Real-time PCR theory and...

Real-time PCR theory and applications

By Dalia M. Sabri, PhD

Lab Manager , BRC, SCU

Biotechnology Research Center, Suez Canal University, 11-11-2015

* PCR History

* What is PCR?

* Endpoint PCR Vs Real-time PCR

* Real-time PCR

* QPCR Detection Chemistry

* Real-time PCR Instruments

* Methods of Quantification

* New technologies

PCR History 1985 PCR first published

Late1980’s PCR used for quantification, but required electrophoresis to detect product at

end-point of PCR

1991 Use of 5’-> 3’ exonuclease activity of Taq polymerase to detect specific PCR

activity (“TaqMan” approach).

1992 Discovery that EtBr (dsDNA probe) can be added to the PCR mix and that the

fluorescence of EtBr will increase at each cycle of PCR due to product formation.

No need to cool to room temperature or to open tubes to measure the increased

EtBr fluorescence. First real-time experiments – in closed PCR tubes on UV-

trans-illuminator. Higuchi and others at Roche and Chiron .

1993 First real-time PCR detection experiments to show utility for DNA quantitation.

Improved EtBr detection (illumination, CCD camera detection). Higuchi and

others at Roche .

1996 TaqMan detection methods used, instead of EtBr, for real-time detection of PCR.

Improved specificity. Heid and others at ABI and Genentec.

1996-7 ABI introduces first real-time qPCR (“Sequence Detection System”) instrument

(the ABI 7700).

Since then Many more instrument manufacturers (Roche, BioRad, Stratagene, Corbett,

Cepheid, MJ) and many more detection chemistries.

What is PCR?

* The Polymerase Chain Reaction (PCR) is a revolutionary technology

developed by Kary Mullis in the 1980s. PCR is based on using the

ability of DNA polymerase to synthesize new strand of DNA

complementary to the offered template strand, that results in generating

thousands to millions of copies of a particular DNA sequence from a

single copy or a few copies of a piece of DNA .

*The method relies on thermal cycling, consisting

of cycles of repeated heating and cooling of the

reaction for DNA melting and enzmatic replication

of the DNA.

The three-step PCR process

1. Denaturation step (Separating the Target DNA)

DNA template is heated to more than 90 ºC, which separates the double-

stranded DNA into two separate single strands.


2. Annealing step (Binding Primers to the DNA Sequence)

The reaction is cooled between 40 and 60 ºC. PCR oligonucleotides bind

(anneal) to sequences on either side of the target DNA region, marking off

the sequence for step three


3. Extension step (Making a Copy)

The temperature is increased to 72 ºC, nucleotides in the solution are added to the

annealed primers by the DNA polymerase to create a new strand of DNA

complementary to each of the single template strands. After completing the

extension, two identical copies of the original DNA have been made.

PCR amplification curve

• Exponential

- Exact doubling of product is accumulating at every cycle.

- The reaction is very specific and precise.

- All of the reagents are fresh and available, the kinetics of the reaction push the reaction to favor doubling of amplicon.

• Linear (high variability)

- As the reaction progresses, some of the reagents are being consumed in amplification process.

- The reactions start to slow down and the PCR product is no longer being doubled at each cycle.

• Plateau (End-point)

-The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade.

Endpoint PCR Vs Real-time PCR


Endpoint PCR Real-time PCR

Measures the amount of

accumulated PCR product at the

end of the PCR cycles

Measures PCR amplification as it

occurs

Reaction endpoint

data collection

Ct



Semi-quantitative

Though comparing the intensity of

the amplified band on a gel to standards of a known

concentration.

Quantitative

Data collection during the exponential (log) phase makes the

quantity of the PCR product is directly proportional to the amount

of template nucleic acid.



Applications

Amplification of DNA for:

Sequencing

Genotyping

Cloning

Applications

Gene expression quantification

Microarray verification

Quality control and assay validation

Pathogen detection

SNP genotyping

Copy number variation

MicroRNA Analysis

Viral quantification



Disadvantages:

- Low sensitivity.

- Post-PCR processing.

- Low resolution and Poor Precision.

-Non-automated.

-Size-based discrimination only &

results are not expressed as numbers.

-Ethidium bromide for staining is not very

quantitative.

Advantages:

- Increased dynamic range of detection

- No post PCR processing.

- Detection is capable down to a 2-fold

change.

- Collects data in the exponential growth

phase of PCR.

-An increase in reporter fluorescent signal is

directly proportional to the number of

amplicons generated.

-The cleaved probe provides a record for

amplified amplicons.

Real-time PCR

Real-time PCR *Real-time PCR combines PCR amplification and detection into a

single step.

*With Real-time PCR, fluorescent dyes are used to label PCR

products during thermal cycling with the capacity to illuminate

each sample with a beam of light of at least one specified

wavelength .

*Real-time PCR instruments measure the accumulation of

fluorescent signal emitted by the excited fluorophore.

QPCR Detection Chemistry

1- Non-specific detection (DNA binding dyes): SYBR Green, BEBO, BOXTO, EvaGreen.

2- Specific detection (Probe based test): TaqMan, Molecular Beacons, Hybridization Probes (Scorpions, Amplifluor Primers, LUX Primers, FRET probe pairs).

SYBR Green I SYBR Green I dye binds to double-

stranded DNA and emits fluorescence

only when bound, DNA-dye-complex

absorbs blue light (λ= 497 nm) and emits

green light (λ=520 nm). The stain can also

binds to single-stranded DNA and RNA

but with lower performance.

SYBR Green I SYBR Green I dye binds to double-

stranded DNA and emits fluorescence

only when bound, DNA-dye-complex

absorbs blue light (λ= 497 nm) and emits

green light (λ=520 nm). The stain can also

binds to single-stranded DNA and RNA

but with lower performance.

*Advantages:

1- Low cost assay.

2-Easy to design and set up.

*Disadvantages:

1-Non specific system as it detects all ds

DNA , in poorly designed PCR reaction

primer-dimer product will be detected

and quantified

2-Not adapted to multiplex .

TaqMan probe-based assay TaqMan probes consist of a fluorophore

reporter attached to the 5’-end of

the oligonucleotide probe and a quencher at the

3’-end. The quencher molecule quenches the

fluorescence emitted by the fluorophore when

excited by the cycler’s light source.







1-

2-

3-

4-







Advantages:

1- very specific

2- Can be used in multiplex qPCR assays

Disadvantages:

1- difficult to design

2- More expensive than SYBR Green

assay

Applications: *Gene expression assays

*Determine the viral load in clinical specimens

*Bacterial Identification assays

*SNP genotyping

*Verification of microarray results

1-

2-

3-

4-

Real-time PCR Instruments

Any Real-time PCR Instrument consists of two main components


Thermal Cycler

(PCR machine)

Optical module ( to detect fluorescence in

the tubes during the run)

1- Halogen lamp

2- Filter /detector :

(5 dyes detectors)

FAM/SYBR Green I, VIC/JOE,

NED/TAMRA/Cy3,

ROX/Texas Red, Cy5

3- CCD camera


Methods of Quantification Absolute Quantification Relative Quantification

Quantification of unknown samples based on a known quantity. First you create a standard curve; then you compare unknowns to the standard curve and extrapolate a value.

This method analyze changes in gene expression in a given sample relative to another reference sample (such as an untreated control sample).

Ct (cycle threshold) is defined as the number of cycles required

for the fluorescent signal to cross the threshold (exceeds

background level).

Threshold is the point of detection.

Cycle-Threshold (Ct) cycle at which sample crosses threshold

Rn

Cycle

Melting curve: The melting curve analysis is based on the Tm, in which half of the DNA molecules are

in the form of single strand and the other half in the form of a double helix.

The Tm is dependent on DNA composition, since each amplified fragment has a specific

size and base composition, they can be identified by melting curve analysis.

FIGURE : Melting curve analysis for HBV genotyping.

A (68 bp), D (119 bp) and F (97 bp)

Ref. : BECKER, Carlos Eduardo et al . MELTING CURVE ANALYSIS FOR THE SCREENING OF HEPATITIS B VIRUS GENOTYPES A, D AND

F IN PATIENTS FROM A GENERAL HOSPITAL IN SOUTHERN BRAZIL. Arq. Gastroenterol., São Paulo , v. 50, n. 3, p. 219-225, Sept. 2013 .

Why we should collect results during exponential phase?

Plateau effect

Rn

Exponential Phase

Real-time PCR Applications

New technologies

Digital PCR Digital PCR works by partitioning a sample into many individual real-

time PCR reactions; some portion of these reactions contain the target

molecule (positive) while others do not (negative). Following PCR

analysis, the fraction of negative answers is used to generate an absolute

answer for the exact number of target molecules in the sample, without

reference to standards or endogenous controls.

Digital PCR QX100™ Droplet Digital

Bio-Rad

(A)

Samples partitioning

into 20,000 nanoliter-

sized droplets

(B)

PCR on a thermal cycler

(C)

The detector reads each

droplet on turn at the

rate 1000 droplet per sec

• No need to rely on references or standards

• Ability to increase precision by using more PCR replicates

• High tolerance to inhibitors

• Capability to analyze complex mixtures

• Linear detection of small-fold changes

Digital PCR Advantages

Digital PCR Applications

• Copy number variation with unrivaled accuracy.

• Rare sequence detection with unmatched sensitivity.

• Mutation detection.

• Gene expression analysis with the highest precision.

• miRNA analysis.

• Next generation sequencing sample quantification.

PCR is a tool

It is your decision how to use it!!?

Real-time PCR theory and...

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