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We analyze DNA sequences with pyrosequencing technology, using the latest generations of Qiagens ID and Q24 instruments.

 

Pyrosequencing is a highly reliable technique for sequencing a single strand of DNA by synthesising the complementary strand along it. After complementation of each individual unpaired base of the template strand, a chemiluminescent enzyme cascade is activated, resulting in an on-line detectable and quantifiable light signal (see Fig. 1). The detected light signals give insight into the DNA sequence and the grade of methylation.

 

Pyrosequencing step by step:

 

Step 1:

The DNA of interest is extracted and treated with bisulphite to convert unmethylated cytosines into uracils. After PCR amplification by which all uracils result in thymidine, the sample is denatured to form single-stranded DNA (ssDNA). Following hybridisation of a sequencing primer to the ssDNA, the complementary strand is synthesised in the presence of adenosine-5´-phosphosulphate (APS), luciferin and the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase.

 

Step 2:

The pyrosequencing process is initiated by addition of one of the four deoxynucleotide triphosphates (dNTPs). If it is complementary to the first unpaired base in the template strand, DNA polymerase catalyses its incorporation into the DNA strand, releasing pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide.

 

Step 3:

With APS as a substrate, ATP sulfurylase quantitatively converts the released PPi to ATP, which drives the luciferase-catalysed conversion of luciferin to oxyluciferin. By this reaction, a visible light signal proportional to the amount of ATP is generated and can be detected by a camera. The resulting peaks, corresponding to the numbers of nucleotides incorporated, produce a so-called pyrogram® (see Fig. 2).

 

Step 4:

Apyrase, a nucleotide-degrading enzyme, continuously degrades ATP and unincorporated dNTPs, “switching off” the light and regenerating the reaction solution. The reaction steps 2 and 3 can restart with the next dNTP.

 

Step 5:

The dNTPs are added one at a time. It should be noted that deoxyadenosine-alpha-thio-triphosphate (dATPaS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP) since it is efficiently used by the DNA polymerase, but not recognised by the luciferase. As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peaks in the pyrogram.

 

Based on comprehensive experience in assay design and application as the case at varionostic, pyrosequencing provides premium results for methylation analysis. Advantages of pyrosequencing include:

  • Pyrosequencing offers a great flexibility in primer placement. Therefore it is easy to design a pyrosequencing assay to analyse virtually any genetic marker.
  • Pyrosequencing assays are mutation tolerant. Unlike hybridisation-based assays, pyrosequencing generates a correct sequence regardless of the appearance of a new, unexpected mutation.
  • Using pyrosequencing the data received are fully quantitative.
Fig. 1: The principle of pyrosequencing
Fig. 2: Pyrogram of a pyrosequencing reaction with seven incorporated nucleotides. Double signal height means incorporation of two nucleotides of the same kind.