Qiagen PyroMark Q96 ID User Manual page 100

Appendix B
The recommended annealing temperature is 60°C and 56°C
for genomic DNA and bisulfite treated DNA, respectively,
when using PyroMark ADSW 2.0.
Addition of Q-Solution
may improve PCR yield and specificity for difficult templates
that, for example, have a high degree of secondary structure
or templates that are GC-rich.
For all PCR optimization tests, analyze 5 µl of a 25µl PCR on
an agarose gel and aim for one strong specific band with
minimal excess of primers.
Please refer to the PyroMark PCR Handbook for further
troubleshooting.
Equal amplification of both alleles in AQ and CpG
assays
Reliable results in quantification assays depend on equal
amplification of both alleles and this must be carefully tested.
To ensure equal amplification in a CpG assay, unmethylated
DNA can be mixed with increasing proportions of completely
methylated DNA. We recommend using EpiTect Control DNAs
(Cat. no. 59695), which provide bisulfite-treated completely
methylated and unmethylated DNA in ready-to-use solutions.
Regression analysis of the frequency of one allele measured in
the PyroMark Q96 ID as a function of the input (expected)
allele, should give an R
For an AQ assay, the allelic variants, including the variable
position, can be mixed at different ratios similar to the
procedure for a CpG assay. If the variable position in an AQ
assay is a SNP, the easiest way to test for equal amplification
is to compare the peak heights from a heterozygote. If the
SNP is represented by single base incorporations, e.g.,
AAC/TGG, the two alleles (C and T peaks) should give peaks
of equal height. An InDel heterozygote should give 50%
deletion.
B-4
® (provided with the PyroMark PCR Kit)
2 value greater than 0.9.
PyroMark Q96 ID User Manual 01/2016