Integrated systems for the multiplexed amplification and detection of six and greater dye labeled fragments
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.
1. An optical detector comprising;
one or more light sources positioned for illuminating one or a plurality of detection positions on a substrate, said substrate comprising a thermal cycling chamber adapted for multiplexed amplification in one PCR reaction, such that primers labeled with 6 or more fluorescent dyes each having a unique peak emission wavelength are used to generate amplified nucleic acid fragments labeled with said 6 or more fluorescent dyes, said thermal cycling chamber in fluid communication with at least one channel on said substrate, wherein each channel comprises a detection position;
a mirror to scan said one or more light sources sequentially between detection positions;
one or a plurality of first optical elements positioned for collecting and directing light emanating from the detection positions on the substrate; and
a light detector positioned to accept light directed from the one or plurality of first optical elements, wherein the light detector comprises a wavelength dispersive element to disperse the light from the one or plurality of first optical elements according to light wavelength into at least 6 wavelength components and, the wavelength dispersive element is positioned to provide at least a portion of the dispersed at least 6 wavelength components to at least 6 detection elements, wherein
each of the detection elements are in communication with a first control element for simultaneously collecting detection information from each of the detection elements and, wherein said light detector detects fluorescence from at least 6 dyes labeled to one or more biological molecules, each dye having a unique peak emission wavelength.
2. The optical detector of claim 1 , wherein the biological molecules are nucleic acids.
3. The optical detector of claim 2 wherein the nucleic acid is DNA.
4. The optical detector of claim 3 , wherein said light detector detects fluorescence from at least 8 different dyes, each dyes being members of at least two 4-dye containing subsets, such that the subsets are capable of distinguishing at least two DNA sequences at a single detection position on the substrate, wherein the number of dyes is a multiple of four, and the number of DNA sequences to be detected is equal to that multiple, such that each of the different dyes is present in only one subset.
5. The optical detector of claim 1 , wherein the biological molecules are proteins.
6. The optical detector of claim 1 , wherein the biological molecules are nucleic acid fragments.
7. The optical detector of claim 1 , wherein said at least one light source is a laser.
8. The optical detector of claim 1 , wherein each detection element is capable of detecting ultraviolet light, visible light, infrared light, or a combination thereof.
9. The optical detector of claim 1 , further comprising a two dimensional optical detector element, said element comprising at least two rows for the detection of optical spectra, wherein a first row detects the optical spectra of a first independent lane and said second row detects the optical spectra of a second independent lane.
10. The optical detector of claim 1 , wherein each of said at least 6 detection elements is a single-anode photomultiplier tube and the wavelength dispersive element is composed of one or a plurality of dichroic mirrors positioned to provide at least a portion of the dispersed at least 6 wavelength components from said one or a plurality of first optical elements to each of said at least 6 detection elements, wherein each dichroic mirror reflects an independently predetermined wavelength of light.
11. The optical detector of claim 10 , further comprising a bandpass filter wherein each independently predetermined wavelength of light essentially corresponds to a fluorescence emission maximum of a fluorescent dye present in at least one of the detection positions.
12. The optical detector of claim 10 , wherein the wavelength dispersive element is a prism, diffraction grating, transmission grating, spectrograph or holographic diffraction grating.
13. The optical detector of claim 1 wherein each of the said at least 6 detection elements is a linear multi-anode photomultiplier tube.