Our methods

Our methods

Our analytical methods have a modular structure and can be combined in a beneficial manner. 

Choose one of our services to learn more about the method.

Our mission is to answer your questions.

The basic principle of PCR

The polymerase chain reaction (PCR) is the most fundamental and important working method of IDENTXX.

Four components are required to successfully carry out a PCR

PCR procedure

Qualitative PCR detection

For qualitative detection, also referred to as end-point PCR, the enzyme reaction is first carried out completely. After the end of the reaction, the products formed are separated according to size in an agarose gel and made visible using fluorescent dyes.

Example

Specific endpoint PCR detection of Peronospora belbahrii

The example shows an agarose gel of a detection of Peronospora belbahrii
downy mildew of basil from seeds. Next to the
Green marker (L) is the positive control (+) followed by the negative control (-).
In the lane of the positive control you can see a clearly white glowing area,
which is also known as ?gang? referred to as. This is the stained specific PCR product. In the lane of sample 1, a band can also be seen at the same height. The samples are therefore assessed as positive. Samples 2 and 3 show no bands and are therefore evaluated as negative.

Our qualitative PCR detections are safe and robust, and are used in the detection of a wide variety of pathogens.

Gel image Xap Test 3

Quantitative PCR detection

Another important PCR method is quantitative PCR (qPCR), also known as real-time PCR. It follows the same reaction principles as qualitative PCR. However, the reaction solution is supplemented by another component, the so-called probe. This is located between the primers and is also specific for the DNA section sought.

The qPCR principle

If a PCR reaction now takes place, the polymerase enzyme breaks down the probe. A fluorescent dye (fluorophore) released in the process can be detected in real time during these degradation processes. The more PCR products are formed, the more fluorophore is released. The thermal cycler displays these measured values in the form of a curve. The earlier there is an exponential rise in the curve, the more DNA was present in the starting solution.

Multiplex analyses

There is another advantage to using probes. By using different fluorophores, we can carry out up to 5 PCR reactions in one reaction tube (multiplex analyses). This makes it possible to compare the content of specific pathogen DNA (shown in blue in the upper curve) to the DNA of the host plant (shown in green).

This approach has several advantages:

DNA sequencing

Sanger sequencing

We also used endpoint PCR as a baseline reaction for DNA sequencing, distinguishing between Sanger sequencing and Pyrosequencing. Two important analysis tools that we used for different analytical purposes.

DNA sequencing using the didesoxy method (Sanger sequencing)

This method was developed by Frederick Sanger and Alan Coulson in the 1970s and forms the basis of modern molecular biology alongside the PCR reaction. The method is as simple as it is ingenious. Special oligonucleotides marked with fluorescent dyes are added to the finished endpoint PCR reaction solution. In a subsequently carried out PCR reaction, DNA fragments of different lengths are obtained, each of which carries a fluorescently labeled oligonucleotide at the end, which can be evaluated with the aid of appropriate detectors. The result is a sequence of colored peaks with the corresponding allocation to the four bases of the DNA.

DNA sequencing according to the Sanger method is used for the following analyses:
Pyrosequencing

An endpoint PCR serves as the basis for pyrosequencing. By using a biotinylated primer, one strand of each of the resulting PCR products carries biotin at the end (labeling). This labeled DNA strand can be separated and purified by covalent binding to streptavidin. During the sequencing reaction, the four bases are added separately to the reaction solution. If incorporation takes place, an enzyme reaction-based flash of light is released, which is detected in real time, as in qPCR.

Advantages of pyrosequencing

The key point is the quantification of nucleotide incorporation. This allows the degree of zygosity of a TSR position to be determined precisely.

Pyrosequencing is used in the following analyses:
Pyrosequencing

An endpoint PCR serves as the basis for pyrosequencing. By using a biotinylated primer, one strand of each of the resulting PCR products carries biotin at the end (labeling). Through the covalent binding to streptavidin, the labeled DNA strand can be separated and cleaned. During the sequencing reaction, the four bases are added separately to the reaction solution. If incorporation takes place, an enzyme reaction-based flash of light is released, which is detected in real time, as in qPCR.

Advantages of pyrosequencing

The key point is the quantification of nucleotide incorporation. This allows the degree of zygosity of a TSR position to be determined precisely.

Pyrosequencing is used in the following analyses:

Fragment length analysis

Fragment length analysis

An endpoint PCR serves as the basis for the fragment length analysis. By using a fluorophore-marked primer, each strand of the resulting PCR product carries a fluorescent dye at the end (labeling).
With the aid of high-resolution capillary electrophoresis, which is equipped with a detector unit for fluorescence signals, the lengths of the PCR fragments can be determined down to the nucleotide.

A fragment length analysis is used in the following analyses:

Methylation analyses

Methylation analyses

In order to identify methylated cytosines, the DNA has to be subjected to a bisulfite reaction before end-point PCR. In this reaction, a uracil is incorporated for all cytosines that are not methylated and are directly in front of a guanine base (CpG dinucleotide).

In the subsequent endpoint PCR, the uracil is replaced by thymine, resulting in a base sequence that differs from the original.

The sequence can be determined after the end of the reaction either by the Sanger method or by the Pyro method.

Methylation analysis is used in the following applications:

greenhouse studies

In cooperation with two leading institutions, IDENTXX greenhouse biotest to determine metabolic resistance, also known as nTSR.

The partners have both a modern spray booth and sufficient greenhouse capacity and specialist staff.
The following points are particularly worth mentioning:

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