Genomic Copy Number Analysis Using Droplet Digital PCR: A Simple Method with EvaGreen Single-Color Fluorescent Design

Takeaki Tamura; Taichi Imaizumi; Keiko Shimojima Yamamoto; Toshiyuki Yamamoto

doi:10.1007/978-1-0716-3810-1_24

Genomic Copy Number Analysis Using Droplet Digital PCR: A Simple Method with EvaGreen Single-Color Fluorescent Design

Methods Mol Biol. 2024:2794:293-304. doi: 10.1007/978-1-0716-3810-1_24.

Authors

Takeaki Tamura^{1

2}, Taichi Imaizumi³, Keiko Shimojima Yamamoto^{4

5}, Toshiyuki Yamamoto^{6

7}

Affiliations

¹ Department of Pediatrics and Child Health, Nihon University School of Medicine, Tokyo, Japan.
² Division of Gene Medicine, Graduate School of Medical Science, Tokyo Women's Medical University, Tokyo, Japan.
³ Department of Pediatrics, St. Marianna University School of Medicine, Kawasaki, Japan.
⁴ Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan.
⁵ Department of Transfusion Medicine and Cell Processing, Tokyo Women's Medical University, Tokyo, Japan.
⁶ Division of Gene Medicine, Graduate School of Medical Science, Tokyo Women's Medical University, Tokyo, Japan. yamamoto.toshiyuki@twmu.ac.jp.
⁷ Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan. yamamoto.toshiyuki@twmu.ac.jp.

PMID: 38630238
DOI: 10.1007/978-1-0716-3810-1_24

Abstract

Droplet digital PCR (ddPCR) is an emerging method for the absolute quantification of PCR products, and it can detect DNA copy numbers accurately. It analyzes the end-point absolute fluorescence signals of the PCR-positive droplets and calculates the target concentration. EvaGreen is a nonspecific double-stranded DNA-binding fluorescent dye, and the ddPCR system also supports assays using this cost-effective hydrolysis probe. Here, we describe a simple method of quantification for DNA copy numbers using the EvaGreen single-color fluorescent design.

Keywords: DNA copy numbers; Droplet digital PCR; EvaGreen; QuantaSoft software; Single color; ddPCR.

MeSH terms

DNA / genetics
DNA Copy Number Variations*
Fluorescent Dyes
Genomics*
Polymerase Chain Reaction

Substances

Fluorescent Dyes
DNA