分子诊断学概论htw.pptx
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TheApplicationofProteomicsToDiseaseDiagnostics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格里夫茲格里夫茲(Griffith)肺炎雙球菌轉形肺炎雙球菌轉形試驗試驗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：http:/fig.cox.miami.edu/cmallery/150/gene/sf11x1b.jpg參考資料：http:/fig.cox.miami.edu/cmallery/150/gene/sf11x1b.jpg參考資料：參考資料：http:/biotech.nstm.gov.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 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 revealedmore 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 insertion/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 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.Author SummaryWe 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.Identificationandanalysisoffunctionalelementsin1%of the human genome by the ENCODE pilot project.Nature.2007Jun14;447(7146):799-816TheEncyclopediaofDNAElements(ENCODE)Projectprovideamorebiologicallyinformativerepresentationofthehumangenomebyusinghigh-throughputmethodstoidentifyandcataloguethefunctionalelementsencoded.First,ourstudiesprovideconvincingevidencethatthegenome is pervasively transcribed,such that themajority of its bases can be found in primarytranscripts,including non-protein-coding transcripts,andthosethatextensivelyoverlaponeanother.Second,systematic examination of transcriptional regulationhasyieldednewunderstandingabouttranscriptionstartsites,includingtheirrelationshiptospecificregulatorysequencesandfeaturesofchromatinaccessibilityandhistonemodification.Third,amoresophisticatedviewofchromatinstructurehasemerged,includingitsinter-relationship with DNA replication and transcriptionalregulation.Finally,integrationofthesenewsourcesofinformation,in particular with respect to mammalianevolution based on inter-and intra-species sequencecomparisons,hasyieldednewmechanisticandevolutionaryinsightsconcerningthefunctionallandscape of the human genome.Together,thesestudies are defining a path for pursuit of a morecomprehensive characterization of human genomefunction.ThehighlightsofourfindingstodateincludeThe human genome is pervasively transcribed,suchthatthemajorityofitsbasesareassociatedwith at least one primary transcript and manytranscripts link distal regions to establishedprotein-codingloci.Many novel non-protein-coding transcripts havebeen identified,with many of these overlappingprotein-codinglociandotherslocatedinregionsofthegenomepreviouslythoughttobetranscriptionallysilent.Numerouspreviouslyunrecognizedtranscriptionstart sites have been identified,many of whichshowchromatinstructureandsequence-specificprotein-bindingpropertiessimilartowell-understoodpromoters.Regulatorysequencesthatsurroundtranscriptionstartsitesaresymmetricallydistributed,withnobiastowardsupstreamregions.Chromatinaccessibilityandhistonemodificationpatternsarehighlypredictiveofboththepresenceandactivityoftranscriptionstartsites.DistalDNaseIhypersensitivesiteshavecharacteristichistonemodificationpatternsthatreliablydistinguishthemfrompromoters;someofthesedistalsitesshowmarksconsistentwithinsulatorfunction.DNAreplicationtimingiscorrelatedwithchromatinstructure.Atotalof5%ofthebasesinthegenomecanbeconfidentlyidentifiedasbeingunderevolutionaryconstraintinmammals;forapproximately60%ofthese constrained bases,there is evidence offunction on the basis of the results of theexperimentalassaysperformedtodate.Although there is general overlap betweengenomic regions identified as functional byexperimentalassaysandthoseunderevolutionaryconstraint,notallbaseswithintheseexperimentallydefinedregionsshowevidenceofconstraint.Differentfunctionalelementsvarygreatlyintheirsequencevariabilityacrossthehumanpopulationand in their likelihood of residing within astructurallyvariableregionofthegenome.Surprisingly,manyfunctionalelementsareseeminglyunconstrainedacrossmammalianevolution.Thissuggeststhepossibilityofalargepool of neutral elements that are biochemicallyactive but provide no specific benefit to theorganism.Thispoolmayserveasawarehousefor natural selection,potentially acting as thesourceoflineage-specificelementsandfunctionallyconservedbutnon-orthologouselementsbetweenspecies遺傳物質：核酸遺傳物質：核酸(nucleic acid)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：http:/academic.brooklyn.cuny.edu/biology 主溝主溝majorgroove小溝小溝minorgroove三個氫鍵三個氫鍵二個氫鍵二個氫鍵二個氫鍵二個氫鍵三個氫鍵三個氫鍵DNA的結構的結構資料來源：http:/academic.brooklyn.cuny.edu/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：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TATAbox大約在大約在-10-35處處CAATbox大約在大約在-75處影響啟動子效率處影響啟動子效率GCbox由由GGGCGG序列組成，也稱為序列組成，也稱為sp1box，為轉錄因子，為轉錄因子sp1結合位置結合位置Sp1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（simianvirus40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silencer與增強子功能相反的調控與增強子功能相反的調控反應元反應元responseelement與轉錄因子結合，作用到啟動子，增強轉錄與轉錄因子結合，作用到啟動子，增強轉錄作用。作用。與特異性刺激因子結合，調節基因表現與特異性刺激因子結合，調節基因表現TRE-TPAresponseelement,SRE-serumresponseelement,HSE-heatshockresponseelement,CRE-Campresponseelement,ERE-estrogenresponseelement,GRE-glucocorticoidresponseelement,MRE-metalresponseelement.阻絕子阻絕子insulator一段一段0.53kb的的DNA，阻斷轉錄因子散佈，阻斷轉錄因子散佈（可能增強子或靜默子）。（可能增強子或靜默子）。轉錄區轉錄區表現子表現子exon（外顯子）（外顯子）具有轉譯成多胜肽鏈或轉錄成具有轉譯成多胜肽鏈或轉錄成RNA分子的序分子的序列，為編碼區列，為編碼區內含子內含子intro（插入子）（插入子）與與exon一起被轉錄，但在一起被轉錄，但在mRNA剪切時會剪切時會被切除。被切除。5端端GT-AG3端端可能與調控有關，基因的穩定有相關可能與調控有關，基因的穩定有相關聚腺苷酸化訊號聚腺苷酸化訊號polyadenylationsignalmRNA3端的端的AAUAAA序列下游約序列下游約15-30bp會被切除，再加上會被切除，再加上polyAtail。遺傳中心法則遺傳中心法則DNARNA蛋白質蛋白質mRNA的剪切由核內的小分子的剪切由核內的小分子RNA(snRNA)和和snRNA蛋白質複合體蛋白質複合體(snRNPs)及及SR蛋白質參與，這些分子形成剪接體結構蛋白質參與，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反轉錄酶的發現顛覆了此中心法則1970HowardTermin和和DavidBaltimore發現發現(1975Nobel)一些一些RNA病毒能利用反轉錄酶將它們的遺傳病毒能利用反轉錄酶將它們的遺傳物質物質(RNA)反轉錄為反轉錄為DNA
