High-efficiency and high-fidelity ssDNA circularisation via the pairing of five 3′-terminal bases to assist LR-LAMP for the genotyping of single-nucleotide polymorphisms
High-efficiency and high-fidelity ssDNA circularisation via the pairing of five 3′-terminal bases to assist LR-LAMP for the genotyping of single-nucleotide polymorphisms
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DOI：
10.1039/d2an01042a
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发表时间：
2022-08-09
期刊：
影响因子：
4.2
通讯作者：
Kageyama, Koji
中科院分区：
文献类型：
--
作者：
Li, Taiwen;Zou, Huayan;Kageyama, Koji
T4 DNA连接酶的保真性差一直限制了单核苷酸多态性（SNP）的简单检测，且仅适用于某些特殊的SNP类型。本研究建立了一种基于高保真单链环化的高灵敏度和特异性的SNP检测方法。它使用T4 DNA连接酶和滚环扩增（RCA）加环介导等温扩增（LAMP）。令人惊讶的是，环化阶段的效率大大提高。通过线性单链DNA（I-DNA）上5 '端的长配对碱基和3 '端的仅5个碱基之间的独特配对模式，连接保真度几乎是完美的。随后，进行LR-LAMP并与环化步骤组合用于SNP的简单检测。结果显示，即使是100 μ M的靶标也可以被正确检测，并且可以分别通过裸眼可视化或荧光检测来分析0.1%或甚至0.01%的突变率。利用基因组DNA样本对该方法进行了验证，结果表明该方法能够有效区分致病疫霉RPA 190-T1145 A的SNPs。这种策略可能在单链DNA的环化和检测任意SNP中发挥重要作用。
The poor fidelity of T4 DNA ligase has always limited the simple detection of single-nucleotide polymorphisms (SNPs) and is only applicable to some special SNP types. This study developed a highly sensitive and specific detection method for SNPs based on high-fidelity single-stranded circularisation. It used T4 DNA ligase and rolling circle amplification (RCA) plus loop-mediated isothermal amplification (LAMP). Surprisingly, the cyclisation stage's efficiency greatly improved. The ligation fidelity was almost perfect via the unique pairing pattern between a long-paired base at the 5 ' terminus and only five bases at the 3 ' terminus on linear single-stranded DNA (l-DNA). Subsequently, LR-LAMP was performed and combined with the circularisation step for the simple detection of SNPs. The results showed that even 100 aM targets could be detected correctly and that a mutation rate of 0.1% or even 0.01% could be analysed via naked-eye visualisation or fluorescence detection, respectively. In addition, genomic DNA samples were used to evaluate the method, which indicated that it could effectively distinguish the SNPs of RPA190-T1145A in Phytophthora infestans. This strategy may play an important role in both circularisation of single-stranded DNA and detecting arbitrary SNPs.