Appunti di Principi-BiolMol CTF - Capitolo3 - Omero

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• Step 2The first deoxribonucleotide triphosphate (dNTP) is addedto the reaction. DNA polymerase catalyzes theincorporation of the dNTP into the DNA strand, if it iscomplementary to the base in the template strand. Eachincorporation event is accompanied by release ofpyrophosphate (PPi) in a quantity equimolar to theamount of incorporated nucleotide.
• Step 3ATP sulfurylase converts PPi to ATP in the presence of adenosine 5'phosphosulfate (APS). This ATP drives the luciferase-me<strong>di</strong>atedconversion of luciferin to oxyluciferin that generates visible light inamounts that are proportional to the amount of ATP. The lightproduced in the luciferase-catalyzed reaction is detected by a chargecoupled device (CCD) chip and seen as a peak in the raw data output(Pyrogram). The height of each peak (light signal) is proportional tothe number of nucleotides incorporated.
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Impieghi della PCR• Strategie di
Page 5 and 6:
TemperaturePCR100Melting94 o C500T
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TemperaturePCR10050Melting94 o CAnn
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TemperaturePCR10050Melting94 o CAnn
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Dopo n cicli il miscuglio di reazio
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Fasi della PCR Denaturazione (melti
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• * Si possono usare al posto del
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Nested PCR
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Hot Start PCR
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Sintesi del cDNA
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• L’elettroforesi su gel dei pr
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IMPIEGHI DELLA PCRDiagnosi infezion
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Test geneticoAmplificazionemediante
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PCR-ARMS (Amplification RefractoryM
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PCR-ARMS (Amplification RefractoryM
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PCR-SSCP (Single Strand Conformatio
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PCR-HAD (HeteroDuplex Analysis)•
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PCR-HAD (HeteroDuplex Analysis)
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PCR-DGGE (Denaturation Gradient Gel
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PCR-DGGE (Denaturation Gradient Gel
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Resa della PCRResa effettiva: effet
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Soluzioni per PCR quantitativa Util
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Perché Real-Time?• Misura l'ampl
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Analisi tramite software
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SYBR greenUtilizzauna molecola fluo
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SYBR green Dopo l’annealing dei p
Page 65 and 66: SYBR green Metodica semplicePossono
Page 67: Sonde TaqMan La sonda di tipo TaqMa
Page 71 and 72: ForwardProbeReverse
Page 73 and 74: Reporter-QuencherDyeQuencher5,6 FAM
Page 75 and 76: Real-Time PCR:attività 5’>3’ e
Page 77 and 78: Real-time PCR: reagenti COMPONENTI
Page 79 and 80: Curve di amplificazioneFluorescenza
Page 81 and 82: QuantificazioneASSOLUTA i campioni
Page 83 and 84: Quantificazione relativaPlot di amp
Page 85 and 86: Sonde FRET(FluorescenceResonanceEne
Page 87 and 88: Molecular Beacons Durante lo step d
Page 89 and 90: Probes : ScorpionsStep 1Lo Scorpion
Page 91 and 92: Step 3Probes : ScorpionsRaffreddand
Page 93 and 94: Real-Time PCR: applicazioni‣ Quan
Page 95 and 96: Analisi della sequenzanucleotidica
Page 97 and 98: ddNTP
Page 99 and 100: • Si suddivide quindi la miscela
Page 101 and 102: ddCTPddCTPddCTPPRIMERDNA Polimerasi
Page 103 and 104: • Le diverse catene polinucleotid
Page 105 and 106: Metodo di sequenziamentodel DNA “
Page 107 and 108: Sequenziamento automatizzato con ma
Page 109 and 110: Le emissioni fluorescenti vengono c
Page 111 and 112: Metodo di Sequenziamento di Maxame
Page 113 and 114: Un altro metodo per la determinazio
Page 115: • Step 1A sequencing primer is hy
Page 119: • Step 5Addition of dNTPs is perf
Page 123 and 124: What is Next-Generation Sequencing?
Page 125 and 126: Illumina sequencing technology• L
Page 127 and 128: Illumina:Sequencing-bysynthesis
Page 129 and 130: Illumina sequencing technology• S
Page 131 and 132: Overview of ABI SOLiD SequencingChe
Page 133 and 134: Bead DepositionDeposit 3’ modifie
Page 136 and 137: SOLiD TechnologyLigation-basedchemi
Page 138 and 139: Exact Call ChemistryUp to 99.99% ac
Page 140 and 141: Latest Platforms:Illumina HiSeq:•
Page 142: Campi di applicazione delle piattaf
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Appunti di Principi-BiolMol CTF - Capitolo3 - Omero

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