Real-time polymerase chain reaction

The real-time quantitative PCR (in short Q-PCR or real-time detection PCR, short RTD- PCR), or quantitative real-time PCR is an amplification method for nucleic acids, which is based on the principle of conventional polymerase chain reaction (PCR), and also allows the quantification of the DNA recovered. The q- PCR is sometimes called ambiguous for reverse transcription followed by q- PCR ( the same approach) as qRT- PCR or RT- qPCR.

Quantitation is carried out by means of fluorescence measurements which are detected during a cycle of PCR in real time ( hence the name " realtime "). The fluorescence increases in proportion to the amount of PCR products. At the end of a run ( which consists of a plurality of cycles ) is carried out to quantify in the exponential phase of the PCR based on the obtained fluorescence signals. Only in the exponential phase of the PCR (which few cycles in a run takes ) is the correct quantification possible because during this phase rule, the optimal reaction conditions. This method thus differs from other quantitative PCR methods (qPCR ), which is subject to the PCR, a quantitative evaluation (eg Competitive PCR), make mostly involving a gel electrophoretic separation of the PCR fragments.

The real-time quantitative PCR can also be used for other purposes, eg to distinguish between homozygous and heterozygous expression.

Sometimes the abbreviation RT - PCR is used for the real -time PCR, but this leads to confusion, because this shortcut is already used for the reverse transcriptase - polymerase chain reaction. The abbreviation qRT -PCR may cause confusion, as well as the quantitative reverse transcriptase - polymerase chain reaction is abbreviated.

• 2.1 Ct or Cp value
• 2.2 Efficiency
• 2.3 Absolute quantification
• 2.4 Relative quantification 2.4.1 calculation with the aid of a standard curve
• 2.4.2 Calculation according to the ΔΔCt method
• 2.4.3 New quantification algorithms
• 2.4.4 reproducibility and comparability of results

Methods

Dyes

The simplest way to quantify PCR products, the use of DNA dyes (such as ethidium bromide or SYBR Green I). These fluorescent dyes are deposited into DNA ( intercalated ) or bind to the minor groove of double-stranded DNA (English minor groove binder ), whereby the fluorescence of these dyes increases. The increase in the colored target DNA, therefore correlates with the increase in fluorescence from cycle to cycle. The measurement takes place in each cycle at the end of elongation.

A disadvantage of this method is the low specificity, as it can not distinguish between different PCR products. Furthermore, no division measurements can be performed. The first disadvantage can be offset by a melting curve analysis is carried out after expiry of PCR, the basis of which the fragment length ( n ) and thus the specificity can be determined.

Wherein a melting curve analysis, the DNA is melted by raising the temperature is slowly increased continuously (50 ° C → 95 ° C). In one specific for the fragment melting temperature of the double-stranded denatured into two single-stranded molecules. In this case, a fluorescent dye is released (for example, SYBR Green I), and registers a change in fluorescence. Since the double-stranded DNA of the specific PCR products having a higher melting point than non-specific primer-dimers formed, a discrimination is possible. The height of the peak of the melting curve are approximate information about the amount of fragment formed.

FRET probes

Another possibility is to take advantage of the Förster resonance energy transfer ( FRET). A donor fluorochrome (Reporter - in connection with the TaqMan probe ) that is excited by a light source, a portion of its energy to a befindliches sufficiently close fluorochrome acceptor (or a dark quencher - in connection with the TaqMan probe ) from. The distance between the acceptor and the donor, so FRET decreases, and thus the fluorescent signal from the acceptor, whereas the increasing of the donor. This method is very complex and expensive, but offers the advantages of high specificity of the assay.

LightCycler probes (including hybridization probes )

The simplest way of the use of FRET for the quantification of nucleic acids is the use of the LightCycler probes. Two, each with a FRET donor and FRET acceptor ( here Reporter) labeled oligonucleotides which bind adjacent to the target sequence, and thus bring the fluorochromes in sufficient proximity for FRET, can be used as probes for the quantitation of PCR products are used. The measurement takes place in each cycle at the end of the annealing phase. Again, a melting curve analysis can follow.

LoopTag probes

The FRET principle also comes at LoopTag probe system used. This system consists of the forward primer, at its 5 ' end a sequence non-specific nucleotide sequence and the fluorescent acceptor is coupled. The other portion, the detection probe ( LoopTag probe) to the sequence- non-specific nucleotide sequence and the donor is coupled to the 3 'end. LoopTag the probe hybridizes to its specific 5'-end of the target sequence and at the 3'- end of the extended specific forward primer. The sequence- non-specific nucleotide sequences of the detection probe, and the forward primer hybridizes with the formation of a probe -primer to form a loop with each strain. The resulting close proximity of the terminal dyes ( donor / probe acceptor / primer) can be the transfer of energy.

TaqMan probes (including hydrolysis probes )

Another commonly used way is to apply the FRET of the probe, at one end with a quencher, is (for example, TAMRA, and FAM) labeled with a reporter fluorescent dye at the other end (Double- Dye oligos TaqMan probe). If the Taq polymerase in addition to the polymerase activity has a 5'-3 ' exonuclease activity, the probe is degraded during the synthesis of the complementary strand at the 5' end to the quencher and fluorophore separate from each other thereby, and a rising reporter fluorescence can be measured. The measurement takes place in each cycle at the end of elongation.

Molecular Beacons

A further possibility of real-time quantification of PCR products by taking advantage of the FRET provides the use of molecular beacons as probes. Molecular beacons are oligonucleotides that are coupled to both a reporter fluorophore and a quencher. Nucleotides at the 5 ' end of the probe which are complementary to the 3' end, so that a characteristic of molecular beacons secondary structure to form. In this as stem -loop ( hairpin ) state designated by the reporter shows his small distance from the quencher no fluorescence. By the addition of the loop region to a complementary DNA sequence, during a PCR cycle the distance between the reporter and quencher is increased. A reporter fluorescence can thus be observed.

Scorpion primer

Scorpion primers are oligonucleotides complex, which combine the properties of real-time PCR probes and PCR primers in a (Uni- Scorpion ) or two molecules (Bi- Scorpion ). Similar to the molecular beacons, they have a characteristic secondary structure with a self-complementary stem region, the ends of which are modified with a reporter fluorophore and a quencher. In addition, these probes carry at the 3 ' end of a PCR primer. During a PCR cycle can be observed by the addition of the loop region to a complementary DNA sequence, and thus increased distance between the quencher and reporter with increasing concentration of a DNA reporter fluorescence.

Lux primers

Lux primers are oligonucleotides labeled with a fluorescent dye whose fluorescence intensity depends on their chemical environment. These primers will be incorporated during the PCR, a DNA, an increase in fluorescence can be observed.

Quantification

Various mathematical models are used for the quantification, with usually a reference gene (e.g., GAPDH, actin, tubulin ) included in the measurement, to perform a relative amount comparison ( relative quantification ). Other, much more sophisticated methods should allow an absolute quantification, in which the exact number of templates present in the sample can be determined.

Ct or Cp value

In the first phase of the amplification of PCR, the template amount is limited and the probability that the template, primer and polymerase meet suboptimal, while in the third stage of amplification, the amount of products (DNA, pyrophosphate, Monophosphatnucleotide ) increases in such a way that it to inhibition by this is, frequent product fragments hybridize with each other, the substrates are consumed slowly and ultimately the polymerases and nucleotides are slowly destroyed by the heat. An exponential and therefore quantifiable increase is found only in the intermediate phase. Exponential remains a PCR at 12 to 400 starting copies for about 30 cycles, at 200-3200 for 25 cycles and, initially at 3200-51200 for a maximum of 20 cycles. To always be able to measure at the beginning of the exponential phase, the Ct value (English Crossing Point) is often (English Cycle Threshold for threshold cycle ) and the Cp value used to describe the cycle at which the fluorescence first time significantly increases above the background fluorescence.

Efficiency

The efficiency can be calculated in different ways, which differ slightly in their result. The simplest way is the following:

Efficiency E can be calculated by the slope m of a standard curve. To this end, a cDNA dilution approach (eg 100 %, 10 %, 1 %, 0.1 %) is used for qPCR, and used the respective Ct values ​​for the graphical structure of the curve. A linear regression line through the curve has the slope -m ( when plotted with increasing DNA concentration ).

A slope of -3.32 m would thus an efficiency of 1 (100% ) indicate that a doubling of the amplified per cycle, a slope of -3.58, an efficiency of 0.9 ( 90%). The formula provides meaningful values ​​below 100 % smaller for slope values ​​than -3.32.

Absolute quantification

Absolute quantification is complex and the results questionable, so this type of quantification is seldom performed. Thus, inter alia, has the efficiency of reverse transcription, which can be between 5 and 95% can be determined, for example by using a known amount of RNA synthesized.

Relative quantification

For this purpose, an internal control is needed. An internal control can be a gene transcript whose signal is used to compensate for variations in the starting amount of RNA used. For housekeeping genes are used, for example. The amount compared with the housekeeping genes is referred to as normalization. Because the overall analysis is based on this signal, the choice of internal control is an important aspect of the experiment.

The ideal internal control is easy to detect, and their expression should not vary during the cell cycle between cell types or in response to the experimental treatment (eg, stress, medications, illness ).

Calculating with the aid of a standard curve

There is a linear inverse proportional relationship between the logarithm of the amount used and the Ct. If the initial amount known, a standard curve can be constructed by plotting the logarithm of the initial amount against the Ct. Due to the linear equation

Can be determined from the standard curve for each unknown sample of the logarithm of the copy number. All samples are normalized by the calculated copy number of the target gene is divided by the number of copies of the internal reference:

Differential expression of two samples relative to each other can be represented as a quotient and gives an n -fold expression:

Calculated by the ΔΔCt method

The differential expression is indicated as n-fold expression using the ΔΔCt value. Important in this process is an equal efficiency of the two involved PCR reactions. The Ct values ​​are here simply subtracted ( ΔCt ), the two ΔCt values ​​of each group (eg, sick / healthy, with / without drug) subtracted from each other ( ΔΔCt value) and in the equation n-fold expression (group A to group B) used = 2 - ΔΔCt.

New quantification algorithms

To improve the accuracy of relative quantification, various, partly based on the ΔΔCt - method approaches have been developed.

Advantages of this new, mostly not yet market-ready methods lie in the detailed analysis and lower variance of the PCR results. In some methods, the efficiency is taken into account in each individual sample approach, allowing a single tube analysis.

Reproducibility and comparability of results

An experiment is worthless if you can not repeat it. For this purpose, it is absolutely necessary to keep the experimental conditions constant. This here is not only the consistency of the assay quality ( primers, probes, polymerase, buffers, etc. ) taken into account, the equipment must meet the requirements. For example, at block systems (96 - or 384 -well ), the so-called the great homogeneity criterion. The assay should be in each well of the block and also run on blocks of identical devices equally well. Furthermore, this uniformity must not vary over time. To make sure whether you can perform with the equipment in the laboratory experiments safe, you should periodically carry out appropriate inspections (some models extremely prone to aging ). This assay many replicas analyzing distributed over the entire block. Clearly: The result should be the same everywhere in the ideal case. Now the experimenter should be aware of the extent to which deviations can also have significant effects. These deviations can be the way not offset by more expensive iterations, as this would be repeated as constant.