Qiagen PyroMark Q24 MDx, Руководство пользователя

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Appendix B
PyroMark Q24 MDx User Manual 01/2016 B-11
PCR optimization
The PyroMark PCR Kit will produce satisfactory results in most
cases. However, if a higher Mg
2+ concentration is required,
we recommend using the 25 mM MgCl
2
provided in the kit.
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
®
(provided with the PyroMark PCR Kit)
can 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,
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 Q24 MDx as a function of the input (expected) allele,
should give an R
2 value greater than 0.9.
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