Charging YOYO-1 on capillary wall for online DNA intercalation and integrating this approach with multiplex PCR and bare narrow capillary-hydrodynamic chromatography for online DNA analysis.
Charging YOYO-1 on capillary wall for online DNA intercalation and integrating this approach with multiplex PCR and bare narrow capillary-hydrodynamic chromatography for online DNA analysis.
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DOI：
10.1021/ac504257b
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发表时间：
2015-02-03
影响因子：
7.4
通讯作者：
Liu, Shaorong
中科院分区：
文献类型：
--
作者：
Chen, Huang;Zhu, Zaifang;Lu, Joann Juan;Liu, Shaorong
多重聚合酶链反应（PCR）已广泛应用于高通量病原体鉴定。通常，用染料插入扩增的DNA片段，并通过DNA熔化曲线分析或凝胶电泳进行病原体鉴定。将DNA扩增和鉴定相结合是实现多重PCR效益最大化的逻辑路径。虽然PCR和凝胶电泳已经集成，但每次运行后补充凝胶是繁琐和耗时的。在本技术说明中，我们开发了一种解决此问题的方法。我们在毛细管内进行多重PCR，将扩增的片段转移到裸窄毛细管，并使用裸窄毛细管-水动力色谱（bac - hdc）在线测量它们的长度，这是我们实验室最近开发的一种用于自由溶液DNA分离的新技术。为了用YOYO-1（一种用于bac - hdc的荧光染料）插入DNA，我们用YOYO-1溶液冲洗毛细管柱；带正电的YOYO-1被吸附（或带电）到带负电的毛细管壁上。当DNA分子沿着色谱柱被分离时，它们与储存在毛细管壁上的YOYO-1发生反应，并与染料在线嵌入。单次YOYO-1充电，色谱柱可运行40次以上，但DNA峰的荧光信号强度逐渐降低。虽然在分离过程中会发生染料- dna插层，但它不影响保留时间、分离效率或分辨率。
Multiplex polymerase chain reaction (PCR) has been widely utilized for high-throughput pathogen identification. Often, a dye is used to intercalate the amplified DNA fragments, and identifications of the pathogens are carried out by DNA melting curve analysis or gel electrophoresis. Integrating DNA amplification and identification is a logic path toward maximizing the benefit of multiplex PCR. Although PCR and gel electrophoresis have been integrated, replenishing the gels after each run is tedious and time-consuming. In this technical note, we develop an approach to address this issue. We perform multiplex PCR inside a capillary, transfer the amplified fragments to a bare narrow capillary, and measure their lengths online using bare narrow capillary–hydrodynamic chromatography (BaNC-HDC), a new technique recently developed in our laboratory for free-solution DNA separation. To intercalate the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary column with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) onto the negatively charged capillary wall. As DNA molecules are driven down the column for separation, they react with the YOYO-1 stored on the capillary wall and are online-intercalated with the dye. With a single YOYO-1 charging, the column can be used for more than 40 runs, although the fluorescence signal intensities of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs during the separation, it does not affect the retention times, separation efficiencies, or resolutions.
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