Capture and Direct Amplification of DNA on Chitosan Microparticles in a Single PCR-Optimal Solution

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• 作者：Kunal R. Pandit ; Imaly A. Nanayakkara ; Weidong Cao ; Srinivasa R. Raghavan ; Ian M. White
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：November 3, 2015
• 年：2015
• 卷：87
• 期：21
• 页码：11022-11029
• 全文大小：310K
• ISSN：1520-6882

文摘

While nucleic acid amplification tests have great potential as tools for rapid diagnostics, complicated sample preparation requirements inhibit their use in near-patient diagnostics and low-resource-setting applications. Recent advancements in nucleic acid purification have leveraged pH-modulated charge switching polymers to reduce the number of steps required for sample preparation. The polycation chitosan (pK_a 6.4) has been used to efficiently purify DNA by binding nucleic acids in acidic buffers and then eluting them at a pH higher than 8.0. Though it is an improvement over conventional methods, this multistep procedure has not transformed the application of nucleic acid amplification assays. Here we describe a simpler approach using magnetic chitosan microparticles that interact with DNA in a manner that has not been reported before. The microparticles capture DNA at a pH optimal for PCR (8.5) just as efficiently as at low pH. Importantly, the captured DNA is still accessible by polymerase, enabling direct amplification from the microparticles. We demonstrate quantitative PCR from DNA captured on the microparticles, thus eliminating nearly all of the sample preparation steps. We anticipate that this new streamlined method for preparing DNA for amplification will greatly expand the diagnostic applications of nucleic acid amplification tests.