This detailed volume explores digital polymerase chain reaction (dPCR), a third generation PCR technology that enables absolute quantification by counting positive and negative partitions after endpoint amplification. The book aims to provide an overview of the various possibilities currently available, both in terms of instrumentation and applications. The selected protocols cover a wide range of uses, including fundamental research, preclinical studies, human applications, and regulatory contexts. Additionally, protocols that further extend potential applications, such as protein detection, the development of custom dPCR systems, and alternative PCR strategies like hpPCR, which, while not digital, are relevant in similar contexts, are covered. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible protocols, and tips for troubleshooting and avoiding known pitfalls.

Authoritative and practical, Digital PCR: Methods and Protocols serves as an ideal guide to this mature technology that will continue to enhance target quantification, notably through its multiplexing capabilities, tolerance to inhibitors, and increased precision.

Includes cutting-edge techniques Provides step-by-step detail essential for reproducible results Contains key implementation advice from the experts

Inhalt

A Beginner Step-by-Step Guide for Gene Expression Analysis Using QIAcuity Digital PCR.- Multiplex Quantification of 19 GM Soybean Lines Using Digital PCR.- Multiplex Digital PCR Assay Targeting DNA Methylation for Early Detection of Cancer.- Multiplexed Digital PCR for Evaluation of Karyotype Quality of Mouse Embryonic Stem Cells.- Optimizing Digital PCR Assays for Multiplexed Microbial Source Tracking in Surface Waters.- Crystal Digital PCRTM-Based Single Nucleus Pollen Genotyping in Barley (Hordeum vulgare L.) to Measure Meiotic Crossover Rates in Defined Chromosomal Intervals.- Robust dPCR Hyper-Plexing Approach by Color Combination for Detection of 17 ESR1 Mutations in Breast Cancer Patient Samples.- Digital PCR Analysis of Methylated Circulating Tumor DNA.- Method for Validation Multiplexed Droplet Digital PCR for the Detection of Copy Number Variants in Cultured Human Pluripotent Stem Cells.- Digital PCR Methods for Discovering Molecular Markers for Hanwoo Cattle Breed Genotyping.- A Duplex Digital PCR Method for Quantification of Transgene Soybean Line MON40-3-2 in Complex Matrices.- DNA Methylation Analyses of Tissue Samples and Cell-Free DNA by Droplet Digital PCR.- Quantification of Tissue and Circulating MicroRNAs by Droplet Digital PCR.- Digital Droplet PCR (ddPCR) for Absolute Quantification of 16S rRNA Copy Number in Metagenomic Data.- Protein Interaction Coupling (PICO): Exploiting dPCR for the Reference-Free Absolute Quantification of Proteoforms and Protein-Protein Interactions.- Extending the Potential of Digital Droplet PCR by Expressing Amplified Genes and Assaying Their Phenotype.- 20-Plex Gene Expression Analysis Using Hyperplex PCR (hpPCR) Toward the Development of Salt-Tolerant Wheat.