Simultaneous Detection of the T790M and L858R Mutations in the EGFR Gene by Oligoribonucleotide Interference-PCR

Abstract

A de novo single-nucleotide mutation in the EGFR gene can cause the development of lung cancer. EGFR tyrosine kinase inhibitors (EGFR-TKIs) are used for clinical treatment of such lung cancers, but acquired resistance often mitigates their efficacy. Accordingly, monitoring of de novo and acquired nucleotide mutations is essential for clinical treatment of lung cancers with EGFR-TKIs. Previously, we reported that oligoribonucleotide interference-PCR (ORNi-PCR) can accurately and cost-effectively detect single-nucleotide mutations. In this study, we applied ORNi-PCR to simultaneous detection of the de novo L858R and acquired T790M mutations in the EGFR gene in lung cancer cells. First, we established optimal experimental conditions for ORNi-PCR to simultaneously detect the two single-nucleotide mutations in genomic DNA from lung cancer cells. The conditions we established could also be used for ORNi-PCR using complementary DNA reverse-transcribed from extracted RNA. We found that ORNi-PCR could detect lung cancer cells possessing both single-nucleotide mutations among a large number of cells harboring wild-type sequences, even when the cancer cells constituted less than ~0.2% of all cells. Our findings demonstrate that ORNi-PCR can simultaneously detect multiple single-nucleotide mutations in a gene of interest and might therefore be useful for simultaneous detection of EGFR mutations in clinical examinations.

Sequence-specific inhibition of reverse transcription by recombinant CRISPR/dCas13a ribonucleoprotein complexes in vitro

“…3A and B; Supplementary Table S1 ). Because we previously confirmed that wild-type EGFR mRNA is expressed in various lung cells, such as a normal lung fibroblast cell line, MRC-5, and a nonsmall cell lung carcinoma cell line, NCI-H1299 [ 11 ], total RNA was extracted from MRC-5 and used as a model RNA. RT reactions were performed using total RNA and the oligo(dT) primer in the presence of the dCas13a/gRNA_EGFR RNP complex, the dCas13a protein alone, or the gRNA alone.…”

New Insights into LINC00346 and its Role in Disease

“…Molecular targeted therapy shows advantages for many diseases, especially malignancies (Baba et al, 2019;Becker et al, 2020;Xu P. et al, 2020). LncRNAs contribute to disease progression through the regulation of cellular pathways (Li Z.-W. et al, 2020;Sun and Wu, 2020).…”

Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences

“…Previously, we developed an oligoribonucleotide (ORN)-mediated blocking PCR named ORN interference-PCR (ORNi-PCR) (Figure A) . ORNi-PCR clearly discriminates nucleotide differences such as indel mutations (which are introduced by genome editing events), single-nucleotide mutations in cancer cells, and nucleotide polymorphisms. − In general, DNA polymerases lacking 5′–3′ exonuclease activity are used for ORNi-PCR because those retaining the 5′–3′ exonuclease activity, such as Taq DNA polymerase, can remove a hybridized ORN during DNA extension. In fact, we observed that DNA amplification by Taq DNA polymerase is not inhibited by an ORN .…”