Digital microfluidics is not limited to PCR and has inspired researchers in other areas of genomic research. One researcher has extended the use of digital microfluidics to the application of whole-genome haplotyping. Christina Fan, Ph.D., formerly at Stanford University, discussed her work with digital microfluidics on determining the fetal genome noninvasively from maternal blood.
“To achieve whole-genome haplotyping, one needs to look at many loci across the chromosome, and in our case, we focused on digital amplification of metaphase chromosomes and looked at 1 million loci using digital MDA.” Unlike digital PCR, which amplifies specific targets in the genome present in the digital compartment, digital MDA (multiple-strand displacement amplification) amplifies all DNA within the compartment.
The current limitation in digital amplification of a single metaphase chromosome has been “the amplification bias, yielding biased representation across the chromosome,” said Dr. Fan. However, she added, “we can pool together amplified materials from replicates of single chromosomes with our solution” to overcome this.
“Noninvasive determination of fetal genome is one application enabled by whole-genome haplotyping, which may ultimately facilitate the diagnosis of all inherited diseases,” says Dr. Fan. She highlighted one example of the importance of haplotyping.
“Given that a person has two mutations at two different loci with the same gene, either mutation would abolish function of the gene. In one scenario, the two mutations can be on the same strand, yielding still one functional copy of the gene. In another scenario, the two mutations can be on different strands, yielding no functional copy. The two scenarios can only be differentiated by haplotyping while genotyping alone would only inform that the person is heterozygous at the two loci,” explained Dr. Fan.
As droplet digital microfluidics becomes more accessible to researchers, the technology is expected to deliver on answering questions that previously could hardly have been imagined.
“One of the things scientists are discovering is that the more they interrogate genomes, the more complexity they find. At this point, we are still at the tip of the iceberg in fully appreciating the biological complexity of our cells. But, technologies such as droplet-based digital PCR are opening up new possibilities for this research and will help scientists piece together these biological puzzles,” said Dr. Link.
The next wave of droplet microfluidic technology can be found at the Micro/Nano Fluidics Fundamentals Focus (MF3) Center at the University of California at Irvine. There, Abraham Lee, Ph.D., oversees the development of a 1 million droplet array platform that simultaneously improves the dynamic range of digital PCR detection and integrates digital PCR with qPCR.
His lab has developed a platform that enables real-time PCR that not only gives absolute count of gene copies but also has the potential to determine starting copy numbers in each droplet when there is more than one DNA molecule. “This should further increase the dynamic range of digital PCR and may potentially detect the subtle variations in gene sequences that influence the extension efficiency of the enzymes,” Dr. Lee added.
“While digital PCR is an exciting technology that provides a new tool to identify absolute copies of target DNA, there is so much more that droplet microfluidics can provide in conjunction with this platform,” said Dr. Lee.
“Droplet microfluidics can provide solutions to sample processing and sample preparation, multiplexing, and in-line or parallel sample detection. Moreover, droplet manipulations that provide pre- and postprocessing steps allow for more automation and enable applications such as point-of-care diagnostics.”
Currently, his students are working on label-less detection schemes (nonoptical, nonfluorescence), some multiplex gene detection techniques, and some next-generation sequencing related applications using digital PCR.
For more on digital PCR, be sure to check out the Expert Tips "8 Reasons Labs Rely on Digital and Quantitative PCR".