Adaptive PCR Based on Hybridization Sensing of Mirror-Image l-DNA

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• 作者：Nicholas M. AdamsWilliam E. Gabella ; Austin N. Hardcastle ; Frederick R. Haselton
• 刊名：Analytical Chemistry
• 出版年：2017
• 出版时间：January 3, 2017
• 年：2017
• 卷：89
• 期：1
• 页码：728-735
• 全文大小：516K
• ISSN：1520-6882

文摘

Polymerase chain reaction (PCR) is dependent on two key hybridization events during each cycle of amplification, primer annealing and product melting. To ensure that these hybridization events occur, current PCR approaches rely on temperature set points and reaction contents that are optimized and maintained using rigid thermal cycling programs and stringent sample preparation procedures. This report describes a fundamentally simpler and more robust PCR design that dynamically controls thermal cycling by more directly monitoring the two key hybridization events during the reaction. This is achieved by optically sensing the annealing and melting of mirror-image l-DNA analogs of the reaction’s primers and targets. Because the properties of l-DNA enantiomers parallel those of natural d-DNAs, the l-DNA reagents indicate the cycling conditions required for effective primer annealing and product melting during each cycle without interfering with the reaction. This hybridization-sensing approach adapts in real time to variations in reaction contents and conditions that impact primer annealing and product melting and eliminates the requirement for thermal calibrations and cycling programs. Adaptive PCR is demonstrated to amplify DNA targets with high efficiency and specificity under both controlled conditions and conditions that are known to cause traditional PCR to fail. The advantages of this approach promise to make PCR-based nucleic acid analysis simpler, more robust, and more accessible outside of well-controlled laboratory settings.