CE combined with rolling circle amplification for sensitive DNA detection

被引：14

作者：

Li, Ni ^{[1
]}

Li, Jishan ^{[1
]}

Zhong, Wenwan ^{[1
]}

机构：

[1] Univ Calif Riverside, Dept Chem, Riverside, CA 92521 USA

来源：

ELECTROPHORESIS | 2008年 / 29卷 / 02期

关键词：

CE; DNA detection and quantification; enzyme digestion; rolling circle amplification;

D O I：

10.1002/elps.200700410

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Here we describe an assay which combines CE with rolling circle amplification (RCA) for sensitive DNA detection and quantification. RCA is an isothermal DNA replication technique that generates a long ssDNA with tandem repeats. It requires simpler temperature control in reaction and offers higher sequence specificity and greater quantitation capability compared to other amplification technologies. In this study, RCA amplified the DNA target via a circular template, and the product was digested into monomers for CE analysis. Less than 2 fmol of the DNA target could easily be detected using this RCA-CE assay and the assay has a dynamic range of two orders of magnitudes. Moreover, simultaneous detection of both the target DNA and the internal standard was achieved by designing two padlock probes with different sizes, which could significantly improve the quantification accuracy. The RCA-CE assay is easy to perform, readily adaptable for detection of multiple targets because of the high resolution power of CE, and is compatible with other applications employing RCA as a signal amplification tool. Additionally, this assay can be used with a capillary array system to perform sensitive, high-throughput genetic screening.

引用

页码：424 / 432

页数：9

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