Class Note for MCB 411 with Professor Fares at UA 4
Class Note for MCB 411 with Professor Fares at UA 4
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Date Created: 02/06/15
DNA replication DNA repair genetic recombination DNA RNA synthesis ltranscriptiunl RNA 3W3 protein synthesis ltranslationl PROTEIN HzNWmCOOH amino acids Figure 572 Molecular Biology of the Cell 4th Edition Isolating RNA from cells Brmk open cells in the moo or RNAse inhibitors 1 Isolate RNA 1 oligodT RNADNA fragments separated by gel electrophoresis Slots tor Bands visible after samples suitable treatment A f39 Electrode i Butter solution Direction ol movement You isolate all the Total RNA and mRNA in a cell and you run each in a lane of a gel You stain the gel so you can see all the RNAs in each lane What will you see Total RNA m RNA Total RNA mRNA Supposing starting from this gel you want to quotseequot a specific RNA corresponding to a gene you care about How do you think you can do this NORTHERN BLOTS RNADNA fra ments se arated b el electro h0resis Al stack of paper towels unlabeled RNA or DNA nitrocellulose paper labeled RNA or DNA of known sizes as size markers Slots for Bands visible after samples suitable treatment Gel Electrode Buffer s solution if I t a Direction of g t E movement gt 39 a J j C9 o C remove nitrocellulose paper with tightly bound nucleic aci S LABELED PROBE lDl HVBRIDIZED TO SEPARATED DNA in buffer LABELED PROBE HYBRIDIZED TO COMPLEMENTARY DNA BANDS E VISUALIZED BY AUTORADIOGRAPHY posgions labeled labeled bands markers Figure 8727 part 2 of 2 Molecular Biology of the Cell 4th Edition NUCLEIC ACIDS SEPARATED ACCORDING TO SIZE BY AGAROSE GEL ELECTROPHORESIS SEPARATED NUCLEIC ACIDS BLOTTED ONTO NITROCELLULOSE PAPER BY SUCTION OF BUFFER THROUGH GEL AND PAPER Figure 8727 part1 of 2 Molecular Biology of the Cell 4th Edition DNA replication DNA repair genetic lt recombination DNA RNA synthesis transcription RNA 9W3 protein synthesis translation PROTEIN amino acids COOH Probe Can be singlestranded 0r doublestranded DNA How does the probe bind to the RNA run on the gel If probe is double stranded DNA do both strands bind to the RNA Figure 6 2 Molecular Biology of the Cell 4th Edition Two eukaryotic genes A and B B has intr0n Size of A mRNA is 1 kb Size of B mRNA is 3 kb I ITB l IPAml Probes 1 ds 2 ds 3 ds 4 ds 5 ds 6 ds 7 55 Top Strand 8 55 Bottom Strand Draw the results of the Northern blot using each of these probes and assuming that you can only detect mRNA not premRNA E coli chromosome gene b gene c 5000 nucleotide pairs RNAtranscrlpls gene 1 gene a 3 I genef gene 9 3 From The Art of Maui l 995 Garland Publishing Inc gene A gene B IBM 7 lTRANSCRIPTION l TRANSCRIPTION RNA RNA TRANSLATION Figure 643 Molecular Blology of the Cell 4th Edition What information are we getting here A EUCARYOTES cytoplasm nucleus exons introns transcription unit u TRANSCRIPTION primary RNA transcript 539 CAPPING grx gfsucwo Mica ADENYLATION mRNA AAAA mRNA AAAA gt deg adation lTRANSLATION protein 7 Figure 6 21 part I of 2 Molecular Biology of the Cell 4th Edition AI EUCARYOTES What will you see if you probed with a gene that makes an mRNA cytoplasm nucleus introns exons DNA m transcription unit total RNA oligodT TRANSCRIPTION RNA transcript 539 CAPPING glrx iag ucwe RN cap ADENYLATION mRNA AAAA EXPORT mRNA AAAA gt deradation lTRANSLATION protein El Figure 21 pani ol2 Molecular Biology ofthe Cell 4th Edition A EUCARYOTES What if you probed with a tRNA or a rRNA gene cytoplasm nucleus introns exons DNA m transcription unit total RNA oligodT quot TRANSCRIPTION rimaw RNA transcript 5 ngPIESF LICING RNA cap ADENYLATION mRNAh AAAA EXPORT L 39 CAPPING mRNAAAAA gt de lTRANSLATION protein radation Figure 6 21 part 1 of 2 Molecular Biology olthe Cell 4th Edition Al EUCARYOTES If a drug blocks CAPPING 0r PolyA addition What would you see cytoplasm nucleus introns exons DNA lI transcription unit No drug drug primer JTRANSCRIF TION RNA trarit 5 CAPPING RrNA SPLICING mRNA AAAA gt de radation lTRANSLATION Figure 6 21 part 1 of 2 Molecular Biology ofthe Cell 4th Edition Total RNA A EUCARYOTES When you add the drug no protein is made Where is the effect of the drug cytoplasm nucleus introns exons DNA transcription unit No drug drug H TRANSCRIPTION primar RNA transcript 5 CAPPING LENA SPLICING A RNA cap 7 V V mRNA AAAA gt de mutation lTRANSLATION protein Figure 6 21 part I of 2 Molecular Biology of the Cell 4th Edition Total RNA How can you differentiate between these two A EUCARYOTES options cytoplasm nucleus introns axons DNA m transcription unit quot JTRANSCWFTION prImar RNA transcriptquot 1 Measure transcription Runon experiments 2 Block transcription and measure degradation 5 CAPPING RINA SPLICING 3 P Y RNA cap ADENYLATION mRNA AAAA EXPORT mRNA AAAA gt de TRANSLATION protein Figure 6721 part1 ol2 Molecular Biology oithe Cell 4th Edition A EUCAHYOTES cytoplasm nucleus axons introns transcription unit u TRANSCRIPTION prlmar FlNAtranscriptquot E CAPPING EPLICING RNACaP ADENYLATION mRNA AAAA EXPORT lTRANSLATION protein El Figure 641 part1 o12 Molecular Biology olthe Cell 4th Edition mRNA AAAA gt deradation When you add the drug no protein is made Where is the effect of the drug No drug drug oligodT RNA A EUCABYOTES When you add the drug no protein nucleus is made Where is the effect of the drug introns exons cytoplasm Real Time qPCR 0 What is realtime quantitative PCR A PCRbased method to measure the number of copies of a particular DNA fragment in a given sample Amplification products are labeled by a DNA binding dye or probe chemistry that emit uorescent signal When excited The signal strength of the emitted light is directly proportional to the amount of PCR product in the reaction The uorescence intensity is detected and recorded every cycle DNA amplification is monitored as the reaction occurs transcription unit No drug drug LYRANSCRIPTION primar RA trscript v V V V 539 CAPPING ENDAOEPLICING RNAfaP ADENYLATION mRNA AAAA mRNA AAAA gt de radation lTRANSLATION protein Figure 6721 part1 of 2 Molecular Biology olthe Cell 4th Edition Total RNA II LYSE CELLS AND issue PURIFY mRNA 99 brain 39 quot 539 mRNA 339 NMAAAAAAA HYBRIDle WITH POLYtT PRIMER 5 339 AAAAAAA MAKE DNA COPY WITH TITITTr mRNA REVERSE THANSCRIPTASE 3 f WSWWWE 3 CENA DEGRADE RNA WITH RNase H 5 rAAAAAAg SmWAmmg SYNTHESIZE A COM PLE ME NTARY DNA STRAND USING DNA POLYMERAS 39 RNA FRAGMENT ACTS AS PRIMER 3 doublestranded cDNA copy of original mRNA Figure 8734 Molecular Biology oi the Cell 4th Edition IIl HEATTO HYBRIDIZATION SEPARATE OF PRIMERS 1 doublestranded DNA STRANDS DNA polymerase DNA dA SYNTHESIS dGTP FROM dCTP PRIMERs dT39TP l l g STEP 1 STEP 2 FIRST CYCLE STEP 3 Figure 8 39 part 1 of 3 Molecular Biology of the Cell 4th Edition Bl separate DNA strands and anneal primer DNA separate DNA SYI39ItheSIS strands and add primer DNA synthesis F E i 39 t a E as DNA oligonucleotide primers ll m region of 39 doublestranded chromosomal DNA to be amplified I 39 FIRST CYCLE SECOND CYCLE DFOdUCIHQ tWO dOUble tranCIEU producing four doublestranded DNA molecules DNA molecules Figure 8 39 part 2 of 3 Molecular Biology of the Cell 4th Edition separate DNA strands and anneal primer f v quotE us a I a E El 7 77 E E etc E I 3 vi quot39 It i El DNA synthesis THIRD CYCLE producing eight doublestranded DNA molecules Figure 8 39 part 3 of 3 Molecular Biology of the Cell 4th Edition TaqMan Chemistry During PCR Illumination of intact probe Reporter FAM L9 results in FRET no fluorescence 0 Quencher from reporter F U l During PCR probe hybridizes Egg to target sequence R J Probe is displaced by polymerase during extension Reporter cleaved from quencher via 539 gt339 nuclease activity of I polymerase Illumination of tree reporter results in unquenchable I uorescence v I Real Time qPCR vsTraditiona1 PCR Starting 75 7 Tem plate g t 539 PC R g Round 1 2quot 2X m cm AD Fluorescence Tem late p Cycle Realtime anal sis Endpoint analysis PC R EE E Detection and constant Agarose gel for Pm mct h monitoring of ampli cation deteCtiOI l Round 2 products 15 pOSSlble during the i entire run Analysis and quanti cation can be made in the logarithmic phase of the 4X reaction rather than at the end Tem pl ate of the reaction I gt 30 40 more cycles Fluorescence Curve As in traditional PCR amplification during real time qPCR occurs in distinct stages Initial Logarithmic Plateau 0 25 7 3 7 027 2 g Logarithmic airs phase 1 Initial phase a DS Plateau J phase I I I I IVI IV I I r I I I I I I I I I I I I I Fluorescence Curve Initial Phase 0 Product is generated but uorescence remains at background levels Initial phase Fluorescence Curve Logarithmic hase 0 Fluorescence rises above background levels during exponential accumulation of product 0 Fluorescence increases in direct proportion to the amount of amplified product present uzg 39 Logarithmic phase Fluorescence a a l P l m llllllll FIUOI396SC611C6 CUI V6 Plateau Phase No significant increase in DNA quantity or uorescence due to the fact that one or more of the reaction components has been depleted Flllolescence 25 a m llllllll Microarraxs After adding a drug you want to check which genes in the Whole human genome 30000 genes and counting produce more RNA less RNA or do not show a change in expression How would you do it collection of geneespeci c DNA molecules PCR amplification robotic printing39 onto glass slide mRN rom rnRN rorn sample 1 labeled sample 2 labeled wit re with reen uorchrome fluorchrorne HYBRIDIZE WASH l SCAN RED AND GREEN SIGNALS AND COMBINE small region of microarray representing expression of 110 genes from yeast Figure 8762 Molecular Biology of the Cell 4th Edition I39D N MuumPILQM ILJAO wound healing cell cycle cholesterol ene genes biosynthesis genes Figure 8e63 Molecular Biology of the Cell 4th Edition
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