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Tecniche di amplificazione quantitative, Real-Time PCR. Mauro Pistello Dipartimento Patologia Sperimentale Università di Pisa. F luorescence (F örster) Resonance Energy Transfer. Quencher. Reporter. Laser. Light quenching. 5’. 3’. Light emission. 3’. 5’.

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Presentation Transcript
slide1

Tecniche di amplificazione

quantitative, Real-Time PCR

Mauro Pistello

Dipartimento Patologia Sperimentale

Università di Pisa

slide2

Fluorescence (Förster) Resonance Energy Transfer

Quencher

Reporter

Laser

Light quenching

5’

3’

Light emission

3’

5’

slide4

Heating Block

Optical Fiber

Lens

Cap

Tube

Thermal

Cycler Block

slide5

Fluorescence Resonance Energy Transfer

Quencher

Reporter

Laser

Light quenching

5’

3’

Light emission

3’

5’

slide6

Raw Spectra

Starting cycle

Quencher

Reporter

Reporter

End point

Quencher

slide7

Increment of Fluorescence

Positive

Sample

Fluorescence Intensity

Negative

Control

Quencher

emission

Reporter

emission

Wavelength

slide10

Variability of PCR(96 replicates)

C.V. 20 - 50%

2Rn

Number of Cycles

slide11

Variability of PCR(96 replicates)

2Rn

C.V. 6 - 12%

Number of Cycles

slide15

Efficiency of PCR

E = 10(-1/S) – 1

where

E = PCR efficiency

S = slope

slide16

HBV DNA

E = 0.893

slide17

TTV DNA

E = 0.959

slide19

Taqman PCR (1)

Denaturation

Annealing

R = Reporter

Q = Quencher

  • Polymerization

Q

Q

R

5’

5’

3’

5’

3’

5’

slide20

Taqman PCR (2)

. Cleavage

R = Reporter

Q = Quencher

R

Q

Q

5’

5’

3’

5’

3’

5’

slide21

Scorpions

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

slide24

Molecular Beacons

Double-dye probe with a stem-loop structure that changes its conformation when the probe hybridizes to the target

slide25

Hybridization 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

slide26

Dye-alone

a

Double stranded DNA intercalating dyes

(e.g. SYBR GreenTM 1)

b

c

slide27

Primer-dimer results from extension of one primer using the other one as template, even though no stable annealing between primers is possible

Primer 1

5’

3’

Primer 2

Once such an extension occurs, primer-dimer is amplified with high efficiency

slide28

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

  • Yield of fluorescence at “plateau” in the growth curve
  • Tm analysis of the DNA products

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.

Rate of increase in fluorescence

Temp

slide29

Factors for Optimal Probe Performance

  • 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
slide31

Advantages 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
slide33

Disadvantages 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
the dna microarray process
The DNA Microarray Process

Technological needs for DNA microarrays

slide37

Capture Molecules

for Protein Arrays

slide38

Potential Virus Targets for Blood Testing Chips

a No disease association.

Petrik, Vox Sanguinis 2001 (mod.)