Multiplex PCR

The multiplex PCR is a form of application of diagnostic PCR . Originally, PCR was mainly used for amplifying (duplicating) DNA for sequencing (determining the base sequence) of the amplified DNA. With the progress of sequencing projects, it became possible to use PCR as a diagnostic method. With the knowledge of the base sequence in the genome of an organism, it became possible to determine the specific sequences for this organism or the sequence changes associated with a disease. A diagnostic PCR is then developed on these specific sequences. Such a PCR for the detection of a section in the genome is also referred to as singleplex PCR or single PCR. For many diseases, however, different viruses or bacteria or genomic changes can be the cause. Therefore, to determine the cause of the disease, it is necessary to perform several singleplex PCR tests. Multiplex PCR is useful under these conditions.

principle

In a multiplex PCR, the individual PCR processes are no longer carried out in separate PCR reactions, but all together in one PCR reaction. This reduces the effort involved in determining the cause of the disease, especially the cost and time. A multiplex PCR is a PCR approach with the potential to detect more than one genome segment. The development of a multiplex PCR is associated with increased effort. Due to the increased number of primers and probes (when designed as real-time PCR) interactions between these oligonucleotides are much more likely and also occur much more frequently in reality. These interactions would reduce the sensitivity of the multiplex PCR and must therefore be ruled out.

was standing

Under real conditions it is very difficult to completely exclude these interactions between the oligonucleotides, which is why sensitive multiplex PCR applications are currently mainly duplex PCR. In turn, the detection of the genome sequence to be determined is very often combined with an internal amplification control. An amplification control is required for many diagnostic PCR applications in order to rule out false negative results. False negative results would arise if the DNA to be examined contained inhibitors for the PCR detection. In reality, these are mainly inhibitors for the enzymatic activity of Taq polymerase, such as iron ions from red blood cells . The amplification control is a PCR detection that detects the absence of inhibitors for the PCR. For this purpose, a second PCR reaction is constructed and a small amount of the target DNA for this PCR is added to the PCR mixture. If the result for the detection of the genome sequence to be determined is negative, this amplification control must be positive in order to confirm the absence of inhibitors and thus the correctness of the negative result. Although duplex PCR reactions are the most widespread, methods with up to four PCR detections per batch are no longer uncommon.

Note

From a marketing point of view, some companies sell detection kits as multiplex PCR, which contain several separate PCR approaches. This is not really a multiplex PCR.

outlook

Because of the large number of real diagnostic problems with a large number of possible causes, it can be assumed that multiplex PCR methods will continue to spread. Examples include such important areas as sepsis analysis, the diagnosis of respiratory diseases, cancer diagnostics, the detection of genetically modified organisms ( GMOs ) or the cause of diarrheal diseases. A further increase in the number of multiplexes and detection using real-time PCR is to be expected.

Web links

• Protocols for Multiplex PCR
• Multiplex PCR software
• Utah University Multiplex TM software

literature

• G. Zangenberg, RK Saiki, R. Reynolds: PCR Applications: Protocols for Functional Genomics . Ed .: Michael A. Innis, David H. Gelfand, John J. Sninsky. Academic Press, San Diego 1999, ISBN 978-0-08-091963-8 , Chapter: Multiplex PCR: Optimization Guidelines ( limited preview in Google Book Search).