It uses the 5' nuclease
assay to discriminate between 2 alleles of a specific SNP. Each assay
is a 10x mix of forward primer, reverse primer, 6FAMTM dye - MGB labeled
probe, and VICTM dye- MGB labeled probe. Control DNA for each allele
is included in the kit. The AD control reactions control for the amplification
process as well as acting as a qualitative measure of the optics of
the 7900HT detection system and also as a control of the autocalling
algorithms of the analysis software. In addition, these samples are
set up alongside the samples to be analysed by the EpMotion 5075 and
therefore act as a control for the mechanics of the robotic set-up.
Additionally, customers are requested, if possible, to send a positive
control DNA sample for which they know the genotype for each assay,
along with the samples to be processed.
SNPlex
SNPlex is a multi-step protocol and is provided by Applied Biosystems
with a comprehensive troubleshooting guide to pinpoint any potential
problem areas. For example, any problems arising from capillary electrophoresis,
such as resolution or signal, can be identified by studying raw electrophoresis
data. A review of the Genemapper software analysis can pinpoint any
errors with file type assignment, size standard peak assignment, or
binning of allelic ladders. A study of the analysed results and assay
controls can help to pinpoint chemistry-related issues, such as DNA
quality, liquid handling, or thermal cycle programming. Inspecting
assay controls, cluster-plot patterns and electropherograms can help
pinpoint the cause.
Every SNPlex run has the following controls: No template controls
reaction (NTC), positive control reactions (control DNA provided with
SNPlex kit to control for the whole SNPlex process) and positive and
negative hybridization controls (PHC and NHC, respectively). The PHC,
which is added to the binding buffer when PCR products are bound to
streptavidin plates, is used to monitor the efficiency and reproducibility
of the ZipChute probe hybridization and elution steps. The NHC is
used to monitor non-specific hybridization that may occur during the
hybridization step.
Quality Control (QC) for Fragment Analysis
In order to assess the accuracy of the electrophoresis and subsequent
DNA fragment sizing analysis, SUPAMAC run an internal control sample
with each batch of samples. The control sample used is the Applied
Biosystems, GeneScan Installation Standard DS-33 (P/N 4330397). This
sample contains pooled PCR products of known sizes, generated by amplification
of CEPH individual 1347-02, using 8 fluorescent PCR primer pairs which
amplify selected microsatellite loci.
Quality Control (QC) for Denaturing High Performance Liquid Chromatography
(DHPLC)
For DHPLC, two controls are run before any customer samples are processed
to assess the performance of the oven and DHPLC itself. These include
a WAVE High-Range Mutation Control Standard (Cat No: 562001) which
is run at 700C, and a WAVE Low-Range Mutation Control Standard (Cat
No: 560077) which is run at 560C.
In addition to the above, regular maintenance is carried out on all
equipment to ensure the continued optimal performance of each service
offered by SUPAMAC.
Validation of In Vitro Diagnostic Devices (IVDs)
SUPAMAC is not a primary DNA testing laboratory, although, as mentioned
above, it does provide sequencing to DNA testing laboratories. Our
tests are not validated for diagnostic purposes, and if used for this
purpose, the appropriate precautions are required when interpreting
the results. If it is particularly important for a customer to validate
a test for their particular needs, SUPAMAC is happy to work with the
customer to validate their test at an additional cost. Please contact
Nicole Sawyer at SUPAMAC on support@supamac.com or on 9515 8403 if
you require assistance with validation of your amplicon on our 3730XL
DNA Analyzer or WAVE DHPLC.
SUPAMAC is NATA ISO15189 accredited.
