Qiagen PyroMark Q24 MDx, User Manual

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Appendix B
B-12 PyroMark Q24 MDx User Manual 01/2016
of equal height. An InDel heterozygote should give 50%
deletion.
Sample preparation
Use 5–20 µl of a 25 µl PCR for immobilization to Streptavidin
Sepharose High Performance (GE Healthcare) according to
the instructions in Section 5.3.3. This will be equivalent to
approximately 0.5–4 picomoles of PCR product, depending
on the PCR efficiency.
Note : When using the PyroMark PCR Kit, 5–10 µl of the PCR
product gives satisfactory Pyrosequencing results in most
cases. This volume should be adjusted to achieve single peak
heights of at least 40 RLU in the Pyrogram.
Pyrosequencing analysis
Use default settings in the software for all assay setups if not
otherwise stated.
Assay setup
AQ and CpG assays
When creating an AQ or CpG assay, the sequence to
analyze should contain a sufficient number of bases to
generate at least five nonvariable reference peaks. For
InDels, a few reference peaks should be included before the
variable position.
If the sequencing primer is placed adjacent to the position to
analyze, include part of the sequence following the variable
position in the “Sequence to Analyze” text box. Ensure that
the last base in the text box is not part of the variable
position.
Blank dispensations are automatically generated by the
software and serve as built-in quality controls for the assay.
Never exclude the blank dispensations as they function as
excellent indicators of unspecific nucleotide incorporation.
When manually generating a dispensation order, include an
appropriate number of blank dispensations. If possible, start