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

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Single-nucleotide variants (SNVs) are crucial for understanding human health and disease.
• Current genotyping methods often involve lengthy procedures, including nucleic acid extraction, and extended analytical times.
• Rapid and efficient detection of specific SNVs is needed for timely diagnosis and research.

Purpose of the Study:

• To develop a fast and efficient protocol for allelic discrimination of isocitrate dehydrogenase 1 R132 (IDH1-R132) variants.
• To integrate locked nucleic acids (LNAs) with loop-mediated isothermal amplification (LAMP) for enhanced genotyping.
• To evaluate the assay's performance in purified DNA and complex biological samples like tumor lysates.

Main Methods:

• Utilized self-annealing loop primers incorporating locked nucleic acids (LNAs).
• Employed loop-mediated isothermal amplification (LAMP) for isothermal DNA amplification and detection.
• Assessed specificity using synthetic DNA and glioma tumor lysates with known IDH1-R132 mutational status.
• Evaluated a pH-based colorimetric indicator and an absorbance ratio end-point format for visual and objective interpretation.

Main Results:

• Demonstrated specific single-nucleotide variant (SNV) discrimination for five IDH1-R132 variants.
• Achieved genotyping results in approximately 35 minutes directly from glioma tumor lysates, bypassing nucleic acid extraction.
• Showcased the assay's ability to detect variants in the presence of excess wild-type DNA.
• Confirmed high reproducibility with no false-positive or false-negative results.

Conclusions:

• The LNA-LAMP assay offers a rapid, specific, and reproducible method for genotyping IDH1-R132 variants.
• This protocol eliminates the need for nucleic acid extraction, significantly reducing assay time.
• The assay is suitable for detecting single base differences in various sample types, including challenging samples with mixed genotypes.