公开(公告)号：US20140005066A1

公开(公告)日：2014-01-02

申请号：US13932719

申请日：2013-07-01

Applicant: ADVANCED LIQUID LOGIC INC.

Inventor： Deborah Boles ,  Lisa Perkins ,  Allen E. Eckhardt ,  Jennifer Foley

IPC: C12Q1/70 ,  C12Q1/68

CPC classification number: C12Q1/703 ,  C12Q1/686 ,  C12Q1/701 ,  C12Q2537/143 ,  C12Q2561/113 ,  C12Q2563/107 ,  C12Q2563/159 ,  C12Q2565/102

Abstract: The present invention provides a droplet actuator device and methods for multiplexed PCR amplification and detection of target amplicons within a single droplet. The methods of the invention combine quantitative real-time PCR (qPCR) amplification with fluorescence-based sequence specific detection technologies for amplified DNA. In one embodiment, fluorescently-labeled oligonucleotide probes may be used for hybridization-based multiplexed detection of target amplicons. The methods of the invention generally involve combining the necessary reactants to form a PCR-ready droplet and thermal cycling the droplet at temperatures sufficient to result in amplification of one or more target nucleic acids. Fluorescence-based detection techniques may be used for end-point or real-time analysis of DNA amplification. For end-point analysis, the accumulation of a signal, e.g., a fluorescence signal, is measured after the amplification of the target sequence is complete. For real-time analysis, the signal is measured while the amplification reaction is in progress.

Abstract translation: 本发明提供了一种液滴致动器装置和用于在单个液滴内多重PCR扩增和检测靶扩增子的方法。 本发明的方法将定量实时PCR（qPCR）扩增与扩增DNA的基于荧光的序列特异性检测技术相结合。 在一个实施方案中，荧光标记的寡核苷酸探针可用于靶扩增子的杂交多重检测。 本发明的方法通常包括组合必需的反应物以形成PCR准备的液滴并在足以导致一种或多种靶核酸扩增的温度下热循环该液滴。 基于荧光的检测技术可用于DNA扩增的终点或实时分析。 对于终点分析，在靶序列的扩增完成之后测量信号的累积，例如荧光信号。 对于实时分析，在扩增反应进行期间测量信号。