Impact of Bridged Nucleic Acid Positions within Blocking Oligonucleotides on DNA Amplification Inhibition in Wild-Type Blocking PCR

Abstract

Detecting low-frequency genetic mutations is crucial for genetic testing, especially in cancer diagnostics. Wild-type blocking PCR identifies these genetic mutations using a blocking oligonucleotide that is fully complementary to wild-type DNA. The blocking oligonucleotide selectively binds to wild-type DNA, inhibiting its amplification by DNA polymerase and allowing preferential amplification of mutant DNA. Bridged nucleic acids (BNAs), with high binding affinities for cDNA, are often incorporated into the blocking oligonucleotide to enhance inhibition. However, the effects of BNA positioning within the blocking oligonucleotide on wild-type DNA amplification inhibition are poorly understood. To address this issue, we evaluated the effects of different BNA positions on amplification inhibition efficacy by comparing blocking oligonucleotides with varying numbers of BNAs at the 5′ end, 3′ end, and central region. Results indicated that BNAs at the 5′ end enhanced the inhibition efficacy, whereas BNAs at the 3′ end notably diminished the inhibition efficacy. Likewise, increasing the number of BNAs in the central region generally decreased the inhibition efficacy. This is one of the first studies to report the importance of BNA positioning in the amplification inhibition efficacy of blocking oligonucleotides.