Hairpin Structure Facilitates Multiplex High-Fidelity DNA Amplification in Real-Time Polymerase Chain Reaction.
Hairpin Structure Facilitates Multiplex High-Fidelity DNA Amplification in Real-Time Polymerase Chain Reaction.
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DOI：
10.1021/acs.analchem.2c00575
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发表时间：
2022-06
影响因子：
7.4
通讯作者：
Ke Zhang;A. Pinto;L. Cheng;P. Song;P. Dai;M. Wang;Luis Rodriguez;Cailin Weller;D. Zhang-D.
中科院分区：
文献类型：
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作者：
Ke Zhang;A. Pinto;L. Cheng;P. Song;P. Dai;M. Wang;Luis Rodriguez;Cailin Weller;D. Zhang-D.
在临床和生物学上，检测罕见的DNA序列变异对于癌症的早期诊断或耐药突变的鉴定是必不可少的。一些常见的基于定量聚合酶链式反应(QPCR)的变体检测方法受到检测限(LOD)的限制，因为用于这些方法的DNA聚合酶有很高的聚合酶错掺合率，因此检测灵敏度有时不能令人满意。随着校对活动的进行，高保真(HiFi)DNA聚合酶的保真度提高了50到250倍。然而，目前还没有合适的基于探针的设计作为荧光指示剂，从而限制了像变体检测这样的HiFi DNA聚合酶的应用。我们提出了一种封闭体系，它由一个带有荧光团的5‘-悬垂的引物和一个带有短茎发夹的探针和一个3’-猝灭剂的修饰组成。我们证明，该封闭系统允许多路HiFi qPCR反应，并与目前的变体-富集法兼容，可将LOD提高10倍。因此，该封闭体系可以作为一种有效的荧光指示剂应用于HiFi qPCR反应，并使HiFi DNA聚合酶应用于不同的检测方法，以提高检测灵敏度。
Clinically and biologically, it is essential to detect rare DNA-sequence variants for early cancer diagnosis or drug-resistance mutation identification. Some of the common quantitative polymerase chain reaction (qPCR)-based variant detection methods are restricted in the limit of detection (LoD) because the DNA polymerases used for these methods have a high polymerase misincorporation rate; thus, the detection sensitivity is sometimes unsatisfactory. With the proofreading activity, high-fidelity (HiFi) DNA polymerases have a 50- to 250-fold higher fidelity. However, there are currently no proper probe-based designs functioning as the fluorescence indicator allowing multiplexed HiFi qPCR reactions, thus restricting the application of HiFi DNA polymerases like the variant detection. We presented the occlusion system, composed of a 5'-overhanged primer with a fluorophore modification and a probe with a short-stem hairpin and a 3' quencher modification. We demonstrated that the occlusion system allowed multiplexing HiFi qPCR reaction, and it was compatible with the current variant-enrichment method to improve the LoD up to 10-fold. Thus, the occlusion system satisfactorily functioned as an efficient fluorescence indicator in HiFi qPCR reactions and allowed the application of HiFi DNA polymerases in variant detection methods to improve detection sensitivity.