Purification of HIV RNA from Serum Using a Polymer Capture Matrix in a Microfluidic Device

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• 作者：Brian E. Root ; Abhishek K. Agarwal ; David M. Kelso ; Annelise E. Barron
• 刊名：Analytical Chemistry
• 出版年：2011
• 出版时间：February 1, 2011
• 年：2011
• 卷：83
• 期：3
• 页码：982-988
• 全文大小：815K
• 年卷期：v.83,no.3(February 1, 2011)
• ISSN：1520-6882

文摘

In this report, we demonstrate the purification of DNA and RNA from a 10% serum sample using an oligonucleotide capture matrix. This approach provides a one-stage, completely aqueous system capable of purifying both RNA and DNA for downstream PCR amplification. The advantages of utilizing the polymer capture matrix method in place of the solid-phase extraction method is that the capture matrix eliminates both guanidine and the 2-propanol wash that can inhibit downstream PCR and competition with proteins for the binding sites that can limit the capacity of the device. This method electrophoreses a biological sample (e.g., serum) containing the nucleic acid target through a polymer matrix with covalently bound oligonucleotides. These capture oligonucleotides selectively hybridize and retain the target nucleic acid, while the other biomolecules and reagents (e.g., SDS) pass through the matrix to waste. Following this purification step, the solution can be heated above the melting temperature of the capture sequence to release the target molecule, which is then electrophoresed to a recovery chamber for subsequent PCR amplification. We demonstrate that the device can be applied to purify both DNA and RNA from serum. The gag region of HIV at a starting concentration of 37.5 copies per microliter was successfully purified from a 10% serum sample demonstrating the applicability of this method to detect viruses present in low copy numbers.