Bio-Rad SsoAdvanced™ Universal Probes Supermix - Manual (Page 9)

| 3 SsoAdvanced ™ Universal Probes Supermix Instruction Manual | 3
Assay Design Considerations
When using custom designed assays, several important considerations should be noted:
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Biological significance (correct isoform/splice variant chosen)
■ ■
Sequence quality and secondary structure — evaluate using web-based tools to understand
the complexity of the structure, as it can impact the reaction performance
■ ■
Sequence length — use the entire gene sequence, or a specific region of interest, to
optimally design an assay
■ ■
Sequence masking — use web-based masking tools to mask low complexity and repetitive
regions to avoid assay design in these regions
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Uniqueness of the sequence — use BLAST or BLAT to ensure no homology exists and help
avoid mispriming events
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Uniqueness of the assay — use in silico PCR, or Primer-BLAST, to “blast” the primers against
the genome of interest to validate primer design specificity
■ ■
of 60ºC designed using the open source Primer3, Primer3Plus or Primer-BLAST, default
settings. For assays designed using other tools, the primer T
m
should be recalculated using
Primer3. Suggested settings: 50 mM Na + , 3 mM Mg ++ , 1.2 mM dNTPs, 250 nM annealing
oligo, SantaLucia/SantaLucia
Some Key Design Considerations
■ ■
For optimal PCR efficiency, design the amplicon size between 70 and 150 bp (<70 bp may be
needed for degraded/FFPE templates)
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Maintain primer lengths between 18 and 25 bp for good specificity and binding abilities
■ ■
■ ■
The optimal amplicon GC content should be within 40–60% (greater range can be obtained
using Bio-Rad’s Sso7d-based supermixes)
■ ■
Avoiding primer secondary structures reduces potential primer-dimer issues
■ ■
Avoid mispriming by ensuring there are no more than 2 Gs or Cs in the last 5 bases on the 3'
end of the primer
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Design your assay such that at least one primer or the probe spans an exon:exon junction
site to avoid gDNA amplification
■ ■
Alternatively, design the assay such that the primers are in separate exons and the intron
size is >1 kb
■ ■
■ ■
Avoid placing Gs on the 5' end of the probe to avoid quenching of the fluorophore even after
probe cleavage
■ ■
Probe lengths typically range from 18–30 bp, and vary depending on the type of probe
chemistry used and the target sequence
Default settings in the software — ensure they are set correctly (for example, salt conditions,
oligo and amplicon sizes). The SsoAdvanced
™ universal probes supermix and the qPCR
cycling protocols have been optimized for assays with a primer melting temperature (T
m
)
Annealing temperatures between 58 and 62ºC are optimal (greater range can be obtained
using Bio-Rad’s Sso7d-based supermixes); temperatures >60ºC may result in less binding
efficiency and <58ºC may result in less specificity
Probe annealing temperature should be 8–10ºC higher than the primers to
ensure binding to the template prior to extension