I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?

To identify and quantify each amplified gene in a multiplexing EvaGreen-based dPCR assay, it's crucial to have distinct amplicon sizes. Based on the available literature, using amplicons of 60bp, 100bp, and 200bp should be effective, as they fall within the recommended size range for dPCR and maintain good efficiency [1][3]. Additionally, employing melting curve analysis can further enhance discrimination between different amplicons [6].

For non-randomly fragmenting gDNA to improve detection and quantification, consider using methylation-sensitive restriction enzymes, which provide specific cleavage at methylated sites. Enzymes like HpaII and MspI are known to be effective for such purposes [2][4]. Furthermore, Type IIS restriction enzymes offer unique capabilities by cleaving outside their recognition sites, thus providing more predictable and useful fragment patterns for downstream applications [5]. Combining these approaches can significantly improve the accuracy and reliability of your assay.

Reference

[1] Esling, P., Lejzerowicz, F., & Pawłowski, J. (2015). Accurate multiplexing and filtering for high-throughput amplicon-sequencing. Nucleic Acids Research, 43, 2513 - 2524.

[2] Dorazio, R., & Hunter, M. E. (2015). Statistical Models for the Analysis and Design of Digital Polymerase Chain Reaction (dPCR) Experiments.. Analytical chemistry, 87 21, 10886-93 .

[3] Moniri, A., Miglietta, L., Malpartida-Cardenas, K., Pennisi, I., Cacho-Soblechero, M., Moser, N., Holmes, A., Georgiou, P., & Rodriguez-Manzano, J. (2020). Amplification Curve Analysis: Data-Driven Multiplexing Using Real-Time Digital PCR.. Analytical chemistry.

[4] Hou, P., Chen, Z., Ji, M., He, N., & Lu, Z. (2007). Real-time PCR assay for ultrasensitive quantification of DNA-binding proteins.. Clinical chemistry, 53 4, 581-6 .

[5] Nell, R., Steenderen, D. v., Menger, N., Weitering, T. J., Versluis, M., & Velden, P. A. v. d. (2020). Quantification of DNA methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital PCR. Human Mutation, 41, 2205 - 2216.

[6] Lundin, S., Jemt, A., Terje-Hegge, F., Foam, N., Pettersson, E., Käller, M., Wirta, V., Lexow, P., & Lundeberg, J. (2015). Endonuclease Specificity and Sequence Dependence of Type IIS Restriction Enzymes. PLoS ONE, 10.

Hi,

I can recommend you McDermott et al. https://pubmed.ncbi.nlm.nih.gov/24180464/ and https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf

You can play around with:

- amplicon length (in the paper they go from 60 to 200)

- primer concentration (Rather go down then go up, as your background will go up too; so

Thank you both for the answers. I will look into these, and keep the post updated if something works 👍