分子诊断学重点

moleculardiagnostics：Itrepresentstheapplicationoftheprinciplesofbasicmolecularbiologytotheinvestigationofhumandiseaseprocesses.中心法则isthetheorythatDNAencodesRNA(theprocessoftranscription)whichencodesproteins(translation),butthatinformationdoesnotflowfromproteinstoDNA.DNA复制:theprocessbywhichthedoublehelixisunwoundandeachstrandactsasatemplate.Basesarematchedtosynthesizethenewpartnerstrands.转录:theprocessbywhichanRNAcopyofageneismade.逆转录:synthesisofaDNAusinganRNAtemplate.翻译:theprocessbywhichribosomesusetheinformationinmRNAstosynthesisproteins.基因:thebasicunitofheredity.Usually,thisisequatedwiththeproductionofoneRNAoroneprotein.Agenecontainscodingregions,introns,untranslatedregionsandcontrolregions内含子:aregionthatinterruptsthetranscribedpartofagene.Anintronistranscribed,butisremovedbysplicingduringmaturationofthetranscript.ThewordreferstotheinterveningsequenceinboththeDNAanditsRNAproduct.外显子:aregionofagenethatisultimatelyrepresentedinthatgene’smaturetranscript.ThewordreferstoboththeDNAanditsRNAproduct.RNA剪切：RemovethenoncodingsequencesfromprimaryRNAtranscript.选择性剪切：produceofmanydifferentproteinsfromonegene.从一个mRNA前体中通过不同的剪切方式产生不同mRNA剪接异构体的过程，而最终的蛋白质也会不同。非编码序列：NoncodingsequencescontaininformationthatdoesnotleadtothesynthesisofanactiveRNAmoleculeorprotein.编码序列：CodingsequencesgiverisetoallofthetranscribedRNAsofthecell,includingmRNA.RNA编辑：RNAediting:thetermRNAeditingisusedtodescribethepost-transcriptionalalterationofgenesequencesbydiversemechanismsincludingthedeletion,insertionandmodificationofnucleotides.向导RNA(gRNAs):smallRNAsthatbindtoregionsofanmRNAprecursorandserveastemplateforeditingaregionupstream.CpG岛：aregionofDNAcontainingmanyunmethylatedCpGsequences.Usuallyassociatedwithactivegenes.点突变：Apointmutationisasimplechangeinonebaseofthegenesequence.删除突变：MutationsthatresultinmissingDNAarecalleddeletions.插入突变：MutationsthatresultintheadditionofextraDNAarecalledinsertions.框移突变：inaframe-shiftmutation,oneormorebasesareinsertedordeleted,theequivalentofaddingorremovingletterinasentence.沉默突变：amutationthatdonotaffectthestructureofthegeneproduct.it'spossiblebecausemostaminoacidscanbeencodedbymorethanonetriplctcodon.无意突变：involvenucleotidebasesubstitutionsthatmodifyatripletcodonthatnormalencodesforanaminoacidintoatranslationalstopcondon.错意突变：missensemutationinvolvenucleotidebasesubstimtionthatalterthetranslationoftheaffectedcodontripletSNP:isthemostcommonformofgeneticvariation,asimplechangeofabasepairinthehumangenome.南blotisatechniquefordetectionofspecificsequencesamongDNAfragmentscombineseparationbygelelectrophoresisandhybridizationwithacomplementarylabeledDNAprobe.北blotisatechniquefordetectionofspecificsequencesamongRNAfragmentscombineseparationbygelelectrophoresisandhybridizationwithacomplementarylabeledDNAprobe.有机提取：Awaytoextrationofnucleicacid.ItisimportantthatthephenolPHliewithinarangeof7.8~8.0topreventnucleicacidfromremainingintheorganicphase.Akeystepintheorganicextrationisemulsificationoforganicandaqueousphases.无机提取：inorganictechniquesreduceexposuretohazardousreagentswhileproducingpurifiednucleicacidofomparablequality.Manycommercialkitsemployinrorganicpurificationmethods,includingsaltprecipitation,absorptiontosilicasurfaces,andanion-exchangechromatographyprotocols.PCR:isamoleculartechnologythatfacilitatestheamplificationofrarecopiesofspecificnucleicacidsequencestoproduceaquantityofamplifiedproductthatcanbeanalyzed.热启动PCR：toachieveatemperaturegreaterthantheannealingtemperatureofprimerspriortothemixingofthereactioncomponents.TouchdownPCR:isanothermodificationofconventionalPCRthatmayresultinareductionofnonspecificamplification.ItinvolvestheuseofanannealingtemperaturethatishigherthanthetargetoptimuminearlyPCRcycles.巢式PCR:meansthattwopairsofPCRprimerswereusedforasinglelocus.ThefirstpairamplifiedthelocusasseeninanyPCRexperiment.Thesecondpairofprimers(nestedprimers)bindwithinthefirstPCRproductandproduceasecondPCRproductthatwillbeshorterthanthefirstone.实时PCR:QF-PCRisatechniqueusedtomonitortheprogressofaPCRreactioninrealtime.ItusesfluorophorestodetectlevelsofDNAandarelativelysmallamountofPCRproductcanbequantified.TaqMan探针：utilizethe5'exonucleaseactivityoftheenzymeTaqPolymeraseformeasuringtheamountoftargetsequencesinthesamples.分子信标：molecularbeaconsaresinglestrandedhairpinshapedoligonucleotideprobes.FRET探针：areapairoffluorescentprobesplacedincloseproximity.Fluorophoresaresochosenthattheemissionspectrumofoneoverlapssignificantlywiththeexcitationspectrumoftheother.蝎状引物：Scorpionprimersarebi-functionalmoleculescontainingaPCRprimercovalentlylinkedtoaprobe.ARMS:Theamplificationrefractorymutationsystem(ARMS)exploitsthisbyutilizingaprimerthatisdesignedsuchthatits3′endmatches,forexample,oneoftwoalternativesatamutantnucleotide.ARMSisideallysuitedtothedetectionofpointmutationsandsmallinsertions/deletions引物延伸：以DNA单链为模版，在特定的条件下，根据碱基互补配对原则，不断将四种dNTP的5'端与引物3'端相连生成磷酸二酯键，使引物得到延伸。SSCP:单链构象多态性分析，在聚丙烯酰胺凝胶上，短的单链DNA和RNA分子依其碱基序列不同形成不同的构象，一个碱基得到改变将影响其构象而导致其在凝胶上的移动的速度的改变HA:杂交双链分析，由突变型和野生型DNA形成的异源杂合双链DNA在其错配出形成一个凸起，在非变性胶中电泳时，会产生与相应的同源双链DNA不同的迁移率，从而使两者被分离。DGGE:变