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May 11, 2015
2. WHAT IS GENE EXPRESSION?
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SAGE AND ITS PRINCIPLE
STEPS IN SAGE, ITS APPLICATIONS AND PROBLEMS.
REFERENCES.
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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.
DNARNA Protein
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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 cells complete gene activity.
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5. SAGE invented at Johns Hopkins University in USA (Oncology
Center) by Dr. Victor Velculescu in 1995.
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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.
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7. Steps In Brief
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8. 8
9. SAGE Flowchart
1. Isolate mRNA.
B
2. (a) Add biotin-labeled dT primer:
(b) Synthesize ds cDNA.
B
3.(a) Bind to streptavidin-coated beads.
(b) Cleave with anchoring enzyme.
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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
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B
6. Combine pools and ligate.
7. Amplify ditags, then cleave with anchoring
enzyme.
12. 8. Ligate ditags.
9. Sequence and record the tags and frequencies.
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13. SAGE In Details
Trapping of RNA with beads
14. Molecules that consist of 20 or so dTs acts like a
attractant to capture mRNAs. 15. Coating of microscopic magnetic
beads with TTTTT tails is done. 16. A magnet is used to withdraw
the bead and the mRNA is isolated.13
17. 14
mRNA
mRNA
mRNA
mRNA
mRNA
Microscopic bead coated with TTTTs
mRNA
mRNA
mRNA
mRNA
mRNA
18. 15
mRNA
mRNA
mRNA
mRNA
mRNA
Microscopic bead coated with TTTTs
mRNA
mRNA
mRNA
mRNA
mRNA
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19. cDNA synthesis
20. cDNA synthesis is immobilized to streptavidin beads.16
21. 17
B
B
Biotinylated oligo dT (primers)
B
B
B
B
mRNA
B
Streptavidin beads
B
cDNA
B
B
22. Enzymatic cleavage of cDNA
23. Type II restriction enzyme used (E.g. NlaIII.) 24. Average
length of cDNA 256bp with sticky ends created.18
25. 19
Nla III (Restriction enzyme)
B
B
B
B
26. Ligation of Linkers to bound cDNA
27. Linkers must contain: 28. NlaIII 4-nucleotide cohesive
overhang. 29. Type IIs recognition sequence. 30. PCR primer
sequence.20
31. 21
Linkers
B
B
B
B
Pool A
Pool B
32. Cleaving with tagging enzyme
33. Repair of ends to make blunt ended tags using DNA polymerase
(Klenow fragments) and dNTPs.22
34. 23
Bsm FI
(tagging Enzyme)
B
Linker adapted SAGE tag
B
35. Formation of Ditags
36. Two groups of cDNAs are ligated to each other, to create a
ditag with linkers on either end. 37. Two tags are linked together
using T4 DNA ligase.24
38. 25
Add DNA ligase
39. PCR amplification of Ditags
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40. 27
PCR Amplification
41. Isolation of Ditags
42. Breaking the linker off right where it was added in
beginning. 43. This leaves a sticky end with the sequence GTAC (or
CAGT on the other strand) at each end of the ditag.28
44. Nla III
(Anchoring enzyme)
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29
29
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45. Concatamerization of Ditags
46. Each ditag is having an AE site, allowing the scientist and
the computer to recognize where one ends and the next
begins.30
47. 31
Concatemirize
48. Cloning Concatamers and Sequencing
49. 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
50.
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51. Vast amount of data is produced, which must be shifted and
ordered for useful information to become apparent.
SAGE reference databases:
52. SAGE Geniehttp://www.ncbi.nlm.nih.gov/cgap
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53. How Does The Data Look Like?
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54. 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)
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55. 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.
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56. 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.
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57. 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 tumorsuppresser
genes.
Used colorectal cancer cell lines to discover p53 targets.
Polyak et al.(1997)Nature, 389(6648), 300-305.
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58. 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.
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59. 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.
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60. References
61. Ji-YeonLee 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. 62.
wikipedia.org/wiki/Serial_analysis_of_gene_expression#Overview 63.
KanlayaneeSawanyawisuth, High Throughput Gene Expression Analysis:
a Review.Srinagarind Med J 2009; 24(2): 154-8.42
64.
65. Bioinformatics, Instant Notesby D.R. Westhead, J.H. Parish
and R.M. Twyman.43
66. Thank You.
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