長庚大學中醫系

1. 後基因世代於中醫藥研究應用與展望 潘台龍 老師 長庚大學中醫系 email:pan@mail.cgu.edu.tw

2. 中藥化學研究 • 中藥藥理學研究 • 中藥複方基礎研究 • 中藥基因體計畫 • 中藥資源研究 • 中藥與辨正研究

3. Examples : 冬蟲夏草 真菌界Fungi　真菌群Eumycota　　　　子囊亞菌群Ascomycetes　　　　　核菌綱Pyrenomycetes　　　　　　球殼菌目Sphaeriales　　　　　　　麥角菌科Clavicipitaceae　　　　　　　　冬蟲夏草屬Cordycpes

4. Backgrounds 分佈區：青海及青康藏高原一帶。 藥效：增強免疫力、防止及致療急性腎臟衰竭暨肝硬化。 品管：冬蟲夏草分類及演化因區域不一而有差異，如何科學化鑑定，成為重要課題之一。

5. Isaria japonica Cordyceps nutans Japanese society for cordyceps research

6. Previously strategy 中藥材 活性分析 成份鑑定/農藥檢測 (LC-NMR-MS) (Cell-based HTS)

7. 中藥化學研究

8. Cell proliferation and viability assays are of particular importance for routine applications. Tetrazolium salts (e.g. MTT, XTT, WST-1) are especially useful for this type of analysis. These tetrazolium salts are cleaved to formazan by the "succinate-tetrazolium reductase" system (EC 1.3.99.1) which belongs to the respiratory chain of the mitochondria and is active only in metabolically active cells. The figure below shows an example of this type of reaction. 活性分析

10. The post-genomics era 中藥材 活性分析 成份鑑定/農藥檢測 (LC-NMR-MS) (Cell-based HTS) DNA抽取 標的篩選 DNA擴增 指紋圖譜

11. Classification and polymorphism on Cordycpessinensis Technologies • RFLP : restriction fragment length polymorphism • PCR-RFLP : Polymerase chain reaction RFLP • RAPD : Random amplified polymorphic • AFLP : AmplifiedFragmentLength Polymorphism

12. RFLP RFLP is one of the DNA fingerprinting techniques that is used to determine plant strain and purity in nutraceutical and herb production.

13. The Polymerase Chain Reaction is used to amplify a sample of DNA.

14. PCR-RFLP A H H H H 109bp Plant A H H H 109bp Plant B B C M A B C D E 109 bp 57 bp 35 bp 22 bp 17 bp 12 bp

15. PCR-DNA sequencing The primers used for amplification of the rDNA internal transcribed spacer regions were primer A (forward): 5'-CGTAGGTGAACCTGCGGAAGGATCA-3' (18S rDNA 3' terminal region of eukaryotic organisms) and primer B (reverse): 5'-TTCCCTGTTCACTCGCCGTTACT-3' (position 6080 bp in eukaryotic 25S rDNA)

16. DNA Sequencing • Like PCR, it utilizes DNA polymerase and thermal cycling • Only reads the sequence of 1 strand of DNA using 1 primer • The CEQ 8000 utilizes the Sanger dideoxy termination method • Reaction generates a population of dye-terminated DNA fragments

17. Cycle Sequencing

18. DTCS DeoxyNucloetide DideoxyNucloetide

19. DTCS Extension and Termination

20. Extension and Termination • Simultaneous reactions terminate at different lengths • Reactions generate multiple fragments of all sizes from 1 to 1000+ bases

21. Separation and Detection • Fragments are separated by capillary gel electrophoresis • Laser-induced fluorescence of dye terminators is sequentially read by the PMT sensor

22. Sequencing Results www.ncbi.nlm.nih.gov

23. RAPD Random amplified polymorphic DNA (RAPD), a simple chain reaction (PCR) amplification of genomic DNA by a single synthetic oligonucleotide primer, can generate a complex pattern. RAPD usually reveals considerable polymorphism in genomic DNA and genetics marker for estimating genetic, taxonomic, and phylogenetics relationships of species.

24. Primer 1 Primer 3

25. Each primer had the following sequence: 18S F: 5' CAA CCT GGT TGA TCC TGC CAG T 3 ' and 18S R: 5 ' CTG ATC CTT CTG CAG GTT CAC CTA C 3 '. Ban II Dde I

26. AFLP:AmplifiedFragmentLength Polymorphism Identification and Typing - strain level • useful for both identification (species) and typing (strain) level Taxonomy - confirms prior classifications

27. E E M CAATGAGTCCTGAGTAG AFLP -How It Works: 1. DNA Extraction Collection of 3 Types of Fragments: 2. Digestion with EcoRI (E) and MseI(M) E M M 3. Ligation of Sequence Specific Adaptors CTCGTAGACTGCGTACC CATCTGACGCATGGTTAA AATTCNNN GNNN NNNT NNNAAT TACTCAGGACTCAT GAGTCCTGAGTAGCAG 4. Amplification: CTCGTAGACTGCGTACCAATTCNNN NNNTTACTCAGGACTCAT • Pre-Selective CATCTGACGCATGGTTAAGNNN GACTGCGTACCAATTCA NNNAATGAGTCCTGAGTAGCAG CTCGTAGACTGCGTACCAATTCNNN NNNTTACTCAGGACTCAT • Selective TGCAATGAGTCCTGAGTAG CATCTGACGCATGGTTAAGNNN GACTGCGTACCAATTCACT NNNAATGAGTCCTGAGTAGCAG

28. AFLP Fragment Data from CEQ 2000 XL

29. 中藥材 活性分析 成份鑑定/農藥檢測 (LC-NMR-MS) (Cell-based HTS) DNA抽取 標的篩選 目的基因選擇 目的基因片段擴增 DNA擴增 DNA訂序 限制媒反應 指紋圖譜 特異性PCR擴增 指紋圖譜

30. Given the size and complexity of the plant genomes, and the extent of repetitive elements within them, the assembly of a large plant genome after shotgun sequencing would require extensive computing capacity, certainly exceeding that needed to assemble the human genome. What are the alternatives to approaches involving whole genome sequencing? • Searching fortranscribed mRNAs provides a much more economic wayto gain insight into the gene repository of individualspecies. • ESTs are created by partially sequencing randomly chosen gene transcripts that have been converted into cDNA.

31. Large-scale EST sequencing provides a cost-effective way to obtain highly valuable information on the transcribed genes of individual species. In addition, EST clones can be used to design expression analysis experiments. • ESTs provide a high throughput means for identifying gene transcripts and monitoring complex gene expression patterns. • Intrinsic problems with EST analysis include the inherent unreliable quality of the sequence, the overrepresentation of highly expressed genes and the incomplete, partial nature of the sequences.

32. Summary of cDNA cloning and expressed sequence tag (EST) sequencing. TRENDS in Plant Science Vol.8 No.7 July 2003

33. Functional Classification of the Bupleurum root ESTs molecular function Biological process Cellular location

34. Biosynthesis of Beta-amyrin and cycloartenol 2 11 2 2 1

35. 病理 同氣相求 氣象因素 病症 人(各型體質) 六淫(風寒暑濕燥火) 生物性致病因素

36. 人類各種體質要素及其相互關係模型 氣、血、津、液、五臟、六腑 DNA 阳 阴 體細胞、組織、器官、系統 功能、結構、代謝 反應

37. 草藥 目標確認 目標公證 引導確認 引導最佳化 活性藥效分析 活性藥效篩選 生物晶片尋找相關之基因 最佳效率和毒性輕微

38. What is Single Nucleotide Polymorphism ? • Many of differences among people have a genetic basis - alterations in the DNA that change the way important proteins are made. • Sometimes the alterations involve a single base pair (the smallest building block of DNA) and are shared by many people. Such single base pair differences are called "single nucleotide polymorphisms", or SNPs. However, the majority of the SNPs do not produce physical changes in people with affected DNA. • Estimate ~ 15M SNPs in total throughout human genome (one SNP every 200 bases).

39. Pharmacogenomics Genetics of Drug Efficacy and Toxicity

40. Application – High Through-put Screening

41. Functional genomics approaches are powerful tools to accelerate comprehensive investigations of cellular metabolism in specialized tissues or whole organisms. + + + Metabolomics Transcriptomics Proteomics Genomics Biological Knowledge in Herbal plant

42. 生物晶片尋找草藥反應標的基因/蛋白 • 於對中藥材料及制成品的定性與定量分析，於藥物鑑定、質量保證方面具有極為重要的應用價值，還可用於尋找、發現中藥新基因。 • DNA 微陣列技術協助尋找草藥於組織作用標的，標的確認可能真實地參與疾病的病理生理機制。 • 現在一般充份接受單一基因體能在不同的生物學的情況之下性質上地而且數量地引起不同的蛋白質體，這些包括例如轉譯後的蛋白質分解處理和例如磷酸和乙醯化的後平移的修正的機制。

43. Focus on Technology Microarray & DNA Chips Principle: HYBRIDIZATION Same idea, better use [Bardeen & Shockley] Transistors to Computer Chips [Edwin Southern] Southern Blots to DNA Chips One to One Correspondence: Clone to Hybridization Signal History of Science Other Landmark One to Ones 1941: Beadle & Tatum One gene, one enzyme 1964: Charles Yanofsky DNA sequence colinear with Protein sequence

45. Focus on Technology Microarray & DNA Chips If you get this, then you have got it. That’s all there’s to it. Really! If you get this, then you have got it. That’s all there’s to it. Really! Principle Northern

46. Focus on Technology Microarray & DNA Chips What are the steps? [1] Choose cell population [or sample for diagnosis] [2] RNA extraction, purify [3] Fluorescent label cDNA [4] Hybridize with PROBE on Microarray or DNA Chip [5] Scan [6] Interpret image

47. Focus on Technology Microarray & DNA Chips A Thumb Nail Version Are Microarrays and DNA Chips the same ? They share the same scientific principle May be used with similar TARGETS DIFFER in construction and type of PROBE Nomenclature

48. Gene expression profile before and after- treatment of B.kaol • Cell matrix protein • Inflammation • MMP • Metabolism • Transcription factor • Oncogene

50. The Challenge of Proteomics Complex Proteome(s) • Multiple Proteins for Each Gene • Varied and Fragile Nature of Protein • Quantitative and Qualitative Changes of the Proteome • Structural and Functional Proteomics Studies Same Genome