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

Kristian A Choate; Edward J Raack; Paul B Mann; Evan A Jones; Robert J Winn; Matthew J Jennings

doi:10.1093/biomethods/bpae012

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

Biol Methods Protoc. 2024 Feb 21;9(1):bpae012. doi: 10.1093/biomethods/bpae012. eCollection 2024.

Authors

Kristian A Choate^{1

2}, Edward J Raack^{2

3}, Paul B Mann^{2

3}, Evan A Jones⁴, Robert J Winn^{1

2}, Matthew J Jennings^{2

3}

Affiliations

¹ Department of Biology, Northern Michigan University, Marquette, MI, United States.
² Upper Michigan Brain Tumor Center, Marquette, MI, United States.
³ School of Clinical Sciences, Northern Michigan University, Marquette, MI, United States.
⁴ Applied Research Lab for Intelligence and Security, College Park, MD, United States.

Abstract

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.

Keywords: IDH (Isocitrate dehydrogenase); IDH1 R132H; Locked Nucleic Acids (LNA); Loop Mediated Isothermal Amplification (LAMP); SNV (single nucleotide variants); SNV discrimination; genotyping panel; glioma; mutant IDH1 glioma.