分子诊断学概论

1、分子診斷學概論分子診斷學概論第一章第一章 綜說綜說 overview疾病發生原因的影響層次疾病發生原因的影響層次DNA、RNA或蛋白質或蛋白質分子診斷的目的分子診斷的目的偵測這些致病因子是那個層次發生變化偵測這些致病因子是那個層次發生變化本書著重本書著重DNA、RNA的變化的變化蛋白質層次由原文書章節提供蛋白質層次由原文書章節提供The Application of Proteomics To Disease Diagnostics遺傳分子的基礎遺傳分子的基礎生物巨分子：生物巨分子：DNA、RNA、蛋白質、糖類、脂、蛋白質、糖類、脂質質遺傳物質遺傳物質DNA的發現的發現1928 格里夫茲格里夫

2、茲 (Griffith)肺炎雙球菌轉形試肺炎雙球菌轉形試驗驗1942 艾佛瑞艾佛瑞(Avery)研究格里夫茲轉形的物研究格里夫茲轉形的物質為何質為何?1952 赫希赫希-卻斯卻斯 (Hershey-Chase)以放射線以放射線標示噬菌體的蛋白質（標示噬菌體的蛋白質（S35）和）和DNA（P32），），感染大腸桿菌的實驗感染大腸桿菌的實驗1953 雙螺旋結構的發現雙螺旋結構的發現2003 人類基因體計畫的完成人類基因體計畫的完成參考資料：/cmallery/150/gene/sf11x1b.jpg參考資料：http:/fig.cox.miami.ed

3、u/cmallery/150/gene/sf11x1b.jpg參考資料：參考資料：.tw/advance/a021.asp 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 ba

4、ses (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 Ce

5、nter for Biotechnology Information human reference assembly revealed 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 (2206 bp), 292,102 heterozygous insertio

6、n/deletion events (indels)(1571 bp), 559,473 homozygous indels (182,711 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

7、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 pre

8、cision 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. Author Summary We have generated an independently assembled d

9、iploid 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 segmen

10、ts (.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

11、, 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

12、 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 start

13、ing point for future individual genome comparisons and enables the emerging era of individualized genomic information.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 (ENCO

14、DE) Projectprovide a more biologically informative representation of the human genome by using high-throughput methods to identify and catalogue the functional elements encoded. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its b

15、ases 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 reg

16、ulatory 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

17、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 charac

18、terization of human genome function. The highlights of our findings to date includeThe 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.Many nov

19、el 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

20、 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. Chromatin accessibility and histone modification patterns are

21、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 replicatio

22、n timing 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 as

23、says performed to date.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 i

24、n 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 ele

25、ments 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遺傳物質：核酸遺傳物質：核酸(nucleic a

26、cid)核酸：核酸：DNA（去氧核醣核酸）、（去氧核醣核酸）、RNA（核醣核酸）（核醣核酸）核酸基本單位：核苷酸核酸基本單位：核苷酸(nucleotide)核苷酸：鹼基核苷酸：鹼基(base)、五碳醣、五碳醣(pentose sugar)、磷酸、磷酸(phosphate)DNA的組成的組成(王文姿等，2003)HHDNA去氧核糖核酸去氧核糖核酸RNA核糖核酸核糖核酸鹼基鹼基DNA的結構的結構資料來源：/biology 主溝主溝 major groove小溝小溝 minor groove三個氫鍵三個氫鍵二個氫鍵二個氫鍵二個氫鍵二個氫

27、鍵三個氫鍵三個氫鍵DNA的結構的結構資料來源：/biology 穩定力量來自穩定力量來自氫鍵氫鍵非共價鍵非共價鍵-堆積堆積力量：力量：凡得瓦力、凡得瓦力、斥水性作用力、斥水性作用力、親水性作用力親水性作用力糖骨架：糖骨架：磷酸磷酸雙酯鍵雙酯鍵環境與序列的影響會形成不同形式結構的環境與序列的影響會形成不同形式結構的DNA較寬與緊密較寬與緊密外表成鋸齒狀外表成鋸齒狀http:/nucleix.mbu.iisc.ernet.in/image/abzDNA.JPG基因的一般結構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，基因定義

28、：染色體上一段有功能的特定序列，可轉錄成可轉錄成RNA分子或是轉譯成多胜肽鏈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？定義可能會更改：調控性功能的序列？ 基因的結構基因的結構表現子（表現子（exon外顯子）外顯子）-可轉錄或轉譯出產物，稱為編可轉錄或轉譯出產物，稱為編碼區碼區轉錄調控區轉錄調控區啟動子啟動子promoter：其特殊序列與轉錄因子結合，引：其特殊序列與轉錄因子結合，引導導RNA聚合酶，產生基因轉錄聚合酶，產生基因轉錄轉錄起始點定為轉錄起始點定為+1轉錄進行的方向稱為下游轉錄進行的方向稱為下游相反的方向稱為上游相反的方向稱為上游保留性序列保留性序列TATA box大約

29、在大約在-10 -35處處CAAT box大約在大約在 -75處影響啟動子效率處影響啟動子效率GC box由由GGGCGG序列組成，也稱為序列組成，也稱為sp1 box，為轉錄因子為轉錄因子sp1結合位置結合位置Sp 1分離自人類細胞分離自人類細胞由加州大學柏克萊分校的由加州大學柏克萊分校的Tjian教授的實教授的實驗室分離驗室分離可有效地促進猿猴濾過性病毒（可有效地促進猿猴濾過性病毒（simian virus 40簡稱簡稱SV40）基因的轉錄）基因的轉錄加強子加強子Enhancer加強特殊基因的轉錄活性加強特殊基因的轉錄活性位置不固定位置不固定與調節蛋白結合，與啟動子間形成圈環構造與調節蛋白結合，與啟動子間形成圈環構造靜默子靜默子si