PCR & qPCR Thermal Cycler
Discover our wide product variety of high-performance PCR as well as highly efficient real-time PCR thermal cyclers for DNA amplification and quantification.
Our PCR and real-time PCR thermal cyclers are also available as fully automated systems for robotic setups.
Throughout the entire lifetime of your instruments, we support you with our application consulting, temperature calibration, maintenance and qualification services.
PCR and qPCR explained
Since the Corona pandemic, the abbreviation PCR has been on everyone's lips. PCR stands for Polymerase Chain Reaction. Today, PCR is a standard method for detecting nucleic acids - primarily DNA or RNA - in samples of a wide variety of starting materials. For this purpose, the nucleic acids are amplified by constant heating and cooling. The method is highly sensitive and can detect minute amounts of genetic material. PCR has undergone continuous development since its invention in the 1970s and 1980s. Real-time PCR, for example, can now display the progress of the amplification in real time, and quantitative real-time PCR, called qPCR, can even quantify the proportions of amplified DNA or RNA.
Nucleic acids and their differences
Nucleic acids are generally understood to be the genetic material of all organisms - animals, plants, fungi, bacteria or viruses. They are macromolecules consisting of nucleotides, each of which is composed of a phosphate group, a sugar molecule and a base. Nucleotides are the main building blocks of the nucleic acids deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA and RNA are the carriers of an organism’s hereditary information. The differences between these nucleic acids are mainly in the type of sugar molecule, the organic bases, and the molecular structure. Both use a pentose - a sugar molecule with five carbon atoms - called ribose. However, the ribose of DNA has only one hydrogen atom at the second carbon atom, while RNA has a hydroxyl group (hydrogen-oxygen group) at this position. This small difference has a significant effect on the stability of the DNA and RNA strands. As a result, RNA is easier to split. RNA also uses the base Uracil instead of the base Thymine, as DNA does. The most obvious difference is in the structure of the molecule. DNA is a double helix, a coiled double strand. RNA, on the other hand, is a single strand. The double helix structure of DNA is caused by a complementary base pairing of Guanine with Cytosine and Adenine with Thymine. Although the bases of RNA can also form bonds with each other, this occurs rather rarely. RNA is chemically more reactive due to its more variable structure and can form more bonds with other molecules.
Both nucleic acids play an important role in protein biosynthesis. DNA stores hereditary information as a code via the various combinations of its base pairs. Replication multiplies this information and passes it on to new cells. The information from the DNA serves as a template and is transferred into an RNA sequence (transcription), which then triggers the production of proteins and enzymes in the ribosomes (translation). This type of RNA is called messenger RNA (mRNA). There are other types of RNA - such as tRNA - that perform different functions in the cell but do not encode genetic information. There are also RNA viruses whose entire genome consists of one RNA strand.
Why nucleic acids are detected via PCR
The genetic information is the genetic fingerprint of every organism. This fingerprint can be clearly detected using PCR. Even the smallest traces or scraps of DNA and RNA can be discovered. This makes PCR an effective method for diagnosing viruses, bacteria or genetic diseases. The method is also used in genealogy or forensics - for example, in parentage reports or to identify DNA from perpetrators. PCR is also used to analyze the origin and contents of foodstuffs e.g., the identification of allergens. Also, plant or animal ingredients often can have different genetic information in different regions. DNA is also amplified industrially to obtain proteins for drugs or other medical applications. This is also often done using PCR applications.
PCR and qPCR - amplification and quantification
The methodology of PCR is fundamentally based on natural replication, i.e., the amplification of genetic material in cells. This process is artificially replicated with the help of thermocyclers. PCR amplifies a precisely defined gene sequence by systematic heating and cooling. This process is called amplification. In a first step, the DNA is heated up to 96°C and denatured, i.e., the bonds between the double strands are broken and it disintegrates into single strands. This is followed by primer binding (annealing). Primer DNA attaches to both sides of the gene sequence and serves as a marker for the DNA fragment to be amplified. The temperature is reduced to 55 to 65°C for this purpose. Now follows, at temperatures around 70°C, the elongation phase, in which new chains of DNA are synthesized. Since synthesis usually stops at some point, this is followed by another denaturation step to accumulate more primers, and then a new synthesis of DNA to form more DNA chains. The process is repeated until a sufficient amount of DNA is produced. To detect RNA, laboratories use reverse transcriptase-polymerase chain reaction (RT-PCR), in which the target RNA is first transcribed into a complementary DNA sequence and then amplified.
Quantitative real-time PCR (qPCR) is an advancement of the PCR method. Whereas classical PCR only allows statements to be made after amplification as to whether a particular gene sequence has been amplified or not, qPCR checks the amount of amplified DNA after each amplification cycle. In this way, the course of amplification can also be analyzed in real time. A fluorescence method is usually used for this purpose. A special fluorescent dye attaches itself to the DNA strands. Modern qPCR thermal cyclers such as the qTOWER³ from Analytik Jena use an additional detector to quantify the dye in order to measure the fluorescence and thus the course of the amplification cycles.
Analogous to standard PCR, qPCR can also be used for the diagnosis of pathogens, in food monitoring, forensic medicine or pharmaceutical production. However, thanks to its quantification function, the range of applications is larger, including many emerging research disciplines. For example, qPCR can be applied to quantify gene expression (how a particular gene appears in an organism). Standard PCR methods are too unreliable for this. qPCR thermal cyclers are also used in next generation sequencing (NGS). NGS is a collective term for new, very fast methods for sequencing genetic material. In many areas of NGS, the quantification of DNA and RNA plays an important role. qPCR is used, for example, for various NGS preparation steps, such as the creation of DNA libraries (library preparation). In addition, qPCR enables quantification and genotyping, i.e., characterization of the strain of viruses, for example, in clinical diagnostics. Many viruses often enter the human body through indirect infection or co-infection, which can lead to misdiagnosis. The number of viruses, for example in the case of certain herpes viruses, can provide information on whether a previously inactive virus has broken out. Quantification via qPCR methods creates a more solid data basis for diagnoses.
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