Multiplexing Chem is Tries

8/2/2019 Multiplexing Chem is Tries

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Real-Time PCR

Optimization of Single and MultiplexReactions on the iCycler iQ System



Real-Time PCR

Through the use of fluorescentmolecules, real-time PCR has the

ability to directly measure thereaction while amplification istaking place.



How fluorescence works

Fluorescence is the emission of light at longerwavelengths in response to excitation of light atshorter wavelengths

Every fluorophore has a particular wavelength that is mostefficient at exciting it.



Real-Time PCR

These fluorescent molecules canbe

Non-specific DNA binding dyes

SYBR® Green IEthidium Bromide

Specific Hybridization Probes

TaqManTM

molecular beacons

ScorpionsTM /AmplifluorTM

dual-oligo FRET pairs



Intercalating dyes

d.NTPs

Thermal StableDNA Polymerase

Primers Add Master Mixand Sample

Denaturation

Annealing

Reaction Tube

Intercalation Dyes

Taq IDl



Intercalating dyes

Extension

5’ 3’

Extension Continued Apply Excitation

Wavelength

5’ 3’

5’ 3’

Taq

Taq

3’

5’ 3’

Taq

Taq

Repeat

ID ID

ID IDID

ID ID ID

ID ID

l l l

ll



Taqman

Once freed from the quencher the reporter fluorescence can be detected

indicatingamplification has occurred

•A hybridization probe is constructed with a

fluorescent reporter at one end to a nearby quencher.

•The reporter is excited but its emitted fluorescence

is captured by the nearby quencher.

No reporter fluorescence is detected.

Reporter

During theextension step, thepolymerase

encounters theTaqMan probe andchews off the end.

Fluorescent Tag

Target Sequence

Reporter

Quencher

TaqPolymerase



Molecular Beacons

5’

5’

3’

3’

d.NTPs

Thermal StableDNA Polymerase

Primers Add Master Mixand Sample

Annealing

Reaction Tube

Denaturation

R Q

R Q

MolecularBeacon

Taq



Molecular Beacons

5’ 3’

Extension Step5’ 3’

1. Strand DisplacementTaq

2. Polymerization

CompleteProbe Silent

l

R Q

Detection

R Q

5’ 3’

5’

Taq R Q

MolecularBeacon



Primer based

l

3’

R Q

HeatIncorporation

R Ql



FRET Probes

5’ 3’

l

D R

5’

Detection

Extension Step

5’ 3’

1. Strand DisplacementSystem Silent

2. PolymerizationComplete

System Silent

5’ 3’

5’

Taq

D

1-5 bases

Taq R

5’



Reality vs.Theory

Cycle #

L o g

T a r g e

t D N A

Theoretical

Amplification is exponential, but theexponential increase is limited:

Real-Time PCR allows usto „see‟ the exponential

phase so we can

calculate how much we

A linear increasefollows exponential

Real Life

• Eventually plateaus



End Point Measurements

96 replicates of the identical reaction canhave very different final amounts offluorescence



Threshold Cycle, CT

The point at which the fluorescence risesappreciably above background



Threshold Cycle, CT

of the same 96 replicates showsnearly identical values



Threshold Cycle, CT

The least? Which one

has themost?

Correlates strongly with the starting copy

number

Is linear with the log of starting copy number

over at least 6 orders



Threshold Cycle, CT, is areliable indicator of initial copy

number

C o p y N u m b e r v s. C t - St a n d a r d C u r v e

y = -3 .3 1 92 x + 3 9 .7 7 2

R2

= 0 .9 9 6 7

0

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

0 1 2 3 4 5 6 7 8 9 1 0 1 1

L o g o f c o p y n u m b e r (1 0 n )

C t

r = is a measure of how well the actual datafit to the standard curve.

= (explained variation/total variation)

The slope of the standard curve can bedirectly correlated to the efficiency of thereactions:

Efficiency () = [10(-1/slope) ] - 1



Threshold Cycle, CT

Two-fold serial dilutions of human genomic DNA(gDNA) from 125 to 16,000 genomic equivalentswere assayed for b-actin.



Good Lab Technique

Do not underestimate theimportance of using:

screwcap tubes

aerosol-barrier tips

hot-start polymerase

replicates

master mixes

And the golden rule:

Pipet only once into each well



MultiplexingReactionsObjectives

Maximize Efficiency

Equalize Efficiency

Eliminate Cross-Reactivity



Individual Reactions

1. Choose amplicon of 75-150 bp

Design and order primers

2. PCR dilution series with SYBRGreen I

3. Check for specificity

Use Melt Curve then confirm with gel

4. Design and order specific probe

5. Confirm probe behavior over wide

dynamic range (5 or 6 orders)



Testing with SYBR

IL-1b plasmid with SYBR detection

5-fold dilution series: 10,000 to 16copies

No resolution below 2000copies

r = 0.957

= 153%



Melt Curve: what is it?

Programming a slow change in temperatureto determine the temperature at which the

DNA separates into two strands - or “melts”apart

This temperature is referred to as the “Tm” or

melting temperature for that piece of DNA

Tm is a characteristic, not unlike the isoelectricpoint of proteins, and can be used to makeobservations about the sequence of DNAwithout directly sequencing it



Melt CurveFunctionality

Melt Curve can be used for:

Identification of non-specific products

Characterization of molecular beacons

Mutation detection/allelic discrimination



Identify Primer-

Dimers by Melt Curve

10,000 copies

No template control 400 copies

2,000 copies



Primer-Dimers

IL-1b plasmid with SYBR detection collected at 82

Poor resolution below2000 copies

Poor replicates

= 93%



Primer-Dimers:

No template controlsIL-1b

collected at 60 ºCIL-1b

collected at 82 ºC



Primer-Dimers

Same primers with a specific Texas Red pr

Poor resolution below 200

Poor replicates

= 71%



Primer-Dimers

After primer re-design to eliminate primer-dim

r = 0.999

= 91.3%



Before Multiplexing

Objectives

Maximize individual efficiencies

Equalize individual efficiencies

Demonstrate lack of cross

reactivity



Maximizing Individual

Reaction Efficiencies Target:

If templates differ by < 1000-fold >80% efficiency per reaction

If templates differ by > 1000-fold

(or an unknown amount)

>90% efficiency per reaction



Adjusting Efficiency

Model Secondary Structure of

Amplicon at Anneal/Extend

temperature Move Primers to avoid areas of

secondary structure

Model Secondary Structure ofPrimers at Anneal/Extend

temperature

Redesign Primers



Optimizing Primerlocation

Template with secondary structure

Cyclophilin

Target Amplicon

Forward Primer Reverse Primer

1

110



Optimizing Primer location

= 66.3 %

Reverse primer A

Forward Primer

1 110

Reverse PrimerA



Optimizing Primer location

= 95.8 %

Reverse primer B

Forward

Primer1

110

Reverse Primer

B



Equalizing Individual

EfficienciesTarget:

If templates differ by < 1000-fold

10% differences okay

If templates differ by > 1000-fold

(or an unknown amount) 5% differences OK



Unequal Efficiencies

a-tubulin r= 0.988=93.1%

GAPDH r= 0.997=101.8%

b-Actin r = 0.970=122.4%

cyclophilin r = 0.964=63.6%

TargetGAPDH

Cyclophilin

Tubulinb-Actin

ReporterHEXCy5

FAMTexas Red

QuencherDABCYL

Black Hole

DABCYLBlack Hole

10

15

20

25

30 35

40

45

50

1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08



~ Equal Efficiencies

a-tubulin r= 1.000

=95.3%

GAPDH r= 1.000

=96.8%

b-Actin r = 0.998

=91.3%

cyclophilin r = 0.996=99.3%

T h r e s h o l d

C y c l e

10

15

20

25

30

35

40

2 3 4 5 6 7 8

Copy number (10n)



Cross-reactivity

How we screen for it:

Primer Recognition (BLAST)

Why it may occur:

Competition for Enzyme

Competition for dNTPs



Multiplexing GenomicTargets

Cy5 - Factor VIII

single = 26.15 0.09multiplex = 25.96 0.10

FAM - a-Tubulin single = 24.13 0.08multiplex = 24.24 0.08

HEX - GAPDH single = 23.11 0.11multiplex = 22.98 0.14

Texas Red - IL1-b

single = 23.56 0.07multiplex = 23.62 0.11



An Individual FAMReaction

= 101%

r = 0.997

103, 105 and 107 copies1.25 U Taq , 3 mM MgCl2, 0.8 mM dNTPs



FAM in multiplex with TET

103, 105 and 107 copies1.25 U Taq , 3 mM MgCl2, 0.8 mM dNTPs

= 131%

r = 0.982



Individual FAM Reaction

102 - 106 copies1.25 U Taq , 3 mM MgCl2, 0.8 mM dNTPs

= 97.6%

r = 0.998



FAM in multiplex with TET

102 - 106 copies of each template3.5 U Taq , 5 mM MgCl2, 1.8 mM dNTPs

= 95.8%

r = 0.999

Eff f Addi i l



Effect of AdditionalEnzyme, MgCl2 and

dNTPs

Single Reaction

Multiplex Reaction

Single Reaction

Multiplex Reaction

nothing extra added: additional reagents:



Reaction Conditions

Taq enzyme1.25 U/50 ml rxn

dNTPs G

G

A

T

C

A

T

C

G

T

C

A

800 mM

3.0 mM MgCl2 Mg+2

3.5 U/50 ml rxn

1800 mM

5.0 mM

300 nM 150-400 nMprimers

Single Reaction Multiplex Reaction

200 -500 nM 200-500 nMprobes



Our favorite probes

Are quenched with darkquenchers:

Black Hole Quenchers

DABCYL

This helps us obtain a high signal-to-noise ratio



Choosing a Quencher

Quenchers:

BHQ-3

BHQ-2

BHQ-1

QSY-7

TAMRA

Eclipse

DABCYL

Fluorophores:

FAMTET, VIC

HEX

JOE

CY3TAMRA

CY3.5, Redmond RedTexas Red, ROX

CY5 LC640

CY5.5 LC705



Texas Red, ROX BHQ-2BHQ-3DABCYLEclipse

CY3.5, Redmond Red BHQ-2DABCYLEclipse

Choosing a Quencher

Quenchers:Fluorophores:

TAMRA

BHQ-1

QSY-7Eclipse

DABCYLFAMTET, VIC

BHQ-3BHQ-2CY5

CY5.5, LC705

LC640 BHQ-3

HEX

JOE

BHQ-2

BHQ-1

QSY-7

TAMRAEclipse

DABCYL

CY3TAMRA

BHQ-2

QSY-7Eclipse

DABCYL

4 C l M l i l



4-Color MultiplexReaction

Texas Red - b-actin

r = 0.999

Cy5 - a-tubulin

r = 0.999

4 C l M lti l



4-Color MultiplexReaction

HEX - GAPDH

r = 1.000

FAM - cyclophilin

r = 0.999



Final Multiplex ConditionsThe final concentration of each component in the

50 ml reaction (for genomic template) was asfollows:

FAM - a-tubulin: 300 nM probe, 400 nM primers

HEX - GAPDH: 200 nM probe, 300 nM primers Texas Red - IL1-b: 150 nM probe, 150 nM primers Cy5 - Factor VIII: 300 nM probe, 400 nM primers

3.25 Units Platinum

Taq Polymerase, 5 mM MgCl2and 1.8 mM dNTPs

PCR conditions were 3 minutes at 95 oC, followedby 50 cycles of 10 seconds at 95oC, 60 seconds at

55 oC.



Large Concentration

Differences

105 - 102 copies

of GAPDHalone

105 - 102 copiesof GAPDH with109 copies of a-tubulin



Large Concentration

Differences

109 - 106 copies

of GAPDHalone

109 - 106 copiesof GAPDH with109 copies of a-tubulin

Large



LargeConcentration

DifferencesGAPDH

Starting quantityMean Ct S.D. Mean Ct S.D.

109 11.32 0.10 11.32 0.14108 14.51 0.06 14.41 0.14107 17.89 0.11 17.70 0.18

10

6

21.03

0.06 20.99

0.18105 24.21 0.10 24.25 0.19104 27.65 0.24 27.54 0.11103 30.62 0.10 30.46 0.15

102 32.99 0.14 32.91 0.23

alone In multiplex



Buyer beware!

Different dNTP Vendors

FAM dye layer of 2-color experiment

Two different dNTP sources



Buyer beware!

Different Probe Vendors

Identical HEX probes by two differentvendors



Buyer beware!

Probe lot-to-lot Variation

4 different lots of the same probe from thesame vendor



Buyer beware!

Effect of Different Quenchers

Identical HEX probes made by the samevendor with two different quenchers:

Black Hole 2 and TAMRA



Remember...

Real-Time PCR is not‘cookbook chemistry’ -a real-time instrument

will not optimize yourexperiments for you However, once you do

optimize yourreactions, you will getreproducible, accurateresults

Multiplexing Chem is Tries

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