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Real-Time PCR!. Fundamentals of Real-Time PCR. What is Real-Time PCR?. Real-Time PCR a specialized technique that allows a PCR reaction to be visualized “in real time” as the reaction progresses. This enables researchers to quantify the amount of DNA in the sample at the start of the reaction!.

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Real-Time PCR!

Fundamentals of Real-Time PCR


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What is Real-Time PCR?

Real-Time PCR a specialized technique that allows a PCR reaction to be visualized “in real time” as the reaction progresses.

This enables researchers to quantify the amount of DNA in the sample at the start of the reaction!


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What is Real-Time PCR used for?

Real-Time PCR has become a cornerstone of molecular biology:

  • Gene expression analysis

    • Cancer research

    • Drug research

  • Disease diagnosis

    • Viral quantification

  • Food testing

    • Percent GMO food

  • Transgenic research

    • Gene copy number


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How does real-time PCR work?

To best understand what real-time PCR is, let’s review how regular PCR works...


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The Polymerase Chain ReactionHow does PCR work??

d.NTPs

Primers

Thermal Stable

DNA Polymerase

Add to Reaction Tube

Denaturation

Annealing


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The Polymerase Chain ReactionHow does PCR work??

Extension

Extension Continued

Repeat


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The Polymerase Chain ReactionHow does PCR work??

Cycle 2

4 Copies

Cycle 3

8 Copies


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Imagining Real-Time PCR

…So that’s how PCR is usually presented.

To understand real-time PCR, let’s imagine ourselves in a PCR reaction tube at cycle number 25…


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Imagining Real-Time PCR

What’s in our tube, at cycle number 25?

A soup of nucleotides, primers, template, amplicons, enzyme, etc.

1,000,000 copies of the amplicon right now.


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Imagining Real-Time PCRHow did we get here?

What was it like last cycle, 24?

Almost exactly the same, except there were only 500,000 copies of the amplicon.

And the cycle before that, 23?

Almost the same, but only 250,000 copies of the amplicon.

And what about cycle 22?

Not a whole lot different. 125,000 copies of the amplicon.


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Imagining Real-Time PCRHow did we get here?

If we were to graph the amount of DNA in our tube, from the start until right now, at cycle 25, the graph would look like this:


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Imagining Real-Time PCRHow did we get here?

?

So, right now we’re at cycle 25 in a soup with 1,000,000 copies of the target.

What’s it going to be like after the next cycle, in cycle 26?


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Imagining Real-Time PCRSo where are we going?

What’s it going to be like after the next cycle, in cycle 26?

Probably there will be 2,000,000 amplicons.

And cycle 27?

Maybe 4,000,000 amplicons.

And at cycle 200?

In theory, there would be 1,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000 amplicons…

Or 10^35 tonnes of DNA…

To put this in perspective, that would be equivalent to the weight of ten billion planets the size of Earth!!!!


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Imagining Real-Time PCRSo where are we going?

A clump of DNA the size of ten billion planets won’t quite fit in our PCR tube anymore.

Realistically, at the chain reaction progresses, it gets exponentially harder to find primers, and nucleotides. And the polymerase is wearing out.

So exponential growth does not go on forever!


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Imagining Real-Time PCRSo where are we going?

If we plot the amount of DNA in our tube going forward from cycle 25, we see that it actually looks like this:


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Imagining Real-Time PCRMeasuringQuantities

How can all this be used to measure DNA quantities??

What if YOU started with FOUR times as much DNA template as I did?

I have 1,000,000 copies at cycle 25.

You have 4,000,000 copies!

So… You had 2,000,000 copies at cycle 24.

And… You had 1,000,000 copies at cycle 23.


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Imagining Real-Time PCRMeasuringQuantities

So… if YOU started with FOUR times as much DNA template as I did…

Then you’d reach 1,000,000 copies exactly TWO cycles earlier than I would!


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Imagining Real-Time PCRMeasuringQuantities

What if YOU started with EIGHT times LESS DNA template than I did?

You’d only have 125,000 copies right now at cycle 25…

…and you’ll have 250,000 at 26, 500,000 at 27, and by cycle 28 you’ll have caught up with 1,000,000 copies!

So… you’d reach 1,000,000 copies exactly THREE cycles later than I would!


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Imagining Real-Time PCRMeasuringQuantities

We describe the position of the lines with a value that represents the cycle number where the trace crosses an arbitrary threshold.

This is called the “Ct Value”.

Ct values are directly related to the starting quantity of DNA, by way of the formula:

Quantity = 2^Ct

Ct Values:

25

23

28


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Imagining Real-Time PCRMeasuringQuantities

There’s a DIRECT relationship between the starting amount of DNA, and the cycle number that you’ll reach an arbitrary number of DNA copies (Ct value).

DNA amount = 2 ^ Cycle Number


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How do We Measure DNA in a PCR Reaction?

We use reagents that fluoresce in the presence of amplified DNA!

Ethidium bromide and SYBR Green I dye are two such reagents.

They bind to double-stranded DNA and emit light when illuminated with a specific wavelength.

SYBR Green I dye fluoresces much more brightly than ethidium.


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How do We Measure DNA in a PCR Reaction?

Extension

Apply Excitation

Wavelength

Repeat


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What Type of Instruments are used with Real-Time PCR?

Real-time PCR instruments consist of TWO main components:

Thermal Cycler (PCR machine)

Optical Module (to detect fluorescence in the tubes during the run)


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What Type of Instruments are used with Real-Time PCR?

An example of such an instrument is the Bio-Rad iQ5 real-time PCR instrument.


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Real-Time PCRActual Data

  • This is some actual data from a recent real-time PCR run.

  • Data like this can easily be generated by preparing a dilution series of DNA.

Color key: Green=100X, Red=10000X, Blue=1000000X , Black=NTC.


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Real-Time PCR – the Concept of MELT CURVES…

  • Melt curves can tell us what products are in a reaction.

RFU vs T

dRFU/dT


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Real-Time PCRThe Concept of MELT CURVES

  • Different amplicons will have different melt peaks.

  • Primer-Dimers will have a very different melt peak.

Color key: Green=100X, Red=10000X, Blue=1000000X , Black=NTC.


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GMO Investigator Kit

Can we extend the

GMO Kit to

demonstrate

Real-Time PCR???


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GMO Investigator Kit contents

  • Bio-Rad certified Non-GMO food

  • InstaGene

  • Master Mix

  • GMO primers

  • Plant PSII primers

  • GMO & PSII positive control DNA

  • PCR MW Ruler

  • DPTPs, microtubes, PCR tubes, foam floats

  • Manual

  • Not Included but required:

  • Thermal cycler

  • Water bath/heat block

  • Electrophoresis Module (agarose, TAE buffer & Fast Blast DNA stain)

  • Electrophoresis equipment & power supply

  • 2-20 ul pipettes & barrier tips


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GMO Investigator Kitin Real-Time

  • To run the GMO Kit in real-time, only two additional items are needed…

  • iQ SYBR Green Supermix

  • A real-time PCR instrument


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Control DNA Dilutions, Plant Primer

GMO Investigator Kitin Real-Time


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Control DNA Dilutions, GMO Primer

GMO Investigator Kitin Real-Time


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GMO Investigator Kitin Real-Time

Spiked Bean Samples

Color key: black=NTC, green=plant primers, red=GMO primers. Curves represent pure DNA and DNA-spiked plant samples.


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A Few Food Samples…

GMO Investigator Kitin Real-Time

Color key: black=NTC, green=plant primers, red=GMO primers. Order of curves left to right = negative control beans, tortilla chips, soy sausage, corn nuts.


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A Few Food Samples, Melt Curve

GMO Investigator Kitin Real-Time

Color key: black=NTC, green=plant primers, red=GMO primers. Order of peak heights (highest to lowest) is negative control bean, tortilla chips, and sausage.


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A Few Food Samples, Gels

GMO Investigator Kitin Real-Time

Key: Lane 1=NTC; 2 = positive control DNA 1:1000; 3 = positive control DNA 1:10,000; 4 = non-GMO beans spiked with #2; 5 = non-GMO beans spiked with #3; 6 = non-GMO beans; 7 = tortilla chips; 8 = corn nuts; 9 = soy sausage. Each DNA sample loaded in pairs, plant primers on left, GMO primers on right. Ladders on end are 1000,750,500 and 200bp. Upper row consists of PCR reactions carried out exactly according to kit instructions using kit reagents. Lower row consists of same with exception of iQ SYBR Green substituted for kit supermix.


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Classroom Results,

Contra Costa College

May 2006

GMO Investigator Kitin Real-Time

Plant

GMO

Color key:

blue = pure GMO DNA,

pink = papaya,

red = corn chips,

green = tofu,

brown = soy flour,

purple = non-GMO beans,

black = no template control.


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Classroom Results,

Contra Costa College

May 2006

GMO Investigator Kitin Real-Time

GMO

Plant


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GMO Investigator Kitin Real-Time

  • To run the GMO Kit in real-time, only two additional items are needed…

  • iQ SYBR Green Supermix

  • A real-time PCR instrument


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GMO Investigator Kit

Quantification

and

Normalization


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Quantification and Normalization

  • First basic underlying principle: every cycle there is a doubling of product.

  • Second basic principle: we do not need to know exact quantities of DNA, instead we will only deal with relative quantities.

  • Third basic principle: we have to have not only a “target” gene but also a “normalizer” gene.

  • Key formula:

    • Quantity = 2 ^ (Cta – Ctb)


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Quantification and Normalization

  • Example: Ct values for the plant gene and GMO gene from different food samples.


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Quantification and Normalization

  • First we’ll compare relative amounts of plant DNA.

  • Using the formula that relates relative quantity to 2^(Cta-Ctb), we can calculate amounts of DNA relative to a single sample.


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Quantification and Normalization

  • Second we’ll determine relative amounts of GMO DNA…

  • We can do the exact same calculations for the Ct values from the GMO genes of the same food samples.


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Quantification and Normalization

  • Now we compare the amount of plant to GMO DNA in each sample…

  • This is called delta-delta-Ct analysis, and is the basis of real-time quantification analysis.


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Summary

Quantification and Normalization

  • First we determined how much plant DNA is in each sample,

  • Second we determined how much GMO DNA is in each sample,

  • Finally we corrected / normalized the GMO DNA against the plant DNA to determine relative amount of GMO for each sample.


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Quantification and Normalization

  • Practice! Try this example.

  • What is the GMO content of product A and product B??


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Quantification and Normalization

  • Results…

  • What is the GMO content of product A and product B??


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Real-TimeDemonstration

Real-Time PCR

Demonstration


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Real-TimeDemonstration

Experimental Protocol

  • Standard curve with serial 10-fold dilutions

  • Two replicates on standard curve

  • Multiple unknown samples

  • 25ul reactions

  • iQ SYBR Green Supermix (2x)

  • 94C 3 min

  • 94C 30 sec

  • 59C 60 sec

  • Goto Step 2 40 Times


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