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Appendix B
Amplicon length
The optimal amplicon length for Pyrosequencing assays is
between 80 and 200 bp, although products up to 500 bp
may work well. Amplicons for CpG assays should ideally be
shorter than 200 bp.
Sequencing primer
Design sequencing primers using PyroMark ADSW. When
designing an InDel assay, it is highly recommended that the
sequencing primer is located a few bases before the variable
position.
PCR setup
PCR reactions of 25 µl are set up using the PyroMark PCR Kit.
Ensure that you follow the instructions provided in the
PyroMark PCR Handbook.
Run the PCR at the optimal annealing temperature for 45
cycles. Using fewer cycles may produce insufficient yield of
template DNA and interfere with subsequent Pyrosequencing
reactions due to the presence of unused biotinylated primer.
The PCR product should give one strong band with minimal
excess of primers when analyzed on an agarose gel.
Starting template
The yield and quality of PCR product is affected by both the
quality and quantity of the nucleic acid starting template. This
is particularly true for amplification of long regions from
DNA that has been fragmented by bisulfite-treatment or
extracted from paraffin-embedded material.
Quality of starting template
Since PCR consists of multiple rounds of enzymatic reactions,
it is more sensitive to impurities such as proteins,
phenol/chloroform, salts, ethanol, EDTA, and other chemical
solvents than single-step enzyme-catalyzed processes.
QIAGEN offers a complete range of nucleic acid preparation
systems, ensuring the highest-quality templates for PCR.
B-2
PyroMark Q96 ID User Manual 01/2016
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