eal Time PC and the icycler iq eal Time PC Detection System for Quantitative PC
PC FOMULA Correlazione tra la quantità di amplificato e la quantita iniziale Y = X (1 + E) n -1 Y = PC amplificato DNA quantità X = Quantità ditarget DNA prima della PC E = Efficienza di Amplificazione n = numero di cicli
PC Numbers Cycle elative Amount 1 2 2 4 3 8 4 16 5 32 6 64 7 128 8 256 9 512 10 1024 11 2048 12 4096 13 8192 14 16384 15 32768 16 65536 17 131072 18 262144 19 524288 20 1048576 21 2097152 22 4194304 23 8388608 24 16777216 25 33554432 26 67108864 27 134217728 28 268435456 29 536870912 30 1073741824...
La reazione Primers d. NTPs Thermal Stable DNA Polymerase Addizionare al tubo di reazione Master Mix contenuto Annealing Denaturare 95 C
La reazione Estensione Estensione continua ipetere per n cicli
La reazione Cycle 2 4 Copie Cycle 3 8 Copie
PC Amplification Curve La crescita esponenziale è limitata Effetto plateaus Teorica Log Target DNA eale Threshould Cycle #
Comparazione dei risultati ad end point. Con gel e eal Time Lockey etal. (1998) Biotechniques 24:744-6
eal Time Vs End Point 9,048 9,498 10,180 9,238 9,111 12,885 10,539
Misurazione End Point? elative Fluorescence
96 epliche di una identica reazione, mostrano differenti efficienze al termine della reazione
Threshold Cycle, C t, dei 96 replicati mostra un identico valore
Threshold Cycle (C T )? 2 1.6 1.2 Threshold Baseline Sample 0.8 0.4 C T 0 0 10 20 21 30 40
Threshold cycle, C t Detection of 125 genomic equivalents from 250. Two-fold serial dilutions of human genomic DNA (gdna) from 125 to 16,000 genomic equivalents were assayed for β-actin.
40 Threshold Cycle, C t, è un indicatore del numero di copie iniziali Copy Number vs. C t - Standard Curve 35 30 25 y = -3.3192x + 39.772 2 = 0.9967 C t 20 15 10 5 0 0 1 2 3 4 5 6 7 8 9 10 11 Log of copy number (10 n )
Intercalating Dyes Intercalating Dyes are inexpensive compared to hybridization probes (depending on the reactions performed). A dye based strategy allows one to take a big picture - that is - get a general confirmation of amplification. uss Higuchi demonstrated the key principle of eal Time PC using Ethidium Bromide - EtBr fluoresces 25 times more brightly when bound to dsdna SYB Green, a more sensitive intercalating dye is an even more attractive approach SYB Green fluoresces 200 times more brightly when bound to dsdna
Intercalating Dyes Primers eaction Tube d. NTPs Intercalation Dyes Thermal Stable DNA Polymerase Add Master Mix and Sample λ Annealing ID Denaturation
Intercalating Dyes Extension ID ID ID ID ID ID λ λ λ ID ID ID ID Extension Continued Apply Excitation Wavelength λ λ epeat
Hybridization Probes Today Hybridization Probe Strategies fall into three main categories: Cleavage Based Assay - Man Assays Displaceable Probe Assays Molecular Beacons Dual oligo FET probes Probes incorporated directly into the primers Amplifluor Scorpions
man Probe Before enzyme cleavage After enzyme cleavage Q Q Forster type energy transfer
Man TM Primers eaction Tube d. NTPs Thermal Stable DNA Polymerase Probe Q Add Master Mix and Sample λ Annealing Q Denaturation
Man TM Q Q Extension Step 1. Strand Displacement Q 2. Cleavage Q Q λ 3. Polymerization Complete 4. Detection
Man TM Eight log orders of dynamic range. Ten-fold serial dilutions from 10 1 to 10 9 copies of a beta- Actin containing plasmid were prepared and amplified in eal-time using a man designed probe.
Cleavable Hydrolysis Probe - Multiplex C t = 23.672 Single Multiplex FAM Factor 8 Single C t = 23.254 Multiplex Texas ed IL-1 beta
Cleavable Hydrolysis Probe - Multiplex Log elative Fluorescence 1000 100 10 Tubulin GAPDH beta Actin Cyclophilin Hex Texas ed FAM Cy5 1 10 13 4 7 31 34 16 19 22 25 28 37 40 43 46 49 4 Color/4 Probe
Cleavable Hydrolysis Probe - Multiplex Threshold Cycle Cycle 45 40 35 30 25 20 15 10 Tubulin GAPDH beta Actin Cyclophilin 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 Concentration Concentration Tubulin r 2 = 0.988 y = -3.50x + 39.67 GAPDH r 2 = 0.997 y = -3.28x + 37.30 b-actin r 2 = 0.970 y = -2.88x + 33.89 Cyclophilin r 2 = 0.964 y = -4.68x + 49.74 Target GAPDH Cyclophilin Tubulin b-actin eporter HEX Cy5 FAM Texas ed Quencher DABCYL Black Hole DABCYL Black Hole
34 40 46 Cleavable Hydrolysis Probe - Multiplex Log elative Fluorescence 1000 100 Tubulin Cyclophilin beta Actin GAPDH Texas ed FAM Hex Cy5 10 1 7 10 13 4 16 19 22 25 28 31 37 43 49 Cycle
Cleavable Hydrolysis Probe - Multiplex 40 35 Tubulin Cyclophilin beta Actin GAPDH Tubulin r 2 = 0.994 y = -3.36x + 39.42 Threshold Cycle 30 25 20 15 Cyclophilin r 2 = 0.996 y = -3.65x + 38.43 b-actin r 2 = 0.995 y = -3.82x + 38.89 GAPDH r 2 = 0.995 y = -3.93x + 37.64 10 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 Concentration
Cleavable Hydrolysis Probe - Multiplex FAM/Tubulin
Cleavable Hydrolysis Probe - Multiplex HEX/Cyclophilin
Cleavable Hydrolysis Probe - Multiplex Tex ed/b-actin
Cleavable Hydrolysis Probe - Multiplex Cy5/GAPDH
Molecular Beacons Primers eaction Tube d. NTPs Thermal Stable DNA Polymerase Molecular Beacon Q Add Master Mix and Sample Q Denaturation Annealing
Molecular Beacons λ Q Q Detection Extension Step 1. Strand Displacement Molecular Beacon Q 2. Polymerization Complete Probe Silent
Molecular Beacons VIC Labeled FAM Labeled A serial dilution from 1x10 3 to 1x10 8 plasmids copies of IL1-b (ATCC#581768) and beta-actin were assayed with hil1-b man PDA kit (Perkin-Elmer) and hb-actin PDA (Perkin-Elmer).
FET Probes λ D 1-5 bases D Detection Extension Step 1. Strand Displacement System Silent D 2. Polymerization Complete System Silent
Primer Based FET λ Heat Incorporation Q λ Q
Scorpions Primer This sequence is complementary to a sequence of the amplified fragment λ Q 3
Scorpions Primer after the Extention The tail of the Scorpion is complementary to a sequence of the amplified fragment Q λ 3
Amplifluor Primer Q Annealing/Extension 1 Q Extension 2 Q λ Detection
Simple folded light path