Dr. Naglaa Ibrahim Azab

Assistant Professor of Medical Biochemistry

Faculty Of Medicine –Benha University

REAL TIME PCR ………A step forward in medicine

BY

PCR IS AN IN VITRO AMPLIFICATION OF

DNA

•PCR is amplification of DNA in a tube

What to put in the PCR tube??

• Template DNA

DNA

cDNA obtained by reverse

transcription of mRNAOr Cell free DNA

What to put in the PCR tube??

• Template DNA• Pair of primers specific to the DNA segment

to be amplified• Thermostable DNA polymerase • dNTPs• Buffer• Cations required for the activity of the

enzyme usually Mg ions

Steps of PCR• Put PCR tubes in the PCR machine (Thermal cycler)

Thermal cycler heats and cools

the reaction tubes

After many cycles of denaturation , annealing & extention Millions of copies of DNA are obtained

There are two main types of detection method of the DNA amplified products

End-point PCR detection –Occurs when the amplification process is complete and typically involves gel electrophoresis, followed by staining to detect the amplified DNA fragments.

Real-time PCR detection –Allows for the direct detection of PCR products combining the amplification and detection in a single step (detection while the reaction is occurring (Real-Time), so that amplification is monitored Continuously.

End-point PCR detection is – quite time consuming vulnerable to contamination can only give a qualitative or semiquantitative result. Real-time PCR detection is – reduces the time required for PCR amplification and

analysis reduced carry over contamination the result can also be quantified more accurate more sensitive: The sensitivity of this method permits the

reliable detection of small amounts of initial template, while delivering a linear range of up to ten orders of magnitude in copy number

There are three main fluorescence-monitoring

systems for DNA amplification in real time

PCR

(1) Hybridizing probes: FRET (fluorescence resonance energy transfer) PROBES: FRET Probes rely on the transfer of Energy from one fluorescent dye to another.

-Fret probes are : oligonucleotide probes that are• Two separate probes• fluorescently labeled: The

upstream probe has a donor molecule on the 3'- end and the downstream probes has an acceptor molecule on the 5'-end.

• sequence specific: The probes are designed so that they hybridize adjacently to each other on the target sequence and bring the donor and acceptor fluorophores in close proximity . This allows for transfer of energy from the donor to the acceptor fluorophore, which emits a signal of a different wavelength.

• Either the decrease in the fluorescence of the donor or the increase in fluorescence of the acceptor can be detected.

• fluorescence is detected during the annealing phase of PCR and is proportional to the amount of PCR product.

(2) Hydrolysis probes include TaqMan probes molecular beacons scorpions.

They use the fluorogenic 5' exonuclease activity of Taq polymerase to measure the amount of target sequences in DNA samples

The principle of TaqMan real-time PCR : The TaqMan probe is designed to be complementary

to a specific sequence spanned by the PCR primers. The TaqMan probe has a reporter dye at its 5́ end and

a quencher dye at its 3́ end which inhibits fluorescence.

As long as the probe is intact and the reporter and the quencher dyes are in close proximity, no fluorescence signal is emitted due to the quenching effect.

After the annealing of the TaqMan probe and the primers, the primers are extended by the DNA polymerase. As the polymerase reaches the TaqMan probe, it uses its exonuclease activity to remove the probe one nucleotide at the time. This releases the reporter from the proximity of the quencher and allows for the release of a fluorescence signal from the reporter .

Advantages of TaqMan Chemistry Specific hybridization between probe

and target is required to generate fluorescent signal

Probes can be labeled with different, distinguishable reporter dyes, which allows amplification of two distinct sequences in one reaction tube

Post-PCR processing is eliminated, which reduces assay labor and material costs.

Disadvantage of TaqMan Chemistry The primary disadvantage of the TaqMan chemistry is that the synthesis of different probes is required for different sequences.

(3) DNA-binding agents :The cheaper alternative is the double-stranded DNA binding dye chemistry, which quantitates the amplicon production (including non-specific amplification and primer-dimer complex) by the use of a non-sequence specific fluorescent intercalating agent (SYBR-green I or ethidium bromide).

that the PCR specificity is high because nonspecific products and primer-dimers will also contribute to the fluorescent signal if present.

                                                                       

The principle of SYBR Green detection in real-time PCR :

The fluorescent dye SYBR Green is added to the PCR mixture.

SYBR Green is a DNA binding dye that fluoresces strongly when bound to double-stranded DNA.

At the start of the reaction, very little double stranded DNA is present, and so the fluorescent signal detected by the thermocycler is low

As the reaction proceeds and PCR product accumulates, the amount of double-stranded DNA increases and with it the fluorescence signal .

The signal is only detectable during annealing and extension, since the denaturation step contains predominantly single-stranded DNA

Advantages of SYBR Green I Dye • It can be used to monitor the amplification of any double-stranded DNA sequence. • No probe is required, which reduces assay setup and running costs. Disadvantage of SYBR Green I Dye The primary disadvantage of the SYBR Green I dye chemistry is that it may generate false positive signals; i.e., because the SYBR Green I dye binds to any double-stranded DNA, it can also bind to nonspecific double-stranded DNA sequences.

Real-time PCR. (A) Theoretical plot of PCR cycle number against PCR

product amount is depicted. Three phases can be observed for PCRs:

exponential phase, linear phase and plateau phase. (B) shows a

theoretical plot of PCR cycle number against logarithm PCR product

amount. Panel (C) is the output of a serial dilution experiment from an ABI

7000 real-time PCR instrument .

Three phases of the amplification process:Exponential: Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise*((1,2,4,8,16... the quantity of PCR products in exponential phase is in proportion to the quantity of initial template under ideal conditions. It is possible to make the PCR amplification efficiency close to 100% in the exponential phases if the PCR conditions, primer characteristics, template purity, and amplicon lengths are optimal.  * Linear: The reaction components are being consumed, the reaction is slowing, and products are starting to degrade. Plateau: The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade.

The amount of DNA theoretically doubles with every cycle of PCR * after each cycle the amount of DNA is twice what it was before.After one cycle of PCR, the amount of DNA is twice what it was beforeAfter two cycles one has 2x2 times as much, 22

After 3 cycles - 2x2x2 times as much or 23 times as muchAfter 4 cycles 2x2x2x2 times as much or 24 times as much. Thus After n cycles there will be 2n times as much DNA.

The Plateau Effect is caused by:

• Enzyme instability.• Reagents consumption.• Product inhibition. e.g. pyrophosphate

accumulation.

• Competition of re-annealing amplification product with primers.

If we plot these figures in the standard fashion. We cannot detect the amplification in the earlier cycles because the changes do not show up on this scale. Eventually you see the last few cycles of the linear phase (pink) as they rise above the baseline and then the non-linear or plateau phase (red) - Actually this starts somewhat earlier than is shown here. However, if we plot these values on a logarithmic scale, we can see the small differences at earlier cycles. In real time PCR we use both types of graph to examine the data. Note that there is a straight line relationship between the amount of DNA and cycle number when you look on a logarithmic scale. This is because PCR amplification is a exponential reaction.

the reaction can’t go on forever, and it eventually tails off and reaches a plateau phase, as shown by the figures in red.

the results are similar to the theoretical graph except that the transition to the plateau phase is more gradual

same real time PCR trace shown on a logarithmic scale - again it is similar to theoretical curve

• Number of amplified molecules

= Initial number of DNA molecules X (amplification efficiency)n n= number of cycles• The optimum efficiency possible in PCR is two-every PCR , but in reality many PCR parameters influence the PCR efficiency, so the efficiency is constant but often different from two: N=No X (Econst)n

• Only few detectable cycles obey this rule, in the final phase, the exponential curve bends toward a plateau and efficiency becomes variable: N=No X (Evar)n

• Amplification plot in PCR

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