Real Time PCR and the icycler iq Real Time PCR Detection System for Quantitative PCR

Transcription

1 eal Time PC and the icycler iq eal Time PC Detection System for Quantitative PC

2 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

3 PC Numbers Cycle elative Amount

4 La reazione Primers d. NTPs Thermal Stable DNA Polymerase Addizionare al tubo di reazione Master Mix contenuto Annealing Denaturare 95 C

5 La reazione Estensione Estensione continua ipetere per n cicli

6 La reazione Cycle 2 4 Copie Cycle 3 8 Copie

7 PC Amplification Curve La crescita esponenziale è limitata Effetto plateaus Teorica Log Target DNA eale Threshould Cycle #

8 Comparazione dei risultati ad end point. Con gel e eal Time Lockey etal. (1998) Biotechniques 24:744-6

9 eal Time Vs End Point 9,048 9,498 10,180 9,238 9,111 12,885 10,539

10 Misurazione End Point? elative Fluorescence

11 96 epliche di una identica reazione, mostrano differenti efficienze al termine della reazione

12 Threshold Cycle, C t, dei 96 replicati mostra un identico valore

13 Threshold Cycle (C T )? Threshold Baseline Sample C T

14 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.

15 40 Threshold Cycle, C t, è un indicatore del numero di copie iniziali Copy Number vs. C t - Standard Curve y = x = C t Log of copy number (10 n )

16 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

17 Intercalating Dyes Primers eaction Tube d. NTPs Intercalation Dyes Thermal Stable DNA Polymerase Add Master Mix and Sample λ Annealing ID Denaturation

18 Intercalating Dyes Extension ID ID ID ID ID ID λ λ λ ID ID ID ID Extension Continued Apply Excitation Wavelength λ λ epeat

19 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

20 man Probe Before enzyme cleavage After enzyme cleavage Q Q Forster type energy transfer

21 Man TM Primers eaction Tube d. NTPs Thermal Stable DNA Polymerase Probe Q Add Master Mix and Sample λ Annealing Q Denaturation

22 Man TM Q Q Extension Step 1. Strand Displacement Q 2. Cleavage Q Q λ 3. Polymerization Complete 4. Detection

23 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.

24 Cleavable Hydrolysis Probe - Multiplex C t = Single Multiplex FAM Factor 8 Single C t = Multiplex Texas ed IL-1 beta

25 Cleavable Hydrolysis Probe - Multiplex Log elative Fluorescence Tubulin GAPDH beta Actin Cyclophilin Hex Texas ed FAM Cy Color/4 Probe

26 Cleavable Hydrolysis Probe - Multiplex Threshold Cycle Cycle Tubulin GAPDH beta Actin Cyclophilin 1.00E E E E E E E+08 Concentration Concentration Tubulin r 2 = y = -3.50x GAPDH r 2 = y = -3.28x b-actin r 2 = y = -2.88x Cyclophilin r 2 = y = -4.68x Target GAPDH Cyclophilin Tubulin b-actin eporter HEX Cy5 FAM Texas ed Quencher DABCYL Black Hole DABCYL Black Hole

27 Cleavable Hydrolysis Probe - Multiplex Log elative Fluorescence Tubulin Cyclophilin beta Actin GAPDH Texas ed FAM Hex Cy Cycle

28 Cleavable Hydrolysis Probe - Multiplex Tubulin Cyclophilin beta Actin GAPDH Tubulin r 2 = y = -3.36x Threshold Cycle Cyclophilin r 2 = y = -3.65x b-actin r 2 = y = -3.82x GAPDH r 2 = y = -3.93x E E E E E+06 Concentration

29 Cleavable Hydrolysis Probe - Multiplex FAM/Tubulin

30 Cleavable Hydrolysis Probe - Multiplex HEX/Cyclophilin

31 Cleavable Hydrolysis Probe - Multiplex Tex ed/b-actin

32 Cleavable Hydrolysis Probe - Multiplex Cy5/GAPDH

33 Molecular Beacons Primers eaction Tube d. NTPs Thermal Stable DNA Polymerase Molecular Beacon Q Add Master Mix and Sample Q Denaturation Annealing

34 Molecular Beacons λ Q Q Detection Extension Step 1. Strand Displacement Molecular Beacon Q 2. Polymerization Complete Probe Silent

35 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).

36 FET Probes λ D 1-5 bases D Detection Extension Step 1. Strand Displacement System Silent D 2. Polymerization Complete System Silent

37 Primer Based FET λ Heat Incorporation Q λ Q

38 Scorpions Primer This sequence is complementary to a sequence of the amplified fragment λ Q 3

39 Scorpions Primer after the Extention The tail of the Scorpion is complementary to a sequence of the amplified fragment Q λ 3

40 Amplifluor Primer Q Annealing/Extension 1 Q Extension 2 Q λ Detection

41 Simple folded light path