The PSQ-96MA Pyrosequencing System enables real time detection of DNA sequencing events, while eliminating the need for gels, dyes, and specific labels. Purchased by the Institute in 2001, the PSQ-96MA is a user-friendly instrument that is adept at sequencing short strands of DNA, analyzing di-, tri-, and tetra-allelic SNPs, insertions/deletions (InDels) and allele frequency quantification of pooled samples.

 

Preceding analysis, PCR products are converted to single stranded templates, and annealed with primer in a 96-well plate. Pyrosequencing technology is based on an enzyme-cascade system, which consists of four enzymes and specific substrates. Analysis begins with dispensation of the substrate-enzyme reagents into the wells containing test samples.  Light is produced when a nucleotide forms a base pair with its compliment.  The light and base are registered by a charge coupled device (CCD) camera, and is seen as a peak. If the next nucleotide added to the template is not complimentary to the nucleotide base, no light is detected on the CCD.  The height of the peaks is proportional to the number of bases that have been integrated.

 

Software used with the PSQ-96MA automatically analyzes the quantitative data. The data can be evaluated by the user on any computer, thus improving efficiency within the laboratory.  Please visit the website for manuals, procedures, and products available from Pyrosequencing.  

 

 

There is now a CpG software suite, enabling sensitive and quick detection of methylation states.  The CpG software is as sensitive as the bisulphite gold standard, and far faster. Using the same enzyme-cascade system as the SNP software, it is possible to have your results in under an hour.  Data analysis is included in the software, making for a more efficient approach, and the analysis output is in Word and Excel format, for convenient viewing at your own computer. 

 

There are several options for using Pyrosequencing technology through the Gene Expression Resource:

Those interested in utilizing any of the Resource’s equipment must first speak with Jenniffer Rothschild, ext. 3958.  She will arrange a time for you to be trained on the equipment and to receive an account so that you may schedule equipment time. Once an account has been established all equipment scheduling is done via Resource Scheduling in Outlook Express Public Folders.  For pricing please click here.

In addition to providing training on the software, the Gene Expression Resource is now offering the Pyrosequencer as a service.  We will take your sample and run the analysis.  All samples submitted must be accompanied by a Pyrosequencing Request Form. 

 

There are two options available for Pyrosequencing service:

 The above options are available with or without primer design.

 

Pricing for this service is done on an individual project basis. Please contact Jenniffer Rothschild for pricing information.

 

Images reproduced with permission from Biotage.

 

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