资源预览内容
第1页 / 共81页
第2页 / 共81页
第3页 / 共81页
第4页 / 共81页
第5页 / 共81页
第6页 / 共81页
第7页 / 共81页
第8页 / 共81页
第9页 / 共81页
第10页 / 共81页
亲,该文档总共81页,到这儿已超出免费预览范围,如果喜欢就下载吧!
资源描述
中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.comSAGE的原理及其应用新乡医学院免疫学研究中心 王 辉中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.comWhat is SAGE?nSerial Analysis of Gene ExpressionnMethod to quantify gene expression levels in samples of cellsnOpen system nCan potentially reveal expression levels of all genes: “unbiased” and “comprehensive”nMicroarrays are closed, since they only tell you about the genes spotted on the array中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.comHow 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 中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com3. 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. 中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com4. 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. AB中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com5. 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. 中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com6. 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. 中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com7. 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中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com8. 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中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com9. Ligate the 26-bp ditags to form concatemers. Gel purify fractions containing 20-50 tags/concatemer. 中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com10. 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. 中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com11. Sequencing the SAGE library中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.comHow 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). 中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immuneweb.com中国免疫学信息网中国免疫学信息网 http:/http:/www.immuneweb.comwww.immu
收藏 下载该资源
网站客服QQ:2055934822
金锄头文库版权所有
经营许可证:蜀ICP备13022795号 | 川公网安备 51140202000112号