SAGE (Serial analysis of Gene Expression)

Serial analysis of gene expression (SAGE).

1. Mohammed Talha Khatkhatay 1 SAGE (Serial Analysis of Gene Expression) SAGE (Serial Analysis of Gene Expression)

2. WHAT IS GENE EXPRESSION? SAGE AND ITS PRINCIPLE… STEPS IN SAGE, ITS APPLICATIONS AND PROBLEMS. REFERENCES. O U T L I N E 2

3. 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 Protein 3

4. 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

5. SAGE invented at Johns Hopkins University in USA (Oncology Center) by Dr. Victor Velculescu in 1995. 5

6. 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

7. Steps In Brief… 7

8. 8

9. SAGE Flowchart… 1. Isolate mRNA . 2 . ( a) Add biotin-labeled dT primer : (b) Synthesize ds cDNA. 3. (a) Bind to streptavidin-c oated beads. (b) Cleave with “ anchoring enzyme”. 9 B B B

10. (c) Discard loose fragments. 4 . (a) Divide into two pools and add linker sequences (b ) Ligate. 10 B

11. 5. Cleave with “tagging enzyme” 6. Combine pools and ligate . 7. Amplify ditags, then cleave with anchoring enzyme. 11 B

12. 8. Ligate ditags. 9. Sequence and record the tags and frequencies . 12

13. SAGE In Details… Trapping of RNA with beads mRNA’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

14. 14 mRNA mRNA mRNA mRNA mRNA mRNA mRNA mRNA mRNA mRNA Microscopic bead coated with TTTT’s

15. 15 15 Microscopic bead coated with TTTT’s mRNA mRNA mRNA mRNA mRNA mRNA mRNA mRNA mRNA mRNA

16. cDNA synthesis ds cDNA is synthesized from the extracted mRNA by means of biotinylated oligo (dT) primer. cDNA synthesis is immobilized to streptavidin beads. 16

17. 17 mRNA cDNA B B B B B B B Biotinylated oligo dT (primers) Streptavidin beads B B B

18. Enzymatic cleavage of cDNA The 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

19. 19 B B B Nla III (Restriction enzyme) B

20. Ligation of Linkers to bound cDNA Captured 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

21. 21 B Linkers B B B Pool A Pool B

22. Cleaving with tagging enzyme Tagging 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

23. 23 B B Bsm FI (tagging Enzyme) Linker adapted SAGE tag

24. Formation of Ditags The 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

25. 25 Add DNA ligase

26. PCR amplification of Ditags The linker-ditag-linker constructs are amplified by PCR using primers specific to the linkers. 26

27. 27 PCR Amplification

28. Isolation of Ditags The 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

29. Nla III (Anchoring enzyme) 29 29 29 29 29 29 29 29

30. Concatamerization of Ditags Tags are combined into much longer molecules, called concatamers. Each ditag is having an AE site, allowing the scientist and the computer to recognize where one ends and the next begins. 30

31. 31 Concatemirize

32. Cloning Concatamers and Sequencing… Lots of copies are required – so the concatamers are inserted into bacteria, which act like living “copy machines” to create millions of copies from original. 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. 32

33. Analysis will do several things: count the tags, determine which one come from the same RNA molecule, and figure out which ones come from known, well studied genes and which ones are new. 33

34. Vast amount of data is produced, which must be shifted and ordered for useful information to become apparent. SAGE reference databases: SAGE map SAGE Genie http://www.ncbi.nlm.nih.gov/cgap 34

35. How Does The Data Look Like? 35

36. From Tags to Genes… Collect sequence records from GenBank . Assign sequence orientation (by finding poly-A tail) Assign UniGene identifier to each sequence with a SAGE tag. Record (for each tag-gene pair) 36

37. Applications Of SAGE… To analyze differences between gene expression patterns of cancer cells and their normal counter parts. Studied the tumors of pancreatic and colon tumors. Zhang et al.(1997)Science, 276(5316), 1268-1272. 37

38. Examining which transcripts are present in a cell. 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 in diseases. 38

39. By comparing different types of cells, generate profiles that will help to understand healthy cells and what goes wrong in diseases . To identify downstream targets of oncogenes and tumor suppresser genes. Used colorectal cancer cell lines to discover p53 targets. Polyak et al.(1997)Nature, 389(6648), 300-305. 39

40. 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. 40

41. Problems In SAGE… 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. 41

42. References… Hunt, Rick Livesy et al, Functional Genomics. Ji-Yeon Lee and Dong- Hee Lee, “Use of Serial Analysis of Gene Expression Technology to Reveal Changes in Gene Expression in Arabidopsis Pollen Undergoing Cold Stress”. Plant Physiol. Vol. 132, 2003 . wikipedia.org/wiki/ Serial_analysis_of_gene_expression#Overview Kanlayanee Sawanyawisuth , “High Throughput Gene Expression Analysis: a Review” . Srinagarind Med J 2009; 24(2): 154-8. 42

43. T . Yamashita, M. Honda, and S. Kaneko “Application of Serial Analysis of Gene Expression in Cancer Research” Current Pharmaceutical Biotechnology, 2008, 9, 375-382. Bioinformatics, Instant Notes by D.R. Westhead , J.H. Parish and R.M. Twyman . 43

44. Thank You. 44