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

IF 1.7 4区 医学 Q3 PHARMACOLOGY & PHARMACY
Takuma Yamashita, Yoshinori Tsukumo, Takenori Yamamoto, Eriko Uchida, Tokuyuki Yoshida, Yasunori Uchida, Takao Inoue
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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.

阻断寡核苷酸桥接核酸位置对野生型阻断PCR中DNA扩增抑制的影响。
检测低频基因突变对基因检测至关重要,尤其是在癌症诊断中。野生型阻断PCR使用与野生型DNA完全互补的阻断寡核苷酸识别这些基因突变。阻断性寡核苷酸选择性地与野生型DNA结合,抑制其被DNA聚合酶扩增,并允许突变DNA优先扩增。桥接核酸(BNAs)对cDNA具有高结合亲和力,通常被并入阻断寡核苷酸中以增强抑制作用。然而,BNA在阻断寡核苷酸内定位对野生型DNA扩增抑制的影响尚不清楚。为了解决这一问题,我们通过比较5‘端、3’端和中心区域不同BNA数量的阻断寡核苷酸,评估了不同BNA位置对扩增抑制效果的影响。结果表明,位于5‘端的BNAs增强了抑制作用,而位于3’端的BNAs明显降低了抑制作用。同样,增加中央区域BNAs的数量通常会降低抑制效果。这是首次报道BNA定位在阻断寡核苷酸扩增抑制效果中的重要性的研究之一。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
3.50
自引率
5.00%
发文量
247
审稿时长
2 months
期刊介绍: Biological and Pharmaceutical Bulletin (Biol. Pharm. Bull.) began publication in 1978 as the Journal of Pharmacobio-Dynamics. It covers various biological topics in the pharmaceutical and health sciences. A fourth Society journal, the Journal of Health Science, was merged with Biol. Pharm. Bull. in 2012. The main aim of the Society’s journals is to advance the pharmaceutical sciences with research reports, information exchange, and high-quality discussion. The average review time for articles submitted to the journals is around one month for first decision. The complete texts of all of the Society’s journals can be freely accessed through J-STAGE. The Society’s editorial committee hopes that the content of its journals will be useful to your research, and also invites you to submit your own work to the journals.
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