Gene expression difference study
Digital PCR can provide more accurate gene expression difference study than real-time fluorescence quantitative PCR, especially for those cases in small differences in target gene expression, such as: expression analysis of mRNAs, microRNAs, lncRNAs, etc.; unbalanced expression of alleles; single cell gene expression analysis; quantitative analysis of exosomal nucleic acid molecules, etc.
Accurate quantification of low-abundance DNA template molecules
Since most of the microdroplets contain only single or no template molecules, so the amplification of low-abundance DNA template molecules is not inhibited by competition from the amplification of high-abundance template molecules. At the same time, the inhibitors that may be present in the sample are relatively diluted in the process of distribution to the microdroplets, and the number of inhibitors acting on the amplification of templates in a single microdroplet is greatly reduced, so it can be applied to many clinical samples (e.g. blood, urine, stool, sputum, etc.). Therefore, it can be applied to the detection of trace nucleic acid markers in many clinical samples (e.g. blood, urine, feces, sputum, thoracoabdominal fluid, cerebrospinal fluid, etc.). In general, the sensitivity of quantitative PCR is about 1%, NGS can reach 1%, and the lowest limit of detection of digital PCR can easily achieve 0.01%.
Copy number variation (CNV) study
Digital PCR directly reads the positive signal and obtains the absolute copy number of the target gene by Poisson distribution correction, providing absolute detection accuracy for the study of copy number variation, and can distinguish copy number differences within 1-fold. The use of digital PCR can effectively validate the results of experiments such as second-generation sequencing (NGS) and microarray comparative genomic hybridization aCGH, and is characterized by short detection cycle, low cost and high sample throughput.
Methylation content identification
Traditional clone sequencing method after bisulfite treatment Antibody detection method Quantitative PCR detection method, etc. are limited by methodological problems and cannot obtain accurate quantification of methylation level, while digital PCR system provides a reliable technique for accurate quantitative detection of methylation level through microdrop processing of samples and absolute copy number quantification of target molecules.
Validation of gene editing results
The invention of CRISPR-Cas9 technology is a major breakthrough in gene editing technology, but how to verify the success of the experiment still requires a highly sensitive detection method, and digital PCR technology can meet this need.
Gene copy number analysis of genetically modified organisms (GMO)
Digital PCR is a technique for absolute quantification of nucleic acid molecules based on the Poisson distribution principle, and has great potential for application in the field of absolute counting/quantification of nucleic acid molecules. The digital PCR method is a rapid and accurate new method for exogenous gene copy number analysis, with high sensitivity and accuracy, overcoming the defects of Southern blot method, which requires a lot of work, is time-consuming, requires high operation and has poor accuracy, and also overcoming the shortcomings of fluorescence quantitative PCR, which relies on standard substances to establish standard curves, which are not easy to obtain or expensive and cannot be applied to all studies.