Digital Polymerase Chain Reaction Analysis Research Articles

Developments in precision medicine: Studies highlight new applications for cancer chemoprevention, treatment.

Developments in precision medicine: Studies highlight new applications for cancer chemoprevention, treatment.

The nitrogen cycle in cryoconites: naturally occurring nitrification-denitrification granules on a glacier.

Cryoconites are microbial aggregates commonly found on glacier surfaces where they tend to take spherical, granular forms. While it has been postulated that the microbes in cryoconite granules play an important role in glacier ecosystems, information on their community structure is still limited, and their functions remain unclear. Here, we present evidence for the occurrence of nitrogen cycling in cryoconite granules on a glacier in Central Asia. We detected marker genes for nitrogen fixation, nitrification and denitrification in cryoconite granules by digital polymerase chain reaction (PCR), while digital reverse transcription PCR analysis revealed that only marker genes for nitrification and denitrification were abundantly transcribed. Analysis of isotope ratios also indicated the occurrence of nitrification; nitrate in the meltwater on the glacier surface was of biological origin, while nitrate in the snow was of atmospheric origin. The predominant nitrifiers on this glacier belonged to the order Nitrosomonadales, as suggested by amoA sequences and 16S ribosomal RNA pyrosequencing analysis. Our results suggest that the intense carbon and nitrogen cycles by nitrifiers, denitrifiers and cyanobacteria support abundant and active microbes on the Asian glacier.

1-Million droplet array with wide-field fluorescence imaging for digital PCR

Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.

Non‐invasive detection of foetal RhD status and other genetic characteristics by circulating nucleic acids in maternal plasma

Since the discovery of cell‐free foetal DNA in maternal plasma in 1997, many circulating foetal DNA markers have been developed for the non‐invasive prenatal assessment of foetal genetic status. Some of the diagnostic applications, such as foetal sex determination and foetal rhesus D blood group genotyping, have already been introduced as routine services in some clinical centres. To extend the spectrum of foetal traits in which circulating foetal DNA can be worked on, advanced molecular technologies, such as mass spectrometry, microfluidic system and digital polymerase chain reaction analysis, have been employed. These newly developed tools have superior detection sensitivities and specificities over conventional methods. They have allowed researchers to overcome many technical challenges regarding the non‐invasive investigations of the foetal genetic characteristics, especially those involving a single nucleotide change. We envision that after validations by large‐scale studies, these new tools would be useful in a clinical setting in the near future.

Microfluidic digital PCR enables rapid prenatal diagnosis of fetal aneuploidy

The purpose of this study was to demonstrate that digital polymerase chain reaction (PCR) enables rapid, allele independent molecular detection of fetal aneuploidy. Twenty-four amniocentesis and 16 chorionic villus samples were used for microfluidic digital PCR analysis. Three thousand and sixty PCR reactions were performed for each of the target chromosomes (X, Y, 13, 18, and 21), and the number of single molecule amplifications was compared to a reference. The difference between target and reference chromosome counts was used to determine the ploidy of each of the target chromosomes. Digital PCR accurately identified all cases of fetal trisomy (3 cases of trisomy 21, 3 cases of trisomy 18, and 2 cases of triosmy 13) in the 40 specimens analyzed. The remaining specimens were determined to have normal ploidy for the chromosomes tested. Microfluidic digital PCR allows detection of fetal chromosomal aneuploidy utilizing uncultured amniocytes and chorionic villus tissue in less than 6 hours.