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Real-Time PCR in diagnostics

Interesting facts about the polymerase chain reaction (PCR) in molecular biology.

PCR - Polymerase Chain Reaction

The polymerase chain reaction [1]; [2] (PCR) is one of the most important techniques in molecular biology and was discovered in 1983 by Kary B. Mullis, for which he received the Nobel Prize in Chemistry in 1993. It is based on an in-vitro procedure that allows the specific amplification of certain DNA sequences within a very short time. This method has become indispensable in the most diverse areas of biological research and medical diagnostics.

Which variants of the PCR exist?

Diversity of PCR technology

Due to the many applications, different variants [2] of the PCR technology have been developed.
A selection of these optimizations is listed below:

Real-time PCR (quantitativ)

The quantitative PCR method enables the quantitative determination of the amount of amplified genetic material in real time by using fluorescence-labelled DNA oligonucleotides. Quantification is performed by fluorescence signals that are either non-specifically generated by dyes intercalating into double-stranded DNA (e.g. SYBR Green) or by hydrolysis probes (e.g. TaqMan) that bind specifically to DNA single strands.

Reverse Transcriptase PCR

In addition to the actual DNA amplification, this further development of classical PCR initially includes a reverse transcription step in which RNA is transcribed into complementary DNA by reverse transcriptase. This DNA is then amplified in a second step.

If both steps take place in a single reaction vessel, this is called a "one-step" process - if both reactions are separated by two consecutive steps, this is called a "two-step" technology. The form of PCR is used for the detection of RNA viruses and for expression studies.

Multiplex PCR

A very powerful method is the multiplex PCR, which allows the detection of different target sequences within a single PCR approach. The differentiation of the products can be done either by the size of the resulting DNA molecules or by using specific hydrolysis probes with different fluorophores.

Sequence-specific PCR

Sequence-specific PCR can detect single nucleotide polymorphisms. This is possible by the use of primers or probes which specifically attach themselves with their 3' end only to the target polymorphism.

„Hot start“ PCR

The "hot start" PCR is carried out using a specially modified polymerase enzyme, which is inhibited e.g. by the binding of a specific antibody and needs to be activated by heating to 95°C. This modification increases specificity and DNA yield by reducing unspecific DNA amplification at the beginning of a PCR.

Specific real-time PCR (quantitative)

The starting material for the amplification of DNA by real-time PCR is double-stranded DNA molecules present in the blood or mucous membranes of an organism. With this method, the amplification and specific detection of the resulting PCR products is carried out in real time and fully automated in a thermal cycler. [3]-[5]

The required components are:

  • free nucleotides,
  • sequence-specific primers,
  • thermostable polymerase with exonuclease activity,
  • sequence-specific TaqMan probe,
  • and genetic sample material in the form of double-stranded DNA.

Workflow

Before the start of the PCR reaction, the TaqMan probe, which later enables specific detection via fluorescence, is intact.

In this state, due to the spatial proximity of the reporter fluorophore (R) to a quencher (Q) between these molecules, a process known as Förster resonance energy transfer (FRET) takes place, which leads to the suppression of the fluorescence signal of the reporter fluorophore when it is excited.

In general, a PCR protocol consists of three basic steps: denaturation, annealing and extension.

Denaturation

The first step of a PCR is the "melting" of the DNA double strands into single strands by increasing the temperature. This process usually takes place at 90 - 97 °C. A high GC content in the target DNA strand affects the required denaturation time and requires higher temperatures.

Annealing

In this step, the reaction temperature is lowered so that the probes and primers can complement each other by attaching themselves to the DNA single strands or hybridize with them. The fluorescence signal of the reporter fluorophore of the TaqMan probe is further suppressed by the spatial proximity to the quencher and the resulting quenching effect.

Extension

During this PCR step the  reaction temperature is adjusted to the activity optimum of the polymerase, which binds to the 3' end of the hybridized primers and adds free nucleotides complementary to the present DNA strand. If the TaqMan probe has specifically bound and the polymerase with 5'-3' exonuclease activity meets the 5' end of the TaqMan probe, the probe is cleaved (hydrolyzed) in the presence of water. This leads to a spatial separation of reporter fluorophore (R) and quencher (Q), which cancels the quenching effect. Excitation of the reporter now leads to light emission, which can be detected by the detection unit of the thermal cycler.

Reproduction

The unique sequence of denaturation, annealing and extension is called a cycle in which a DNA double strand is doubled under optimal reaction conditions. A PCR protocol usually comprises up to 40 cycles.

Fluorescence emission

The fluorescence emission of the reporter fluorophore excited by a light source correlates quantitatively with the amount of amplification produced and is plotted in real time by the thermal cycler as an amplification curve. In the exponential phase, the PCR products double as described above, whereby a corresponding increase in the fluorescence signal is only detectable when the background signal is exceeded.

The cycle in which the fluorescence signal of the reaction exceeds a threshold value is called threshold cycle*. The threshold value can either be set at a fixed value, such as 200 relative fluorescence units (RFE/ RFU), or it can be calculated automatically by software. The doubling of the amplification products from cycle to cycle corresponds to 100% efficiency and is influenced by various factors after a certain time.

PCR curve

Primers or free nucleotides are no longer present in excess and the DNA polymerase loses activity, inhibiting metabolites may be formed and/or denaturation becomes more inefficient. This finally leads to the plateau phase, in which amplification is almost complete.

Advantages of our qPCR products

We mix various PCR innovations to develop above all safe, efficient and user-friendly diagnostic products.

  • powerful through multiplexing
  • reliable results through internal amplification control
  • low DNA concentrations sufficient for analysis
  • short analysis times
  • simple setup
  • high automation level
  • Real-time monitoring
  • no working with toxic gels and no other toxic waste
  • one step procedure for reverse transcriptase PCR
  • user-friendly software solutions for evaluation

Indications for use in diagnostics

The methodology of qPCR is used in routine diagnostics7 , during the night shift and in the research field for a wide variety of questions:

Genotypisierung

What is your blood type?

  • Transfusion Diagnostics

Are donor and recipient compatible in organ transplantation?

  • Transplantation Diagnostics

Are there genetic predispositions for a disease?

  • Human Genetics

Basic research

Where, when and under what conditions does the expression of genes occur?

  • Expression analyses

Viral diagnostics

Is there a viral disease?

  • Hepatitis A virus
  • herpes simplex virus
  • COVID-19

bibliography

1 Mullis, K. B. (1990): The unusual origin of the polymerase chain reaction. In: Scientific American 262 (4), 56-61, 64-5. DOI: 10.1038/scientificamerican0490-56.

2 Maheaswari, Rajendran; Kshirsagar, Jaishree Tukaram; Lavanya, Nallasivam (2016): Polymerase chain reaction: A molecular diagnostic tool in periodontology. In: Journal of Indian Society of Periodontology 20 (2), S. 128–135. DOI: 10.4103/0972-124X.176391.

3 Applied Biosystems®, life technologies™: Real-time PCR handbook. Basics of real-time PCR. In:. Online verfügbar unter https://www.thermofisher.com/, zuletzt geprüft am 13.05.2020.

4 Joshi, Mohini; Deshpande, J. D. (2011): POLYMERASE CHAIN REACTION: METHODS, PRINCIPLES AND APPLICATION. In: Int Jour of Biomed Res 2 (1). DOI: 10.7439/ijbr.v2i1.83.

5 Calculations for Molecular Biology and Biotechnology (2016): Elsevier.

6 Deepak, S. A.; Kottapalli, K. R.; Rakwal, R.; Oros, G.; Rangappa, K. S.; Iwahashi, H. et al. (2007): Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes. In: Current Genomics 8 (4), S. 234–251.

7 Holzapfel, Bianca; Wickert, Lucia (2007): Die quantitative Real-Time-PCR (qRT-PCR). Methoden und Anwendungsgebiete. In: Biol. Unserer Zeit 37 (2), S. 120–126. DOI: 10.1002/biuz.200610332.

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