primer A or B).
Type IIS restriction enzyme tagging enzyme.

Linker/docking module: PRIMER TE AE
_TAG
CLEAVAGE WITH TAGGING ENZYME Tagging enzyme, usually BmsFI cleave DNA 14-15 nucleotides, releasing the linker adapted SAGE tag from each cDNA.
Repair of ends to make blunt ended tags using DNA polymerase (Klenow) and dNTPs.
FORMATION OF DITAGS
What is left is a 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.
Ligation using T4 DNA ligase.
PCR AMPLIFICATION OF DITAGS The linker-ditag-linker constructs are amplified by PCR using primers specific to the linkers.
ISOLATION OF DITAGS
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
CONCATAMERIZATION OF DITAGS
Tags
are combined into much longer molecules, called concatemers. scientist and the computer to recognize where one ends and the next begins.
Between each ditag is the AE site, allowing the
CLONING CONCATAMERS AND SEQUENCING

put into bacteria, which act like living "copy machines" to create millions of copies from the original These copies are then sequenced, using machines that can read the nucleotides in DNA. The result is a long list of nucleotides that has to be analyzed by computer Analysis will do several things: count the tags, determine which ones come from the same RNA molecule, and figure out which ones come from known, well-studied genes and which ones are new
Lots of copies are required- So the concatemers are
How does SAGE work?

1. Isolate mRNA.
2.(a) Add biotin-labeled dT primer: 2.(b) Synthesize ds cDNA. 3.(a) Bind to streptavidin-coated beads. 3.(b) Cleave with anchoring enzyme.
3.(c) Discard loose fragments 4.(a) Divide into two pools and add linker sequences
4.(b) Ligate
5. Cleave with tagging enzyme. 6. Combine pools and ligate

7. Amplify ditags, then cleave with anchoring enzyme.
8. Ligate ditags
9. Sequence and record the tags and frequencies.
Sage flow chart
Vast amounts of data is produced, which must be shifted and ordered for useful information to become apparent
APPLICATIONS
1. SAGE: A LOOKING GLASS FOR CANCER

Deciphering pathways involved in tumor genesis and identifying novel diagnostic tools, prognostic markers, and potential therapeutic targets. SAGE is one of the techniques used in the National Cancer Institutefunded Cancer Genome Anatomy Project (CGAP). SAGE studies have been performed in patients with colon, pancreatic, lung, bladder, ovarian, and breast cancers.
SAGE experiments validated in multiple tumor and normal tissue pairs using a variety of approaches, including Northern blot analysis, real-time PCR, mRNA in situ hybridization, and immunohistochemistry.
2. p53- TUMOR SUPRESSOR GENE

p53 is thought to play a role in the regulation of cell cycle checkpoints, apoptosis, genomic stability, and angiogenesis. Sequence-specific transactivation is essential for p53mediated tumor suppression.
The analysis of transcriptomes after p53 expression has determined that p53 exerts its diverse cellular functions by influencing the expression of a large group of genes. Identification of Previously Unidentified p53-Regulated Genes by SAGE analysis. Variability exists with regard to the extent, timing, and p53 dependence of the expression of these genes.
3. IMMUNOLOGICAL STUDIES
Only a few SAGE analysis has been applied for the study of immunological phenomena.
SAGE analyses were conducted for human monocytes and their differentiated descendants, macrophages and dendritic cells. cDNA library represented more than 17,000 different genes. Genes differentially expressed were those encoding proteins related to cell motility and structure.
SAGE has been applied to B cell lymphomas to analyze genes involved in BCR mediated apoptosis.- polyamine regulation is involved in apoptosis during B cell clonal deletion.
4. YEAST TRANSCRIPTOME
Yeast is widely used to clarify the biochemical physiologic parameters underlying eukaryotic cellular functions.
Yeast chosen as a model organism to evaluate the power of SAGE technology. Most extensive SAGE profile was made for yeast. Analysis of yeast transcriptome affords a unique view of the RNA components defining cellular life.
5.ANALYSIS OF TISSU TRANSCRIPTOMES

Used to analyze the transcriptomes of renal, cervical tissues etc. Establishing a baseline of gene expression in normal tissue is key for identifying changes in cancer.
Thank you

Serial Analysis of Gene Expression (SAGE)

Uploaded by

Copyright:

Available Formats

Serial Analysis of Gene Expression (SAGE)

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Serial Analysis of Gene Expression (SAGE)

Uploaded by

Copyright:

Available Formats

SERIAL ANALYSIS OF GENE EXPRESSION (SAGE)

NEED FOR SAGE

THREE PRINCIPLES UNDERLIE THE SAGE METHODOLOGY:

4. Clone these chains into a vector which can be taken up by bacteria.

SAGE TECHNIQUE (in detail)

Messenger RNAs end with a long string of "As" (adenine)

ENZYMATIC CLEAVAGE OF cDNA.

LIGATION OF LINKERS TO BOUND cDNA

Type IIS restriction enzyme tagging enzyme.

Two groups of cDNAs are ligated to

Ligation using T4 DNA ligase.

This leaves a sticky end with the sequence GTAC

Between each ditag is the AE site, allowing the

CLONING CONCATAMERS AND SEQUENCING

Lots of copies are required- So the concatemers are

How does SAGE work?

5. Cleave with tagging enzyme. 6. Combine pools and ligate

9. Sequence and record the tags and frequencies.

Sage flow chart

1. SAGE: A LOOKING GLASS FOR CANCER

2. p53- TUMOR SUPRESSOR GENE

5.ANALYSIS OF TISSU TRANSCRIPTOMES

You might also like