分子診斷學概論

1. 分子診斷學概論 • 第一章 綜說 overview • 疾病發生原因的影響層次 • DNA、RNA或蛋白質 • 分子診斷的目的 • 偵測這些致病因子是那個層次發生變化 • 本書著重DNA、RNA的變化 • 蛋白質層次由原文書章節提供 • The Application of Proteomics To Disease Diagnostics

3. 遺傳分子的基礎 • 生物巨分子：DNA、RNA、蛋白質、糖類、脂質 • 遺傳物質DNA的發現 • 1928 格里夫茲 (Griffith)肺炎雙球菌轉形試驗 • 1942 艾佛瑞(Avery)研究格里夫茲轉形的物質為何? • 1952 赫希-卻斯 (Hershey-Chase)以放射線標示噬菌體的蛋白質（S35）和DNA（P32），感染大腸桿菌的實驗 • 1953 雙螺旋結構的發現 • 2003 人類基因體計畫的完成

4. 參考資料：http://fig.cox.miami.edu/~cmallery/150/gene/sf11x1b.jpg參考資料：http://fig.cox.miami.edu/~cmallery/150/gene/sf11x1b.jpg

5. 參考資料：http://fig.cox.miami.edu/~cmallery/150/gene/sf11x1b.jpg參考資料：http://fig.cox.miami.edu/~cmallery/150/gene/sf11x1b.jpg

6. www.accessexcellence.org

7. 參考資料：http://biotech.nstm.gov.tw/advance/a021.asp

11. Presented here is a genome sequence of an individual human. It was produced from ~32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 scaffolds, comprising 2,810 million bases (Mb) of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed

12. more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel) included 3,213,401 single nucleotide polymorphisms (SNPs),53,823 block substitutions (2–206 bp), 292,102 heterozygous insertion/deletion events (indels)(1–571 bp), 559,473 homozygous indels (1–82,711 bp), 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments .200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information.

13. Author Summary • We have generated an independently assembled diploid human genomic DNA sequence from both chromosomes of a single individual (J. Craig Venter). Our approach, based on whole-genome shotgun sequencing and using enhanced genome assembly strategies and software, generated an assembled genome over half of which is represented in large diploid segments (.200 kilobases), enabling study of the diploid genome. Comparison with previous reference human genome sequences, which were composites comprising multiple humans, revealed that the majority of genomic alterations are the well-studied class of variants based on single nucleotides (SNPs). However, the results also reveal that lesserstudied genomic variants, insertions and deletions, while comprising a minority (22%) of genomic variation events, actually account for almost 74% of variant nucleotides. Inclusion of insertion and deletion genetic variation into our estimates of interchromosomal difference reveals that only 99.5% similarity exists between the two chromosomal copies of an individual and that genetic variation between two individuals is as much as five times higher than previously estimated. The existence of a well-characterized diploid human genome sequence provides a starting point for future individual genome comparisons and enables the emerging era of individualized genomic information.

14. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature. 2007 Jun 14;447(7146):799-816 • The Encyclopedia of DNA Elements (ENCODE) Project • provide a more biologically informative representation of the human genome by using high-throughput methods to identify and catalogue the functional elements encoded.

15. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

16. The highlights of our findings to date include • The human genome is pervasively transcribed, such that the majority of its bases are associated with at least one primary transcript and many transcripts link distal regions to established protein-coding loci. • Manynovel non-protein-coding transcripts have been identified, with many of these overlapping protein-coding loci and others located in regions of the genome previously thought to be transcriptionally silent. • Numerous previously unrecognized transcription start sites have been identified, many of which show chromatin structure and sequence-specific protein-binding properties similar to well-understood promoters. • Regulatory sequences that surround transcription start sites are symmetrically distributed, with no bias towards upstream regions.

17. Chromatin accessibility and histone modification patterns are highly predictive of both the presence and activity of transcription start sites. • Distal DNaseI hypersensitive sites have characteristic histone modification patterns that reliably distinguish them from promoters; some of these distal sites show marks consistent with insulator function. • DNA replicationtiming is correlated with chromatin structure. • A total of 5% of the bases in the genome can be confidently identified as being under evolutionary constraint in mammals; for approximately 60% of these constrained bases, there is evidence of function on the basis of the results of the experimental assays performed to date.

18. Although there is general overlap between genomic regions identified as functional by experimental assays and those under evolutionary constraint, not all bases within these experimentally defined regions show evidence of constraint. • Different functional elements vary greatly in their sequence variability across the human population and in their likelihood of residing within a structurally variable region of the genome. • Surprisingly, many functional elements are seemingly unconstrained across mammalian evolution. This suggests the possibility of a large pool of neutral elements that are biochemically active but provide no specific benefit to the organism. This pool may serve as a 'warehouse' for natural selection, potentially acting as the source of lineage-specific elements and functionally conserved but non-orthologous elements between species

19. DNA的組成 H 遺傳物質：核酸(nucleic acid) 核酸：DNA（去氧核醣核酸）、RNA（核醣核酸） 核酸基本單位：核苷酸(nucleotide) 核苷酸：鹼基(base)、五碳醣(pentose sugar)、磷酸(phosphate) (王文姿等，2003)

20. H DNA 去氧核糖核酸 RNA 核糖核酸

21. 鹼基

24. DNA的結構 主溝 major groove 小溝 minor groove 資料來源：http://academic.brooklyn.cuny.edu/biology

25. DNA的結構 二個氫鍵 三個氫鍵 三個氫鍵 二個氫鍵 • 穩定力量來自 • 氫鍵 • 非共價鍵-堆積力量：凡得瓦力、斥水性作用力、親水性作用力 • 糖骨架：磷酸雙酯鍵 資料來源：http://academic.brooklyn.cuny.edu/biology

26. 環境與序列的影響會形成不同形式結構的DNA 較寬與緊密 外表成鋸齒狀

27. http://nucleix.mbu.iisc.ernet.in/image/abzDNA.JPG

28. 基因的一般結構 • 基因定義：染色體上一段有功能的特定序列，可轉錄成RNA分子或是轉譯成多胜肽鏈 • 定義可能會更改：調控性功能的序列？

29. 基因的結構 • 表現子（exon外顯子）-可轉錄或轉譯出產物，稱為編碼區 • 轉錄調控區 • 啟動子promoter：其特殊序列與轉錄因子結合，引導RNA聚合酶，產生基因轉錄 • 轉錄起始點定為+1 • 轉錄進行的方向稱為下游 • 相反的方向稱為上游 • 保留性序列 • TATA box大約在-10 ~ -35處 • CAAT box大約在 -75處影響啟動子效率 • GC box由GGGCGG序列組成，也稱為sp1 box，為轉錄因子sp1結合位置

32. Sp 1分離自人類細胞 • 由加州大學柏克萊分校的Tjian教授的實驗室分離 • 可有效地促進猿猴濾過性病毒（simian virus 40﹐簡稱SV40）基因的轉錄 • 加強子Enhancer • 加強特殊基因的轉錄活性 • 位置不固定 • 與調節蛋白結合，與啟動子間形成圈環構造 • 靜默子silencer • 與增強子功能相反的調控

33. 反應元response element • 與轉錄因子結合，作用到啟動子，增強轉錄作用。 • 與特異性刺激因子結合，調節基因表現 • TRE-TPA response element, SRE-serum response element, HSE-heat shock response element, CRE-Camp response element, ERE-estrogen response element, GRE-glucocorticoid response element, MRE-metal response element…. • 阻絕子insulator • 一段0.5 ~ 3 kb的DNA，阻斷轉錄因子散佈（可能增強子或靜默子）。

34. 轉錄區 • 表現子exon（外顯子） • 具有轉譯成多胜肽鏈或轉錄成RNA分子的序列，為編碼區 • 內含子intro（插入子） • 與exon一起被轉錄，但在mRNA剪切時會被切除。5端 GT-AG 3端 • 可能與調控有關，基因的穩定有相關 • 聚腺苷酸化訊號polyadenylation signal • mRNA 3端的AAUAAA序列下游約15-30 bp會被切除，再加上poly A tail。

36. 遺傳中心法則 • DNA→RNA→蛋白質 • mRNA的剪切由核內的小分子RNA (snRNA)和snRNA蛋白質複合體(snRNPs)及SR蛋白質參與，這些分子形成剪接體結構(splicesome) • 反轉錄酶的發現顛覆了此中心法則 • 1970 Howard Termin和David Baltimore 發現 (1975 Nobel) • 一些RNA病毒能利用反轉錄酶將它們的遺傳物質(RNA)反轉錄為DNA