The Principles Behind Pyrosequencing

May 23, 2010

Pyrosequencing is a technique to sequence DNA, thus to identify the base pairs of DNA, based on the synthesis of a complementary strand. In contrast to the well-known Sanger sequencing approach not the determination of the complementary strand synthesis leads to information about the bases, but the synthesis itself coupled to the amount of free pyrophosphate. The method was developed by Pål Nyrén and his student Mostafa Ronaghi in Stockholm in 1996.

As with Sanger sequencing a DNA polymerase and a primer molecule are used in order to build up a new DNA strand based on the already existing complementary strand. This process is “watched” and consequently used to sequence the present DNA molecule. In addition one of the four existing nucleoside triphosphates (NTPs) is added, followed by another one (after usage and/or degradation of the first one) and so on. The synthesis process of the newly formed strand, thus the integration of the fitting NTP, can be made visible with the help of a complex enzyme system and a light detector because a small light flash is emitted through an enzymatic cascade when the fitting nucleotide is inserted on the strand. If a non-fitting nucleotide is added, there is no enzymatic reaction and no detectable light flash occurs. After this reaction the remaining NTPs, which were not used, are degraded by enzymes. The following NTP is added. If it fits the measurable light flash occurs again. The light flash occurs at the latest after the fourth addition of NTPs because then all possible pairing options have been used. The intensity of the occurring light flash accounts for the amount of NTPs of one sort that were utilized. That means, for example, that the flash of three used adenine NTPs is linearly more intense than the use of one adenine NTPs. Knowledge of the intensity of the emitted light (displayed on the Y-axis) and the used NTP (displayed on the X-axis) per run consequently leads to the sequence of the DNA.

The light emitting enzymatic reaction is based on the following principles: When a complementary NTP is inserted on the DNA strand a pyrophosphate (PPi) is released from the used NTP. PPi then reacts to ATP (the biologically most important energy containing molecule) with help of the enzyme ATP-sulfurylase. The ATP molecule is then used to power the luciferase-reaction. Within this reaction the luciferin molecule is transformed into oxyluciferin. The intensity of the detectable emitted light flash during this chemical reaction is proportional to the first produced and then used amount of ATP molecules.

Pyrosequencing is a process that can be highly automated and is therefore very useful for the frequent and fast analysis of DNA samples. This is especially of high value when unique gene mutations within the DNA, called Single Nucleotide Polymorphisms (SNPs) (which are not necessarily negative), have to be analyzed. For example genetically inherited diseases can be detected using this sequencing approach.

Figure displaying the principles behind the technique of pyrosequencing 1.

An excellent page about the technique of pyrosequencing you can find at

1. Quaigen, “The principle of Pyrosequencing technology,” 2010,


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