Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes. PyroMark Q24 MDx is a fully integrated system that provides real-time sequence information and is highly suited for genetic and epigenetic analysis. The system includes the PyroMark Q24 MDx Instrument, PyroMark Q24 MDx Vacuum Workstation, PyroMark Q24 MDx Software 2.0, PyroMark Gold Q24 Reagents, PyroMark Control Oligo, and PyroMark Q24 Validation Oligo. Sample preparation solutions are also available to enable preparation of single-stranded DNA using the PyroMark Q24 MDx Vacuum Workstation.
Steps of the Pyrosequencing reaction:
Step 1: A DNA segment is amplified, and the strand to serve as the Pyrosequencing template is biotinylated. After denaturation, the biotinylated single-stranded PCR amplicon is isolated and allowed to hybridize with a sequencing primer. The hybridized primer and single-stranded template are incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, and apyrase, as well as the substrates adenosine 5' phosphosulfate (APS) and luciferin (see figure " Principle of Pyrosequencing — step 1").
Step 2: The first deoxribonucleotide triphosphate (dNTP) is added to the reaction. DNA polymerase catalyzes the addition of the dNTP to the squencing primer, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi), in a quantity equimolar to the amount of incorporated nucleotide (see figure " Principle of Pyrosequencing — step 2").
Step 3: ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS). This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by CCD sensors and seen as a peak in the raw data output (Pyrogram). The height of each peak (light signal) is proportional to the number of nucleotides incorporated (see figure " Principle of Pyrosequencing — step 3").
Step 4: Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added (see figure " Principle of Pyrosequencing — step 4").
Step 5: Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alfa-thio triphosphate (dATPαS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP), since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is elongated, and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace (see figure " Principle of Pyrosequencing — step 5").
Dedicated IVD-validated assays
QIAGEN offers an expanding suite of IVD-validated assays to be used with the PyroMark Q24 MDx. Currently, these kits include important cancer-related mutations:
|therascreen KRAS Pyro Kit ||codons 12, 13, and 61 of the human KRAS gene |
|therascreen BRAF Pyro Kit ||codons 600 and 464-469 of the human BRAF gene |
|therascreen EGFR Pyro Kit ||codons 719, 768, 790, 858, and 861 and exon 19 of the human EGFR gene |
|therascreen NRAS Pyro Kit ||codons 12, 13, and 61 of the human NRAS gene |