Tecniche di amplificazioneTecniche di amplificazione

quantitative, Real-Time PCRquantitative, Real-Time PCR

Mauro PistelloMauro Pistello

Dipartimento Patologia SperimentaleDipartimento Patologia Sperimentale

Università di PisaUniversità di Pisa

Laser

5’ 3’

Reporter Quencher

5’

3’

FFluorescence (Fluorescence (Förster)örster) Resonance Energy Transfer Resonance Energy Transfer

Light emissionLight emission

Light quenchingLight quenching

Dye Absorbance(nm)

Emission(nm)

Extinction Coefficient

(cm-1M-1)

Cy3 552 570 150000

Cy5 643 667 250000

6-FAM 494 525 83000

Fluorescein 492 520 78000

Joe 520 548 71000

LC Red 640 625 640 110000

Rox 585 605 82000

Tamra 565 580 91000

Tet 521 536 -

Light Absorbance and Emission of Fluorescent DyesLight Absorbance and Emission of Fluorescent Dyes

TAMRA Dye Spectra

Optical Fiber

Lens

Cap

Tube

ThermalCycler Block

Heating Block

Laser

5’ 3’

Reporter Quencher

5’

3’

FFluorescenceluorescence Resonance Energy Transfer Resonance Energy TransferFFluorescenceluorescence Resonance Energy Transfer Resonance Energy Transfer

Light emissionLight emission

Light quenchingLight quenching

Raw SpectraRaw Spectra

Quencher

Starting cycle

Quencher

End pointReporter

Reporter

0

1000

2000

3000

4000

5000

6000

7000

8000

480 500 520 540 560 580 600 620 640 660

wave length (nm)

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1st cycle

5th cycle

10th cycle

15th cycle

20th cycle

25th cycle

30th cycle

35th cycle

40th cycle

45th cycle

50th cycle

PositiveSample

NegativeControl

Flu

ore

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Inte

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In

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Re

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Qu

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WavelengthWavelength

Increment of FluorescenceIncrement of Fluorescence

HBV DNAHBV DNA

Variability of PCRVariability of PCR (96 replicates)(96 replicates)

C.V

. 20

- 5

0%

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 10 20 30 40

Cycle Number

²Rn

Number of Cycles

2R

n

Variability of PCRVariability of PCR (96 replicates)(96 replicates)

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 10 20 30 40

Cycle Number

²Rn

C.V. 6 - 12%

Number of Cycles

2R

n

Threshold Cycle (CThreshold Cycle (CTT))Threshold Cycle (CThreshold Cycle (CTT))

CCTT

Rn

HBV DNAHBV DNA

HBV DNAHBV DNA

Efficiency of PCR Efficiency of PCR

E = 10E = 10(-1/(-1/SS)) – 1 – 1

wherewhere

EE = PCR efficiency = PCR efficiencySS = slope = slope

Slope Amplification Efficiency

-3.60 1.8957 0.8957

-3.50 1.9307 0.9307

-3.40 1.9684 0.9684

-3.30 2.0092 1.0092

-3.20 2.0535 1.0535

-3.10 2.1017 1.1017

-3.00 2.1544 1.1544

HBV DNAHBV DNA

E = 0.893

TTV DNATTV DNA

E = 0.959

Technology Detection System Manufacturer

PCR TaqMan probe ABI, Roche

PCR Scorpion Eurogentec

PCR Hairpin primer Intergen

PCR Molecular Beacon Stratagene

PCR Dye-alone Roche

PCR Hybridization Probes Roche

NASBA Molecular Beacon bioMerieux

Commercial Real-Time SystemsCommercial Real-Time Systems

Taqman PCR (1)Taqman PCR (1)

• PolymerizationPolymerization• PolymerizationPolymerization

5’

5’

3’5’

3’5’

RR

R = Reporter

Q = Quencher

DenaturationDenaturation AnnealingAnnealing

QQQ

5’

5’

3’5’

3’5’

R = Reporter

Q = Quencher

. . CleavageCleavage. . CleavageCleavage

RR QQQ

Taqman PCR (2)Taqman PCR (2)

ScorpionsScorpionsDouble-dye probe held in a hairpin loop configuration by a complementary stem sequence

ScorpionsScorpions

Hairpin PrimersHairpin Primers

Molecular BeaconsMolecular Beacons

Double-dye probe with a stem-loop structure that changes

its conformation when the probe hybridizes to the target

Hybridization ProbesHybridization Probes

1. Probes hybridize in head-to-tail

arrangement

2. The green fluorescent light

emitted by the Fluorescein excites

the LC Red 640 that subsequently

emits a red fluorescent light

Dye-aloneDye-alone

a

b c

Double stranded DNA

intercalating dyes

(e.g. SYBR GreenTM 1)

Primer-dimer results from extension of one primer using

the other one as template, even though no stable

annealing between primers is possible

Primer-dimer results from extension of one primer using

the other one as template, even though no stable

annealing between primers is possible

Once such an extension occurs, primer-dimer is

amplified with high efficiency

5’ 3’

Primer 1

Primer 2

Methods for Confirming Specificity of Target Methods for Confirming Specificity of Target Detection in Dye-alone Real-Time PCRDetection in Dye-alone Real-Time PCR

Yield of fluorescence at “plateau” in the growth curveYield of fluorescence at “plateau” in the growth curve

TTmm analysis of the DNA products analysis of the DNA products

Yield of fluorescence at “plateau” in the growth curveYield of fluorescence at “plateau” in the growth curve

TTmm analysis of the DNA products analysis of the DNA products

Rat

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Temp

Tm, temperature at which

half the DNA is melted or

annealed. It depends on

DNA sequence and can be

determined by heating the

DNA to 95°C and slowly

cooling.

Double strand DNA-

specific dyes intercalate

with annealed DNA.

• Quenching in the intact probe

• Hybridization conditions

• Cleavage of probe/amplimer hybrids

• Length and GC-content of oligonucleotides

• Tm probe at least 5° higher than Tm primers

• Avoid the G nucleotide at the 3’ end

• Avoid secondary structures

Factors for Optimal Probe PerformanceFactors for Optimal Probe Performance

Real-Time NASBAReal-Time NASBA

Advantages of Real-Time AmplificationAdvantages of Real-Time Amplification

• Test results in short time

• Reduced handling, material and labor costs

• Quantitation over a 5-6 log range

• High throughput

• Simultaneous detection of multiple analytes

• Long shelf-life of labeled probes

• Low risk of contamination

Amplicons Content After PCR

AerosolAerosol

PCR reaction volume ( l ) Total copy number 100 1,000,000,000

10 100,000,000

1 10,000,000

0.1 1,000,000

0.01 100,000

0.001 10,000

0.0001 1,000

0.00001 100

0.000001 10

Disadvantages of Real-Time AmplificationDisadvantages of Real-Time Amplification

• Theoretical and real primer and PROBE

performances can be very different

• Assay set up longer than conventional PCR

• High cost of the real-time instruments

• Cost of reagents (patent royalties)

• Cost of probe synthesis

Ruolo dei microarrays Ruolo dei microarrays

in virologia clinicain virologia clinica

Processes Involved in Making and Using an ArrayProcesses Involved in Making and Using an Array

The DNA Microarray ProcessThe DNA Microarray ProcessTechnological needs for DNA

microarrays

Capture MoleculesCapture Moleculesfor Protein Arraysfor Protein Arrays

Target Transfusion Transmitted Mandatory Testing

HBV + +

HIV-1,-2 + +

HCV + +

HTLV-I, -II + +

HAV + (rarely) -

HGV (GBV-C)a + -

TTVa + -

CMV + + (subset)

HHV-8 ? -

Prion nvCJD ? -

Parvovirus B19 + -

Potential Virus Targets for Blood Testing ChipsPotential Virus Targets for Blood Testing Chips

a No disease association.Petrik, Vox Sanguinis 2001 (mod.)

DNA microarray Real-time PCR

Sample preparation time 4-8 h 1.5-2 h

Minimum sample volume 4 x 106 cells

50-100 g RNA

1 to 1 x 104 cells

0.01-100 ng RNA

Turnaround/data generation time 2 days/sample 1.5-3.0 h/plate

Number of samples per run 1 30-40 per 96-well

150-170 per 384-well

Maximum number of targets/sample 500-40,000 4

Cost/sample $ 2000-8000 $ 2-5

DNA Microarrays Versus Real-Time PCRDNA Microarrays Versus Real-Time PCR