University:
Swansea University
Course:
PM-149 | Genetics, Genomics & Evolution
Academic year:

2023
Views:

436

Pages:

5
Author:

Amal J.

Sangel's dideoxy Termination Method2'3' dideoxy analogeDNA sampleFPDOCH2oBase3'- GAAT CGCTAATGC5' - C TTAA2'11H HPrimersimilarlyIII& IV tube withPDI DNA POI IddctpfddGITPII tobeDeoxyLabelled dATP,T TTP,3'- - GAATTCGICTANTAG-3analog ofdGTP + aCTP5' - CTTAAGCGATdTIPDideoxy analog+(daTTP)of dATP'- Inone3' - GAATTCGCTAATGC -5'(ddATP) tube5' - C TTAAGCGATI3'- GAATTCG CTAATGE5'- CTTAAGCGATTA (terminatddCTP (III tube)+GAATTCG CTAATGG.3' - GAATTCGCTAATGC.CTTAAGE5'- - CTTAAGCGA(termination+GAATTCGCTAATGGddGTPCTTAAGCGATTAC3'-GAAT: T CGCTAATGC-5'+5'- CTTGAATTCGCTAATGGCTTAAAGCGATTA High throughput Sequencing (Next Generation Sequencing)PycosequencingBefore NGSThen attach thefragmentscleavening shortgunadapter (primer sea)viaadapterSephalosebeadoilemulsionPCRATGCTAGC TAGCEmulsiasoilPrimerseq.Beadt DNAottemulsion PCRlinkedvia adapters.load in picotites plate + enzyme beadslightemitcorrect nucleotideadd a dNTP.light doesntwasburing ApyraseemitDNA POI1) Growing DNA fragment + dNTPdNMPpt1 + PPi2) PPi + Adenosine phesphosoljate ATP surjurylase ATP+ solfateATP + locifein to2 Lucif elaseAMP + PPi +oxylocifein+CO2+ LightGeneration of Pycogram.TillingpathmethodGenerationof sequence Gene knock outIdentify the role of thegene in briorganism.20nt flanking seaPrepaingaTarget geneconstruct for discuptionPrimer 2.20ntflankingseaKaoMXPeimel1PCRabresgeneKanMXDisruptionconstractleft& bight borderof taugetgoneSuccesfolly transformedselect GAlE resistanceyeastcan survive inyeast with destructionKanamycin mediumconstruct survive ithemediumSporolation} If the dirupted gene is essentialthe spores will be nonviableKnockout miceHKHSVtk thymidine kinase from Happes simplexGeneReplacementconstructTransformation3ponsibiliti onno recombinationhomdogousnon homologousrecombinationrecombunation0 homologous recombinationpeo(KHSVESCELLPNSGenexEscellDNAotherg enesmutationin goneNomotationingenexselection+ve selection G 14 )2ve selection (Ganciclovir -22Generating knock out niceES cellchromosome from knockout (Brown)blastocystchromosome from Black mouseSurgicallytransfeitomother (Pseudopregnant female - induce2chancesof magary.pregnancy conditionthroughbormones)ChimeicBlackprogenymagenychimaicx blackcurringformarylimesknockout mice.Brown+ Black(x/x - ) Brown NMDA dais of receptor in hipocampesReceptor for learning &memoryknock out NMDA inknockout NMDAhypocampisNconatallearningsmemory defectchdeathmice.LOXP- Cre system - Site specific RecombinationPhage P1LOXbehave as transposible elements.Crerecombinase. (catalase Recombination)Frtsiteflprecombinase.(transposible elementABCDorient in oppositedirection)DCBAInvesionIf transposible elements ale in same direction, the segment willbe deletedCrerecombinase from bacteriophage P1 & FLP from 2e/m plasmicof yeastboth recombinase recognize subpsitesloxPf FRPrespectivelyLoxp x or FRP13bp13b PCentralelement

Sangers dideoxy termination methods in DNA Sequencing

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