Researchers sometimes genetically engineer microorganisms—such as fungi—to convert renewable feedstock materials into useful biofuel or biochemical products. Polymerase chain reaction (PCR), which allows researchers to quickly amplify DNA sequences for analysis, is one of the most essential tools in this process. When working to synthesize biofuels using fungi, PCR is particularly important for evaluating various strains of mutated fungi.
An Onerous Extraction System
A significant bottleneck in this process has been the DNA extraction step. Typically, following genetic modification of a fungus, researchers aim to identify the fungus with the mutation. This requires culturing the fungus into a mature biomass for DNA extraction, a prerequisite for PCR. The traditional DNA extraction from biomass is a tedious, multi-step process involving hazardous chemicals, and can take from three days to a week, significantly delaying biofuel research.
Squash Spore PCR
With support from the U.S. Department of Energy (DOE) Bioenergy Technologies Office (BETO) and Agile BioFoundry (ABF) consortium, Pacific Northwest National Laboratory (PNNL) researchers have found that simply squashing the fungal spores—rather than the fungal biomass—reliably yields high-quality DNA samples suitable for use in PCR. This discovery allows the researchers to amplify genome DNA orders of magnitude faster than conventional methods. The researchers are calling the method “squash spore PCR.”
While it may seem straightforward, squash spore PCR is counterintuitive for a couple reasons. The spores are small, tough to break open, and don’t respond well to the same extraction chemicals used on fungal biomass. In order to circumvent these obstacles, the researchers needed to go shockingly low-tech, mechanically squashing the spores in a buffer on a microscope slide with their thumbs. After squashing, the DNA is released into the buffer, which can then be used for PCR.
Enormous Advantages
Guoliang Yuan, an ABF biologist on PNNL’s Biological Conversion Team and first author of their research paper published in Fungal Biology and Biotechnology, said that this took the DNA extraction process from “at least three days to maximum three minutes.”
The technique is remarkably effective, with only a minor downside: squashing lots of samples by hand can be tiring and time-consuming. To address this, the team created a specialized plastic plate with 96 wells and a corresponding cover with pins to more efficiently squash large volumes of samples. Further refinements to the process are ongoing.
This thousand-fold speedup in the time it takes to extract DNA from fungal samples will help researchers at PNNL and across the bioproducts community iterate and analyze mutated fungi much more quickly, accelerating biofuels development. One of the first applications for squash spore PCR at PNNL has been extracting DNA from mutated strains of Asperillus niger, a fungus species that serves as the basis for important bioproducts like 3-HP—an intermediate chemical used in everything from electronics to clothing.
This work was supported by BETO and the ABF, a consortium of seven DOE national laboratories that operate as a distributed biofoundry in collaboration with industry and academia. Funded by BETO, ABF’s work supports BETO’s goals to develop sustainable aviation fuels and decarbonize energy-intensive industries. ABF collaborates with industry and academia to accelerate innovation and adopt new biomanufacturing methods.