Single-Cell Polymerase Chain Reaction (PCR)
Mira Guo
Single-cell PCR is highly informative in many applications, especially metagenomic studies. Metagenomics analyzes the genomic sequences in mixed populations of microbes to determine what genes are present and how they affect the function of the population as a whole. Interesting populations include collections of diverse but poorly characterized bacteria or virus species from biomedically relevant environmental samples such as soil, sewage wastewater, or the human gut. Billions of unique bacteria species exist in nature, but very little is known about all but 0.1% of these species. The other 99.9% represent a vast reservoir of biodiversity that is poorly characterized because they are extremely difficult to grow in the laboratory. Unculturable species cannot be studied because conventional bulk methods for genetic analysis require thousands of identical bacteria cells per reaction.
Single-Cell PCR for Metagenomics
Typical metagenomic datasets determine all the genes present in such a population by fragmenting the microbial DNA in a sample and sequencing each fragment. This method yields insight into the distribution of species in a population, the metabolic functions that the population can perform, and other biologically relevant information. However, because short DNA fragments from multiple cells are mixed together, all information about which fragment originated from which cell is lost, and there is no way to associate sequences coming from the same cell. Whole-genome assembly from single cells in an environmental sample is therefore impossible. Even if several identical cells in a complex population allow for assembly from overlapping fragments, assembly is still an enormous computational challenge.
Droplets can be used to associate distant portions of the genome from a single cell. Encapsulating single cells or viruses while their genomes are still intact ensures that all the DNA within a given droplet must have originated from the same cell, no matter how fragmented it becomes in later steps, as long as the droplet remains intact. The droplet effectively replaces the cell membrane or virion capsule as a container for the whole genome.
We use microfluidics to encapsulate individual bacteria cells in drops for PCR and genome sequencing. Our microfluidic techniques bypass the difficult step of culturing the bacteria and enable analysis of a single cell, by reducing the reaction volume and thus increasing the concentration of DNA in each reaction. This is critical to studying the diverse populations of mixed species living in environmental samples or the human body. The human gut and other environmental samples contain large populations of diverse bacteria that are poorly characterized and unculturable, yet have many functions relevant to human health. Our goal is to identify exactly which species carry some gene of interest, such as a carbohydrate metabolism gene.
Once single cells or viruses from a population are encapsulated in droplets, single-cell PCR can target an interesting gene previously detected in the population and amplify that gene in every droplet where it is present. Droplet microfluidics can then sort out those droplets containing the PCR products. Each of these droplets must also contain the original genome that carried the gene that produced those PCR products. The selected droplets can then be coalesced and subjected to whole genome sequencing using conventional methods in bulk. Because the selected genomes of interest will constitute a population of much lower complexity than the original environmental sample, whole genome assembly will be much more feasible. In fact, if the selected genomes were very rarely occurring variants within the original population, whole genome assembly would be impossible without screening in droplets. Droplet microfluidics thus enables whole genome sequencing of rare single cells of interest from complex environmental samples.
Figure 1. We encapsulate bacteria cells (green) at a dilute concentration in droplets (gray outlines), and stain the genomic DNA with an intercalating fluorescent dye. Light gray circles have been added to the image to indicate droplet boundaries.
Single-Cell PCR for Sepsis Diagnosis (inactive project)
We have previously explored using emulsion PCR to detect very low levels of pathogenic cells present in a large population. Sepsis, also called blood poisoning, occurs when microbes infect a patient and induce an inflammatory response throughout the whole body. Mortality rates can be as high as 30-60% for advanced stages of infection (severe sepsis and septic shock) [1]. This is partly because it is difficult to identify the specific organism causing sepsis in a patient. It may be one of many species of bacteria or fungi, such as Staphylococcus, Candida, or Aspergillosis, but all species will generally produce the same nonspecific symptoms, such as fever, low blood pressure, and arrhythmia. It is therefore necessary to perform tests that go beyond immediate clinical observations, in order to determine which antibiotic or antifungal drug to use for treatment. These tests generally involve culturing blood samples and take as long as a week to produce results, but mortality rates rise quickly over the course of 2-3 days.
Blood cultures are currently necessary for diagnosis because the infecting microbes can be present in the bloodstream at concentrations as low as a few cells per milliliter of blood, even while causing a life-threatening condition. These concentrations are too low to detect with conventional methods, so cultures are used to multiply the microbes and increase their concentrations to detectable levels. However, emulsion PCR has much higher sensitivity than bulk methods, so it may be possible to use microfluidics to directly encapsulate a blood sample in emulsion drops, run the PCR reaction, and identify the responsible species within a few hours.
[1] Vandijck et al., Dimensions of Critical Care Nursing, 27 (6): 244-248, 2008.