Pyromark Q96 ID

Pyromark Q96 ID instrument for:
• quantitative analysis of genetic or epigenetic DNA modifications using Pyrosequencing technology

Pyromark Q96

PyrosequencingTM is a well established technology for DNA analysis. The Neurogenetics Unit has evaluated the Pyrosequencing method and our previous PSQTM 96 instrument for the analysis of genetic variations [Nordfors et al. 2002. Hum Mut 19(4): 395-401], which showed that the instrument is very robust and has a > 99% accuracy.

The instrument is user-friendly and fast (96 samples are analyzed within 10 min). The Assay Design Software helps you choose your optimal PCR- and pyrosequencing primers.

The core facility will provide start up theoretical and technical assistance for “hands-on” users. Each user reserves a date and time for occupying the instrument via iLab.

 

Prices and bookings

https://karolinska.corefacilities.org/service_center/show_external/3698/

 

Description of the method

The method does not require any gels or dyes. However, one PCR primer needs to be biotinylated for the DNA template amplification.

The Pyrosequencing method is based on sequencing-by-synthesis and on real-time detection of pyrophosphate.

In short, a sequencing primer is hybridized to a single-stranded DNA template, which is then incubated with luciferin as well as with a 4-enzyme mixture of DNA polymerase, ATP sulfurylase, firefly luciferase and the nucleotide degrading enzyme apyrase.

The different nucleotides are added one at a time in a pre-programmed order (the dispensation order) using ink-jet technology. If a nucleotide is complementary to the next nucleotide in the template DNA strand, it will be incorporated by the DNA Polymerase.

For every incorporation event, PPi will be released in proportion to the number of nucleotides that have been incorporated.

The PPi is converted to ATP by the ATP sulfurylase and the ATP in turn will drive the luciferase mediated conversion of luciferin into visible light. Between each cycle, unincorporated nucleotides will be degraded by the apyrase.

The light is detected by a CCD camera and the result is presented as real-time signals in a pyrogram were the peak heights are proportional to the number of nucleotides that have been incorporated.