Assay Design And Validation - Qiagen PyroMark Q96 ID User Manual

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.
PCR
For PCR amplification we recommend using the PyroMark
PCR Kit (Cat. no. 978703) 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 de novo sequencing.
The convenient master mix format enables specific
amplification of various starting materials such as genomic
DNA from a variety of sources, as well as bisulfite converted
DNA, using only one protocol.
PCR primers
One of the primers must be biotin-labeled to enable
immobilization of the PCR amplicons to streptavidin-coated
beads (Streptavidin Sepharose High Performance, GE
Healthcare) for 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
biotinylated PCR product for binding sites on the streptavidin-
coated Sepharose beads.
PyroMark Q96 ID User Manual 01/2016
Appendix B
B-1