Rapid IDH1 -R132 genotyping panel utilizing locked nucleic acid loop-mediated isothermal amplification.

Autor:	Choate KA; Department of Biology, Northern Michigan University, Marquette, MI, United States.; Upper Michigan Brain Tumor Center, Marquette, MI, United States., Raack EJ; Upper Michigan Brain Tumor Center, Marquette, MI, United States.; School of Clinical Sciences, Northern Michigan University, Marquette, MI, United States., Mann PB; Upper Michigan Brain Tumor Center, Marquette, MI, United States.; School of Clinical Sciences, Northern Michigan University, Marquette, MI, United States., Jones EA; Applied Research Lab for Intelligence and Security, College Park, MD, United States., Winn RJ; Department of Biology, Northern Michigan University, Marquette, MI, United States.; Upper Michigan Brain Tumor Center, Marquette, MI, United States., Jennings MJ; Upper Michigan Brain Tumor Center, Marquette, MI, United States.; School of Clinical Sciences, Northern Michigan University, Marquette, MI, United States.
Jazyk:	angličtina
Zdroj:	Biology methods & protocols [Biol Methods Protoc] 2024 Feb 21; Vol. 9 (1), pp. bpae012. Date of Electronic Publication: 2024 Feb 21 (Print Publication: 2024).
DOI:	10.1093/biomethods/bpae012
Abstrakt:	While the detection of single-nucleotide variants (SNVs) is important for evaluating human health and disease, most genotyping methods require a nucleic acid extraction step and lengthy analytical times. Here, we present a protocol which utilizes the integration of locked nucleic acids (LNAs) into self-annealing loop primers for the allelic discrimination of five isocitrate dehydrogenase 1 R132 ( IDH1 -R132) variants using loop-mediated isothermal amplification (LAMP). This genotyping panel was initially evaluated using purified synthetic DNA to show proof of specific SNV discrimination. Additional evaluation using glioma tumor lysates with known IDH1 -R132 mutational status demonstrated specificity in approximately 35 min without the need for a nucleic acid extraction purification step. This LNA-LAMP-based genotyping assay can detect single base differences in purified nucleic acids or tissue homogenates, including instances where the variant of interest is present in an excess of background wild-type DNA. The pH-based colorimetric indicator of LNA-LAMP facilitates convenient visual interpretation of reactions, and we demonstrate successful translation to an end-point format using absorbance ratio, allowing for an alternative and objective approach for differentiating between positive and negative reactions. Importantly, the LNA-LAMP genotyping panel is highly reproducible, with no false-positive or false-negative results observed. (© The Author(s) 2024. Published by Oxford University Press.)
Databáze:	MEDLINE
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