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使用靶标引发的分支滚环扩增对单核苷酸多态性进行均相无标记荧光检测。

Homogeneous and label-free fluorescence detection of single-nucleotide polymorphism using target-primed branched rolling circle amplification.

作者信息

Cheng Yongqiang, Li Zhengping, Zhang Xian, Du Baoan, Fan Yongshan

机构信息

College of Chemistry and Environment Science, Hebei University, Baoding, Hebei Province 071002, People's Republic of China.

出版信息

Anal Biochem. 2008 Jul 15;378(2):123-6. doi: 10.1016/j.ab.2008.03.040. Epub 2008 Mar 28.

Abstract

We present a simple, sensitive, and cost-effective fluorescent assay of single-nucleotide polymorphism (SNP) with target-primed branched rolling circle amplification (TPBRCA). Designed padlock probe is circularized after perfect hybridization to mutant DNA. Then rolling circle amplification (RCA) reaction can be initiated from the mutant DNA that acts as primer and generates a long tandem single-stranded DNA (ssDNA) product. At the same time, the introduction of a reverse primer complementary to the target-primed RCA products leads to the branched RCA and eventually generates the various lengths of ssDNA and double-stranded DNA products, which are sensitively detected using SYBR Green I (SG) fluorescence dye. In contrast, the wild DNA contains a single mismatched base with the padlock probe and primes only a limited extension with the unligated padlock probe, generating weak background fluorescence with the addition of SG. Due to the excellent specificity and powerful amplification of TPBRCA reaction, the mutant DNA was distinctively differentiated from the wild DNA in a homogeneous and label-free manner. The assay is sensitive and specific enough to detect 5-amol (8.6-fM) mutant DNA strands. It was possible to accurately determine the mutant allele frequency as low as 1.0%.

摘要

我们展示了一种利用靶标引发的分支滚环扩增(TPBRCA)对单核苷酸多态性(SNP)进行简单、灵敏且经济高效的荧光检测方法。设计的锁式探针与突变型DNA完美杂交后环化。然后滚环扩增(RCA)反应可从作为引物的突变型DNA起始,生成一条长串联单链DNA(ssDNA)产物。同时,引入与靶标引发的RCA产物互补的反向引物会导致分支RCA,最终生成各种长度的ssDNA和双链DNA产物,使用SYBR Green I(SG)荧光染料可灵敏地检测到这些产物。相比之下,野生型DNA与锁式探针存在单个错配碱基,仅用未连接的锁式探针引发有限的延伸,添加SG后产生较弱的背景荧光。由于TPBRCA反应具有出色的特异性和强大的扩增能力,突变型DNA能以均相且无标记的方式与野生型DNA明显区分开来。该检测方法灵敏且特异,足以检测5个amol(8.6 fM)的突变型DNA链。能够准确测定低至1.0%的突变等位基因频率。

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