A refined two-step oligoribonucleotide interference-PCR method for precise discrimination of nucleotide differences

Abstract

We previously developed oligoribonucleotide (ORN) interference-PCR (ORNi-PCR), in which an ORN hybridises with a complementary DNA sequence and inhibits PCR amplification across the sequence in a sequence-specific manner. Suppression of target amplification by ORNi-PCR can be used to detect nucleotide differences such as mutations in a target sequence. In the present study, we refined the ORNi-PCR method and established a detailed technical protocol to precisely discriminate single-nucleotide differences. We first revealed that a two-step (denaturing and annealing plus elongation) rather than a standard three-step (denaturing, annealing and elongation) method is more suitable for stably hybridising an ORN to its target DNA sequence for sequence-specific suppression of target amplification. We then optimised the ORNi-PCR method using two-step cycles and established a step-by-step technical protocol. The optimised Two-Step ORNi-PCR method could discriminate single-nucleotide differences in genomic DNA or cDNA introduced by genome editing or mutations in cancer cells. In addition, we showed that Two-Step ORNi-PCR can detect the cancer cells possessing a single nucleotide mutation in a target locus mixed with a large number of cells harboring wild-type sequences in the locus so that the number of the cancer cells is only 0.2% of the total cell number. Two-Step ORNi-PCR is useful for simple, precise, cost-effective and positive detection of nucleotide differences in a wide range of molecular biology and medical applications.

Discrimination of CpG Methylation Status and Nucleotide Differences in Tissue Specimen DNA by Oligoribonucleotide Interference-PCR

“…In this experiment, we investigated whether ORNi-PCR could discriminate the polymorphism in DNA extracted from AFPE rat liver specimens. To this end, we designed ORN_GA2 and ORN_GA3, 20-base and 22-base ORNs, complementary to the GA-type sequence, respectively, as shown in Figure 2 A. DNA was extracted from sliced AFPE rat liver specimens and used for ORNi-PCR, as described previously [ 7 ]. As shown in Figure 2 B,C, ORNi-PCR with ORN_GA3 suppressed the amplification of the GA-type but not TT-type Gstm1 .…”

“…We extracted DNA from the FFPE specimens and investigated whether ORNi-PCR could detect mutated EGFR DNA, corresponding to EGFR L858R (CTG → CGG), as shown in Figure 3 A. As shown in Figure 3 A–D, ORNi-PCR with ORN_EGFR_L858 [ 7 ] successfully detected the mutated EGFR, even when it accounted for only 5% of total EGFR . By contrast, ORN_EGFR_L858 did not affect the amplification of the irrelevant GAPDH gene, as shown in Figure 3 E. Thus, ORNi-PCR can detect a nucleotide difference in FFPE specimen DNA, so long as >5% of the corresponding sequence includes the difference.…”

“…By contrast, ORN_EGFR_L858 did not affect the amplification of the irrelevant GAPDH gene, as shown in Figure 3 E. Thus, ORNi-PCR can detect a nucleotide difference in FFPE specimen DNA, so long as >5% of the corresponding sequence includes the difference. We previously studied the sensitivity of ORNi-PCR with genomic DNA (gDNA), purified from cultured cell lines using the same method as employed in Figure 2 D [ 7 ]. In our previous study, ORNi-PCR detected the mutation corresponding to EGFR L858R when the mutated EGFR accounted for >0.1% of the total EGFR .…”

“…ORNs can be flexibly designed and cost-effectively synthesized, representing advantages over other blockers, such as artificial nucleic acids. We showed that ORNi-PCR can discriminate differences in nucleotide sequences, such as single-nucleotide mutations in cancer cells, as well as indel mutations introduced by genome-editing [ 6 , 7 , 8 , 9 ]. Thus, ORNi-PCR is a useful tool for use in the biological and medical fields.…”

“…In previous work, we used DNA extracted from cultured cell lines or complementary DNA (cDNA) reverse-transcribed from freshly prepared RNA for ORNi-PCR [ 6 , 7 , 8 , 9 ]. Such DNA is generally of high quality and can readily be used as a template for ORNi-PCR or conventional PCR.…”