1. 中国免疫学信息网 http://www.immuneweb.com SAGE 的原理及其应用 新乡医学院免疫学研究中心 王 辉

2. 中国免疫学信息网 http://www.immuneweb.com What is SAGE? Serial Analysis of Gene Expression Method to quantify gene expression levels in samples of cells Open system Can potentially reveal expression levels of all genes: “unbiased” and “comprehensive” Microarrays are closed, since they only tell you about the genes spotted on the array

3. 中国免疫学信息网 http://www.immuneweb.com How does SAGE work? 1. Isolate mRNA. 2. Bind RNA sample to oligo (dT) magnetic beads. The beads capture poly A+ RNA directly from your sample. Synthesize double stranded cDNA on the beads containing your mRNA using SuperScript. II reverse transcriptase and E. coli DNA polymerase

4. 中国免疫学信息网 http://www.immuneweb.com 3. Digest the double stranded cDNA with a sequence specific restriction enzyme (an anchoring enzyme) that cleaves most transcripts at least once. Nla III is used as an anchoring enzyme since Nla III sites are known to occur approximately every 250 bp.

5. 中国免疫学信息网 http://www.immuneweb.com 4. Divide the cDNA into two fractions and ligate with two adapters (A and B, ~40 bp each). The adapters contain cohesive 4-bp overhangs complementary to the Nla III digested cDNA, a Type IIS restriction enzyme (tagging enzyme) recognition site at the 3  end, and priming sites for PCR amplification. A B

6. 中国免疫学信息网 http://www.immuneweb.com 5. Cleave with Type IIS restriction enzyme (tagging enzyme), BsmF I. The tagging enzyme binds to the recognition sequence in the adapter and cleaves the cDNA 10-14 bp downstream from the recognition site releasing a ~50-bp tag with a 4-bp overhang at the 5  end. The tag consists of ~40 bp of adapter sequence and 10-14 bp of unique sequence from a single transcript.

7. 中国免疫学信息网 http://www.immuneweb.com 6. Perform a Klenow reaction to fill-in the 5′ overhangs created by BsmF I digestion and ligate the two fractions of tags to form ~100-bp ditags.

8. 中国免疫学信息网 http://www.immuneweb.com 7. Amplify the ~100-bp ditags using primers specific for the primer binding sites in the adapters to produce sufficient ditags for subsequent generation of concatemers. G. 102 bp linker-ditag

9. 中国免疫学信息网 http://www.immuneweb.com 8. Cleave the ~100-bp ditags with the anchoring enzyme that was used to cleave the original cDNA (Nla III) to release a 26-bp ditag. These ditags are comprised entirely of sequences derived from transcript cDNAs. Each ditag is punctuated by Nla III recognition sequence. The 26-bp ditags are purified away from the adapters by polyacrylamide gel electrophoresis. 37 bp linker 28 bp ditag

10. 中国免疫学信息网 http://www.immuneweb.com 9. Ligate the 26-bp ditags to form concatemers. Gel purify fractions containing 20-50 tags/concatemer.

11. 中国免疫学信息网 http://www.immuneweb.com

12. 10. Clone the concatemers into the pZErO®-1 vector to obtain a SAGE. library. Sequence selected clones. Each transcript is identified by its unique 10-14-bp sequence tag and is quantified by the number of times the tag occurs within a given population of clones.

13. 中国免疫学信息网 http://www.immuneweb.com 11. Sequencing the SAGE library

14. 中国免疫学信息网 http://www.immuneweb.com

15. How to analyze the SAGE data? Analyze the sequence data using the SAGE. analysis software. The software extracts tag sequences from the concatemer sequences, tabulates the occurrence of each tag, and creates a report of each tag and its abundance. Following the SAGE-analysis, SAGE-tags will be extracted using the SAGE2000 software (www.sagenet.org).www.sagenet.org

16. 中国免疫学信息网 http://www.immuneweb.com

41. How to get the SAGE information from NCBI

42. 中国免疫学信息网 http://www.immuneweb.com

56. GAAAATAAAG-----------------------------------AAAAAAAAAAAAA TTTTTTTTTTTTT 5 ’ primer 3 ’ primer As a probe to get the new gene cDNA from cDNA library GLGI(generation of longer cDNA fragments from SAGE tags for gene identification)

57. 中国免疫学信息网 http://www.immuneweb.com

64. Thank You!