Qiagen PyroMark Q24 MDx, User Manual

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Appendix B
B-8 PyroMark Q24 MDx User Manual 01/2016
Appendix B
Assay design and validation
Assay design
Pyrosequencing assays can be designed using the latest
version of PyroMark Assay Design Software (ADSW). The
program automatically generates primer sets that include
both PCR and sequencing primers. Each primer set is given a
quality score based on several parameters that are specific
for Pyrosequencing analysis. Ensure you use the correct assay
type in PyroMark ADSW. We recommend the use of QIAGEN
IVD-labeled assays for Pyrosequencing, which include all
necessary optimized primers.
PCR
For PCR amplification we recommend using PyroMark PCR
Kit (QIAGEN) which is specifically optimized for
Pyrosequencing analysis and enables highly specific and
unbiased amplification of template DNA for various
Pyrosequencing applications, such as mutation detection,
SNP analysis, methylation analysis, and base-calling. The
convenient master mix format enables specific amplification
of various starting materials such as genomic DNA from a
variety of species, as well as bisulfite converted DNA, using
only one protocol.
PCR primers
One of the primers must be biotin-labeled to enable
immobilization to streptavidin-coated beads (Streptavidin
Sepharose High Performance; GE Healthcare) during the
preparation of a single-stranded DNA template. The
orientation of the assay can either be forward or reverse. If
designing primers with PyroMark ADSW, the primer that
needs to be biotinylated is indicated.
The biotinylated primer should be purified by HPLC or an
equivalent procedure since free biotin will compete with the