Appunti di Principi-BiolMol CTF - Capitolo3 - Omero

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Latest Platforms:Illumina HiSeq:• ~1 billion clusters• 30x coverage of two human genomesin a single run• ~10K per sample?• 1 x 35bp: ~1.5 days, ~30Gb• 2 x 50bp: ~4 days, 75-100Gb• 2 x 100bp: ~8 days, 150-200GbSOLiD 5500xl:• With microbeads or nanobeads• 20-45 Gb/day• 12 lanes• Similar run times as HiSeq• Up to 180-300Gb per run
Rapid Decrease in Cost•The Human GenomeProject: 13 years and $3billion.• Sequencing of the WatsonGenome by 454 in 2007:$2 million• Illumina: eight days at acost of about $10,000.• ~104 reduction in 5 yrs• Claims: a genome in 15minutes for $1000?
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Impieghi della PCR• Strategie di
Page 5 and 6:
TemperaturePCR100Melting94 o C500T
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TemperaturePCR10050Melting94 o CAnn
Page 9 and 10:
TemperaturePCR10050Melting94 o CAnn
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Dopo n cicli il miscuglio di reazio
Page 14 and 15:
Fasi della PCR Denaturazione (melti
Page 16 and 17:
• * Si possono usare al posto del
Page 18 and 19:
Nested PCR
Page 20:
Hot Start PCR
Page 23:
Sintesi del cDNA
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• L’elettroforesi su gel dei pr
Page 30 and 31:
IMPIEGHI DELLA PCRDiagnosi infezion
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Test geneticoAmplificazionemediante
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PCR-ARMS (Amplification RefractoryM
Page 37 and 38:
PCR-ARMS (Amplification RefractoryM
Page 39 and 40:
PCR-SSCP (Single Strand Conformatio
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Page 43 and 44:
Page 45 and 46:
PCR-HAD (HeteroDuplex Analysis)•
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PCR-HAD (HeteroDuplex Analysis)
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PCR-DGGE (Denaturation Gradient Gel
Page 51 and 52:
PCR-DGGE (Denaturation Gradient Gel
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Resa della PCRResa effettiva: effet
Page 55 and 56:
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
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Curve di amplificazioneFluorescenza
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QuantificazioneASSOLUTA i campioni
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Quantificazione relativaPlot di amp
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Sonde FRET(FluorescenceResonanceEne
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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 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 142: Campi di applicazione delle piattaf
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Appunti di Principi-BiolMol CTF - Capitolo3 - Omero

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