Pyrosequencing is an emerging genotyping method based on allele-specific primer extension (Fig. 2). This real-time sequencing technology comprises a cascade of four enzymatic reactions, yielding a luminometric signal that is proportional to incorporated nucleotides.

The pyrosequencing reaction is performed using a previously amplified target sequence. Prior to the allele-specific reaction, the PCR product should be purified from unincorporated nucleotides and PCR primers because they interfere with subsequent reactions. Generally, one of the PCR primers is biotinylated at the 5'-end to allow immobilization onto streptavidin-coated sepharose or magnetic beads. Following immobilization, the captured DNA template is purified from soluble PCR components and denaturated to obtain single-stranded DNA (ssDNA). Both the immobilized biotinylated and eluted strands can be used for pyrosequencing.

In the pyrosequencing reaction, a ssDNA template with a short annealed sequencing primer is incubated with DNA polymerase, adenosine triphosphate (ATP) sulfurylase, luci-ferase, and apyrase, and the substrates, adenosine 5' phosphosulfate (APS) and luciferin. The four deoxynucleotide triphosphates (dNTPs) are added to the mixture in a defined order. If the added nucleotide is complementary to the base in the template strand, the DNA polymerase catalyzes the incorporation of the nucleotide, and pyrophosphate will be released. The new pyrophosphate will then be converted to ATP by an ATP sulfurylase. In the next step, luciferase mediates the conversion of luciferin to oxyluciferin using the previously generated ATP. The light emitted as a result of this reaction can be detected, where the signal corresponds to the number of nucleotides incorporated. The unincorporated dNTPs and excess ATP will be degraded by apyrase. If the added nucleotide is not complementary to the DNA template, it will not be incorporated, and no signal will be

Figure 2 Pyrosequencing. Abbreviations: PPi, pyrophosphate; ATP, adenosine triphosphate.

generated. When degradation is complete, the primer strand is elongated by sequential addition of the different dNTPs, followed by degradation of excess nucleotides by apyrase.

Current available pyrosequencing instruments utilize 96-well and 384-well plate formats that facilitate analysis of between 5.000 and 50.000 SNPs per day. This technique offers high accuracy, flexibility in primer positioning, and real-time determination of more than 50 bp along the target sequence that allows analysis within 10 minutes. Contrary to other sequencing methods, it circumvents time-consuming electrophoresis and size separation. Sequencing also provides information on the adjacent nucleotides. Thus, one main advantage of this method is the detection of additional insertions and deletions and also novel polymorphisms across the DNA template.

One of the major drawbacks is the time-consuming template preparation due to immobilization and generation of ssDNA and also the use of target-specific biotinylated PCR primers. An enzymatic template preparation scheme has been developed that avoids labeled primers and the use of any solid support, thereby, reducing costs and simplifying automation of template preparation. Direct use of double-stranded DNA (dsDNA), instead of ssDNA, has simplified the template preparation step prior to performing an analysis using pyrosequencing. Recent developments enable single-step preparation of double-stranded templates using blocking PCR primers. Another challenge in pyrose-quencing is the difficulty to determine the number of more than five incorporated identical nucleotides due to nonlinear light response and multiplex genotyping and pooling approaches to further reduce the cost of the analysis (see Special Genotyping Applications).

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