Application of Droplet Digital PCR Technology in Muscular Dystrophies Research

Abstract

Although they are considered rare disorders, muscular dystrophies have a strong impact on people's health. Increased disease severity with age, frequently accompanied by the loss of ability to walk in some people, and the lack of treatment, have directed the researchers towards the development of more effective therapeutic strategies aimed to improve the quality of life and life expectancy, slow down the progression, and delay the onset or convert a severe phenotype into a milder one. Improved understanding of the complex pathology of these diseases together with the tremendous advances in molecular biology technologies has led to personalized therapeutic procedures. Different approaches that are currently under extensive investigation require more efficient, sensitive, and less invasive methods. Due to its remarkable analytical sensitivity, droplet digital PCR has become a promising tool for accurate measurement of biomarkers that monitor disease progression and quantification of various therapeutic efficiency and can be considered a tool for non-invasive prenatal diagnosis and newborn screening. Here, we summarize the recent applications of droplet digital PCR in muscular dystrophy research and discuss the factors that should be considered to get the best performance with this technology.

Keywords: absolute quantification; cffDNA; droplet digital PCR; exon skipping; muscular dystrophy; serum biomarkers.

Figure 1

Schematic illustration of ddPCR (Created with Bio.Render.com). This figure illustrates a typical ddPCR workflow. (1) For the assay, a quantity of ddPCR mix, (buffer, dNTPs, primers, and probes) and the DNA sample are loaded in a multi-channel cartridge with droplet generation mineral oil. (2) The droplet generator creates a vacuum with negative pressure crosswise the cartridge. In this way, the negative pressure subdivides the DNA sample into water-in-oil droplets at the same time. (3) Subsequently, these partitions will be individually amplified under specific thermal cycling conditions. (4) After PCR amplification is complete, the plate is placed in the droplet reader, which analyses each droplet individually.