In run A, PCR products and non-template controls (NTC) were loaded into a Q48 disc in alternating order. The Pyrosequencing results show no cross-contamination between disc wells. Run B was performed with NTC samples only and without replacing the absorber strip from the first run. The results exclude any cross-contamination from one run to another. Results shown in relative light units.
A sequencing primer mixture is loaded into 1 reservoir of the primer cartridge and dispensed to 4 different wells. Each primer only anneals to its specific target sequence and will be elongated during the Pyrosequencing reaction. Primers in each MPD mix should be designed and checked to avoid formation of primer–dimers or binding to another PCR template.
The NRAS Pyro assay was used to measure mutation frequencies in defined mixtures of wildtype and mutated NRAS sequences. Nine different mixtures representing frequencies of 0, 5, 10, 25, 50, 75, 90, 95, and 100% were analyzed using three different PyroMark platforms. The mutation frequencies measured were plotted against the expected frequencies. The data reveals that all three platforms gave the same results, showing that previously designed assays can be transferred among various PyroMark platforms.
ER-alpha methylation was measured in defined mixtures of methylated and unmethylated control DNA, representing methylation degrees of 0, 50 and 100%, using four different PyroMark platforms. The methylation measured for one CpG site was plotted against the expected percentage of methylated DNA. All four platforms gave the same results, showing that previously designed assays can be transferred among various PyroMark platforms.
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.
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.
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.
Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added.
Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alpha-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.