Appunti di Principi-BiolMol CTF - Capitolo3 - Omero

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ABI SOLiD (Seq by Oligo Ligation/Detection)• Clonal bead library via emulsion PCR.• The actual base detection is no longer done by the polymerasedrivenincorporation of labeled <strong>di</strong>deoxy terminators.• SOLiD uses a mixture of labeled oligonucleotides and queriesthe input strand with ligase.• Each base is interrogated twice:– built-in error checking capability that <strong>di</strong>stinguishes betweenmeasurement errors and true polymorphisms;– detection of more complicated variations.
Overview of ABI SOLiD SequencingChemistry• Library Preparation Prepare one of the twotypes of libraries for SOLiD Systemsequencing-fragment or mate-paired. Yourchoice of library depends on the applicationyou're performing and the information youdesire from your experiments.
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Impieghi della PCR• Strategie di
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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
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SYBR green Metodica semplicePossono
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Sonde TaqMan La sonda di tipo TaqMa
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ForwardProbeReverse
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Reporter-QuencherDyeQuencher5,6 FAM
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Real-Time PCR:attività 5’>3’ e
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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 and 116: • Step 1A sequencing primer is hy
Page 117 and 118: • Step 3ATP sulfurylase converts
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: Illumina sequencing technology• S
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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