Rapid and robust squashed spore/colony PCR of industrially important fungi

Abstract

Background Fungi have been utilized for centuries in medical, agricultural, and industrial applications. Development of systems biology techniques has enabled the design and metabolic engineering of these fungi to produce novel fuels, chemicals, and enzymes from renewable feedstocks. Many genetic tools have been developed for manipulating the genome and creating mutants rapidly. However, screening and confirmation of transformants remain an inefficient step within the design, build, test, and learn cycle in many industrial fungi because extracting fungal genomic DNA is laborious, time-consuming, and involves toxic chemicals. Results In this study we developed a rapid and robust technique called “Squash-PCR” to break open the spores and release fungal genomic DNA as a template for PCR. The efficacy of Squash-PCR was investigated in eleven different filamentous fungal strains. Clean PCR products with high yields were achieved in all tested fungi. Spore age and type of DNA polymerase did not affect the efficiency of Squash-PCR. However, spore concentration was found to be the crucial factor for Squash-PCR in Aspergillus niger, with the dilution of starting material often resulting in higher PCR product yield. We then further evaluated the applicability of the squashing procedure for nine different yeast strains. We found that Squash-PCR can be used to improve the quality and yield of colony PCR in comparison to direct colony PCR in the tested yeast strains. Conclusion The developed technique will enhance the efficiency of screening transformants and accelerate genetic engineering in filamentous fungi and yeast.

Development of a whole-cell SELEX process to select species-specific aptamers against Aspergillus niger

“…The bound sequences were recovered by elution and subsequent precipitation in ethanol to remove the conidia from the recovered ssDNA before PCR ampli cation. This step was introduced due to the large amounts of PCR inhibitors present in A. niger conidia, among which melanin, that would otherwise strongly interfere with the ampli cation reaction (Eckhart et al, 2000;Fraczek et al, 2019;Yuan et al, 2023). After recovery, the precipitated ssDNA was subjected to two independent test-PCRs (dnc1 and dnc2) for the determination of the optimal number of cycles for enrichment PCR.…”

Simple and Effective Squash-PCR for Rapid Genotyping of Industrial Microalgae

“…( 4) Collect the cell solution with a pipette and dilute it with 10 µL of ultrapure water or PBS-Tween 20 as required. For more detailed instructions, refer to Yuan et al [7].…”

“…This method is built upon the "squash" technique, where whole cells are flattened on a glass surface and examined under a microscope. The effectiveness of Squash-PCR has been demonstrated across distinct filamentous fungal and yeast strains, and has consistently yielded high efficiency [7].…”

“…In contrast, we have recently developed a novel approach known as Squash-PCR for microbial spore/colony PCR [7]. This method is built upon the "squash" technique, where whole cells are flattened on a glass surface and examined under a microscope.…”