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Global Health Technologies

Global Health Technologies - Poster Session E

Poster Y3 - Simplify and Amplify: Streamlining Multiplex Target Detection with One-Pot Ambient Enzymatic Digestion on Digital PCR

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Digital PCR (dPCR) enables the absolute quantification of multiple gene targets for applications ranging from genomic analysis to gene expression quantification. One such application is copy number variation (CNV) analysis, which analyzes polymorphic genes for the presence of duplications, deletions, or hybridizations to determine their functional activity level.

For microfluidics-based dPCR systems, genomic DNA (gDNA) samples are enzymatically digested to promote confluent sample distribution, reduce target co-dependent amplification, and to validate Poisson statistical assumptions [1]. Hence, careful enzyme selection and robust digestion protocols are critical to the success of gDNA analysis on dPCR.

Conventional digestion is performed as a pre-step, whereby aliquots of whole gDNA are heated and incubated with selected restriction enzyme(s). Because digested gDNA is not ubiquitously compatible with downstream analyses, any unused aliquots are sources of undesirable sample waste, e.g. for limited-volume samples. Elimination of the pre-digestion step simplifies the user workflow, reduces DNA discard, and maximizes whole DNA availability for biobanking needs. Our solution is to perform ambient enzymatic digestion in the prepared dPCR reaction mixture for one-pot, direct CNV analysis of multiplexed gene targets in 2 hours.

[1] J. F. Regan et al. (2015). PLoS One 10: e0118270.

Materials and

Methods:  CYP2D6, a highly polymorphic gene that encodes the cytochrome P450 2D6 enzyme, was selected to undergo CNV analysis and evaluate the efficacy of one-pot enzymatic digestion. A custom TaqMan® four-target multiplex assay was designed to detect three gene loci (Exon9, Intron6, 5’ untranslated region [UTR]) plus the telomerase reverse transcriptase (TERT) reference gene [2]. gDNA samples were digested using the Anza™ BglI restriction enzyme to achieve independent targets (Figure 1). Samples were run at 10 ng/reaction concentration on the QuantStudio™ Absolute Q™ dPCR System with automated CNV analysis.

The concordance of digestion methods was evaluated by comparing three conditions on Coriell Institute purified gDNA: 1) 15-min 37°C pre-digestion, 2) 30-min ambient (20-25°C) one-pot digestion, and 3) no-enzyme negative control. Data was taken on the Absolute Q system.

To assess the clinical compatibility of the one-pot method, donor gDNA samples were procured from human liver tissue (n=8), blood (n=4), and saliva (n=4). The samples underwent CNV analysis using the 1) one-pot digestion with the 4-target multiplex assay (3 loci + reference) on the Absolute Q system, and 2) pre-digestion with the 2-target simplex assays (1 locus + reference) on the Bio-Rad QX200 system for comparison.

The efficacy of ambient digestion was evaluated at twelve timepoints between zero (no incubation) and 60 minutes. Ambient benchtop stability of the one-pot enzymatic digestion was assessed at four timepoints between 60 minutes and 72 hours. Both studies tested four Coriell gDNA samples.

[2] V.M. Pratt et al. (2021). J. Mol. Diagnostics 23: 1047-1064.

Results, Conclusions, and Discussions: Sixteen Coriell gDNA samples were selected to test a range of copy numbers (0- to 6-copy) and variant types (homogenous or hybrid). The samples were previously analyzed using real-time PCR and the comparative Ct method to document baseline CNV copy numbers [3]. For dPCR runs, CNVs were automatically calculated by the instrument software and rounded to their nearest integer values. Copy calls were accepted if they accurately rounded to their expected baseline values and were ±0.25 of the integer value (e.g. 1.75 to 2.25 were accepted as 2-copy).

Table 1 lists the results of the concordance study. The CNVs of both the pre-digest and one-pot digest data matched their baseline values, therefore establishing concordance of digestion methods. For the no-enzyme control, three gDNA samples exhibited discordant or obscure copy calls, suggesting that enzymatic digestion is critical to the accuracy of CNV results.

Table 2 compares the one-pot digested + multiplex Absolute Q copy numbers (CNs) with the pre-digested + simplex QX200 CNs. The Absolute Q data achieved comparable copy call clarity against the QX200 data, verifying compatibility of the extracted donor samples with both the one-pot digestion method and the multiplex assay.

Four (4) of 16 gDNA samples were subjected to digestion efficacy and benchtop stability experiments. Temperature was monitored for the full duration to be within 20-25°C. Figure 2 depicts the data. Copy calls were found to be accurate as early as 0-minute incubation, suggesting sufficient enzymatic activity at ambient temperatures. Most samples achieved excellent ambient stability up to 72 hours. The 6-copy hybrid gene (NA18107) exhibited a drop in copy call clarity after 24 hours, but maintained correct numerical rounding to its baseline value (5.6 → 6).

In summary, one-pot enzymatic digestion was demonstrated as a simple, direct, and robust method for multiplex dPCR analysis and broader genomics research. Ambient digestion plus 72 hours of tested benchtop stability can streamline workflows, reduce sample waste, and enable scale-up towards high-throughput or automated liquid-handling systems.

[3] T.D. Schmittgen and K.J. (2008). Livak. Nat. Protoc. 36: 1101-1108.

Acknowledgements (Optional):  Donor samples were procured from the Liver Tissue Cell Distribution Center System (CTCDS), Discovery Labware, Inc. (Corning Inc.), and the Children’s Mercy Research Institute (CMRI) repository for research purposes. The use of de-identified samples by W.W., E.B., and A.G. was approved by the CMRI Institutional Review Board (IRB).