Integrated nucleic acid analysis

Inventors

Tan, Eugene • Lam, Heung Chuan • Bogdanov, Valery Leonidovich • Kellogg, Gregory John • Wright, John A. • Thomann, Ulrich Hans • Selden, Richard F.

Assignees

Ande Corp

Abstract

The present disclosure provides fully integrated microfluidic systems to perform nucleic acid analysis. These processes include sample collection, nucleic acid extraction and purification, amplification, sequencing, and separation and detection. The present disclosure also provides optical detection systems and methods for separation and detection of biological molecules. In particular, the various aspects of the invention enable the simultaneous separation and detection of a plurality of biological molecules, typically fluorescent dye-labeled nucleic acids, within one or a plurality of microfluidic chambers or channels. The nucleic acids can be labeled with at least 6 dyes, each having a unique peak emission wavelength. The present systems and methods are particularly useful for DNA fragment sizing applications such as human identification by genetic fingerprinting and DNA sequencing applications such as clinical diagnostics.

Core Innovation

The invention is directed to DNA analysis of at least two samples on a biochip using multiplex PCR amplification followed by liquid flow and electric-field separation of amplified nucleic acid fragments, and subsequent optical detection. Multiplex PCR amplification is induced in separated reaction reservoirs within a first region of the biochip, each reservoir being designated for multiplex PCR amplification based on a respective sample. The amplified nucleic acid fragments are moved to separation units in a second region of the biochip, where electric fields separate the fragments by size within respective, fluidically separated separation channels.

The multiplex PCR primers are labeled with six or more fluorescent dyes, each having a unique peak emission wavelength, so that amplified nucleic acid fragments are generated labeled with said 6 or more fluorescent dyes. The detection architecture detects fluorescence from at least six dyes labeled to one or more labeled nucleic acid fragments, and the separated nucleic acid fragments labeled with the at least 6 fluorescent dyes are detected in each of the separation channels using a wavelength-dispersive optical detector.

In the optical detector, a wavelength dispersive element selected from prisms, diffraction gratings, transmission gratings, holographic diffraction gratings and spectrographs disperses light according to light wavelength into at least six wavelength components over a physical space according to light wavelength. Detection elements corresponding to the wavelength components, or a multi-anode photomultiplier tube, simultaneously collect detection information for each wavelength component, enabling wavelength-discriminated fluorescence readout of multiple dyes with unique peak emission wavelengths.

Claims Coverage

The independent claim coverage describes a biochip-based method for DNA analysis of at least two samples with multiplex PCR using six-or-more uniquely dyed primers, followed by electric-field size separation in fluidically separated channels and wavelength-dispersive optical fluorescence detection using at least six dye channels. Additional refinements mention at least eight dyes and detector architectures using dichroic mirrors or bandpass filters.

The inventive features combine multiplex PCR on a biochip, size separation in fluidically separated channels, and wavelength-dispersive fluorescence detection for multi-dye readout, with further refinements to template handling, detector architecture, sample multiplexing, and higher dye count.

Stated Advantages

Documented Applications

No documented applications found