Experimental design Drop-off digital PCR method two: gene mutation and gene deletion calculation method

How to use Crystal Miner analysis software to analyze Drop-off digital PCR results

Only two channels of Crystal Digital PCR can be used to perform absolute quantification of mutant alleles (MAF) with high sensitivity

The first channel: The Drop-Off probe is only complementary to the wild-type sequence, and the mutant sequence is skipped

The second channel: the reference probe hybridizes to the adjacent site of the mutation site and complements the mutation and wild-type alleles (the green channel on the horizontal axis in Figure 1)

When a wild-type allele is present, both the drop-off and the reference probe will hybridize with the template, and a double positive droplet will appear

When there is a mutant allele, even a single nucleotide mutation is enough to destroy the instability of the drop-off probe hybridization, so only the reference probe can hybridize to the template, thereby generating a single positive spot

For more information on Drop-off design and analysis, please refer to our manual "Drop-off Crystal Digital PCR Detection of NRAS, KRAS and EGFR Mutations", website, https:// /

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Use Crystal Miner analysis software to quantitatively analyze Drop-off results step by step

The wild-type concentration Cwt (double positive droplets) can be calculated directly by the ratio of Pwt (regardless of whether the third channel is positive or negative), and the concentration of drop-off mutation Cmut (mutant allele) can be calculated by Pmut

To perform quantitative calculations using Crystal Miner analysis software, you need to follow the steps below:

1.

Follow the steps, ANALYZE DATA> Plots&Populations> 2D dot plot

2.

Steps: ANALYZE DATA> Plots&Populations> Population Editor

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hint:

The LOD value relative to the drop-off mutant increases exponentially with the expected wild-type concentration (at the 95% confidence level, the LOD increases from 0.

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