SAGE TECHNOLOGY AND ITS APPLICATIONS PRESENTED BY Dr. R.A.Siddique & Dr.Anand Kumar Animal Biochemistry Division N.D.R.I., Karnal (Haryana)India, 132001 E-mail: firstname.lastname@example.org
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. • An overview of a cell’s complete gene activity. • 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.
PRE REQUISITES: • Extensive sequencing techniques • Deep bioinformatic knowledge • Powerful computer software (assemble and analyze results from SAGE experiments) Limited use of this sensitive technique in academic research laboratories
STEPS IN BRIEF….. • Isolate the mRNA of an input sample (e.g. a tumour). • Extract a small chunk of sequence from a defined position of each mRNA molecule. • Link these small pieces of sequence together to form a long chain (or concatamer).
Clone these chains into a vector which can be taken up by bacteria. • Sequence these chains using modern high-throughput DNA sequencers. • 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.
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.
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. • Between each ditag is the AE site, allowing the scientist and the computer to recognize where one ends and the next begins.
CLONING CONCATAMERS AND SEQUENCING • Lots of copies are required- So the concatemers are 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
Quantitation of gene expression And data presentation
2.(a) Add biotin-labeled dT primer: 1. Isolate mRNA. 2.(b) Synthesize ds cDNA. 3.(a) Bind to streptavidin-coated beads. 3.(b) Cleave with “anchoring enzyme”. 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. How does SAGE work? 3.(c) Discard loose fragments. 9. Sequence and record the tags and frequencies.
Vast amounts ofdata is produced, which must be sifted and ordered for useful information tobecome apparent. Sage reference databases: • SAGE map • SAGE Genie http://www.ncbi.nlm.nih.gov/cgap
FROM TAGS TO GENES…… • Collect sequence records from GenBank • Assign sequence orientation (by finding poly-A tail or poly-A signal or from annotations) • Extract 10-bases -adjacent to 3’-most CATG • Assign UniGene identifier to each sequence with a SAGE tag • Record (for each tag-gene pair) • #sequences with this tag • #sequences in gene cluster with this tag Maps available at http://www.ncbi.nlm.nih.gov/SAGE
DIFFERENTIAL GENE EXPRESSION BY SAGE • Identification of differentially expressed genes in samples from different physiological or pathological conditions. • Application of many statistical methods • Poisson approximation • Bayesian method • Chi square test.
SAGE software searches GenBank for matches to each tag • This allows assignment to 3 categories of tags: • mRNAs derived from known genes • anonymous mRNAs, also known as expressed sequence tags (ESTs) • mRNAs derived from currently unidentified genes
SAGE VS MICROARRAY • SAGE – An open system which detects both known and unknown transcripts and genes.
SAGE Detects 3’ region of transcript. Restriction site is determining factor. Collects sequence information and copy no. Sequencing error and quantitation bias. MICROARRAY Targets various regions of the transcript.Base composition for specificity of hybridization. Fluorescent signals and signal intensity. Labeling bias and noise signals. COMPARISON……
RECENT SAGE APPLICATIONS • Analysis of yeast transcriptome • Gene Expression Profiles in Normal and Cancer Cell • Insights into p53-mediated apoptosis • Identification and classification of p53-regulated genes • Analysis of human transcriptomes • Serial microanalysis of renal transcriptomes • Genes Expressed in Human Tumor Endothelium • Analysis of colorectal metastases (PRL-3) • Characterization of gene expression in colorectal adenomas and cancer • Using the transcriptome to analyze the genome (Long SAGE)
LIMITATIONS • Does not measure the actual expression level of a gene. • Average size of a tag produced during SAGE analysis is ten bases and this makes it difficult to assign a tag to a specific transcript with accuracy • Two different genes could have the same tag and the same gene that is alternatively spliced could have different tags at the 3' ends • Assigning each tag to an mRNA transcript could be made even more difficult and ambiguous if sequencing errors are also introduced in the process
Quantitation bias: • Contamination of of large quantities of linker-dimer molecules. • low efficiency in blunt end ligation. • Amplification bias. • Depending upon anchoring enzyme and tagging enzyme used, some fraction of mRNA species would be lost.
Advances over SAGE • Generation of longer 3` cDNA from SAGE tags for gene identification (GLGI) • Long SAGE • Cap Analysis of Gene Expression (CAGE) • Gene Identification Signature (GIS) • SuperSAGE • Digital karyotyping • Paired-end ditag
Long SAGE • Increased specificity of SAGE tags for transcript identification and SAGE tag mapping. • Collects tags of 21bp • Different TypeII restriction enzyme-Mmel • Adapts SAGE principle to genomic DNA. • Allows localisation of TIS and PAS.
CAGE (Capped Analysis of Gene Expression) • Aims to identify TIS and promoters. • Collects 21 bp from 5’ ends of cap purified cDNA. • Used in mouse and human transcriptome studies. • The method essentially uses full-length cDNAs , to the 5’ends of which linkers are attached. • This is followed by the cleavage of the first 20 base pairs by class II restriction enzymes, PCR, concatamerization, and cloning of the CAGE tags
Reverse transcription AAAAA • Full strand DNA synthesis • ssDNA release Biotin AAAAA Biotin + • ssDNA capture • Second strand synthesis x Biotin MmeI digestion of dsDNA Mmel + Biotin Ligation to second linker Xma JI Biotin Mmel-PCR PCR amplification Biotin Uni-PCR • Concatenation • Cloning • Sequencing XmaJI tag1 tag2 XmaJI
Micro SAGE • Requires 500-5000 fold less starting input RNA. • Simplifies by the incorporation of a ‘one tube’ procedure for all steps. • Characterization of expression profiles in tissue biopsies, tumor metastases or in cases where tissue is scarce. • Generation of region-specific expression profiles of complex heterogeneous tissues. • Limited number of additional PCR cycles are performed to generate sufficient ditag.
An expression profile can be obtained from as little as 1-5 ng of mRNA. • Comparison between the two…
SuperSAGE • Increases the specificity of SAGE tags and use of tags as microarray probes. • Type III RE EcoP15I – tag releasing • Collects 26 bp tags • Has been used in plant SAGE studies. • Study of gene expression in which sequence information is not available.