Li-cor, Inc.

University of Nebraska

Lambda Instruments Company

Lyle R Middendorf

John A Brumbaugh

Gabor Patonay

Robert D Eckles

William W Biggs

Robert Clark Bruce

Narasimhachari Narayanan

Han-Wei Wang

John H Wurm

Michael D Furtaw

Donald Thomas Lamb

Lucjan Strekowski

Malgorzata Lipowska

Kay M Parkhurst

Gi Young Jang

Lawrence J Parkhurst

Jerome E Johnston

W Walter Biggs

Devices and methods are provided for the separation and dispensing of material using a microfluidic separation column connected via an exit channel to one or more sheath flow channels. The flow of separated material through the separation column is at least partially driven by a voltage potential between a first electrode within the separation column and a terminating electrode within at least one of the sheath flow channels. The separation column, exit channel, sheath flow channels, and electrodes are all within a single monolithic chip. The presence of an on-chip terminating electrode allows for separated material to be entrained in the sheath fluids and ejected onto a surface that can be non-conductive. The presence of multiple sheath flows allows for sheath flow fluids to have different compositions from one another, while reducing the occurrence of sheath flow fluids entering the separation column.

Methods for using multi-sheath flow and on-chip terminating electrode for microfluidic direct-blotting

Methods and systems are provided for the detecting and presenting of faint signals from a real-time interactive imager. A real-time view of a subject is controlled by a user by entering commands that move the subject or a camera used to record the real-time view. The rate of this movement is monitored, and when the rate descends below a preset value, a trigger is sent to the camera. In response to the trigger, the camera switches to a lower frame frequency for capturing the real-time view of the subject, thereby increasing the amount of light collected in each frame, and increasing the sensitivity of the camera to faint signals. The methods and systems are particularly relevant for surgical or biopsy imaging, in which both a higher frame rate preview image of a subject for 3D visualization, and a higher sensitivity fluorescence image of the subject for tumor margin assessment, are desired.

Motion-adaptive interactive imaging method

Methods and devices are disclosed for the imaging of a biological sample with a top-down camera and a side-view camera. A biological sample is held on an imaging stage that is capable of rotation in two orthogonal axes. The top-down and side-view cameras can record a series of images of the sample using multiple imaging modalities at different rotational positions of the imaging stage. The top-down camera can be translated along its optical axis to affect the camera zoom and influence the resolution and field of view of the recorded images. Fluorescence excitation light sources can be positioned proximate to each of the top-down and side-view cameras to provide substantially uniform illumination of the sample for imaging with each camera.

Top-down and rotational side view biopsy specimen imager and methods

Multi-sheath flow and on-chip terminating electrode for microfluidic direct-blotting

A method for detecting point mutations in DNA using a fluorescently labeled oligomeric probe and Forster resonance energy transfer (FRET) is disclosed. The selected probe is initially labeled at each end with a fluorescence dye, which act together as a donor/acceptor pair for FRET. The fluorescence emission from the dyes changes dramatically from the duplex stage, wherein the probe is hybridized to the complementary strand of DNA, to the single strand stage, when the probe is melted to become detached from the DNA. The change in fluorescence is caused by the dyes coming into closer proximity after melting occurs and the probe becomes detached from the DNA strand. The change in fluorescence emission as a function of temperature is used to calculate the melting temperature of the complex or T . In the case where there is a base mismatch between the probe and the DNA strand, indicating a point mutation, the T has been found to be significantly lower than the T for a perfectly match probe/stand duplex. The present invention allows for the detection of the existence and magnitude of T , which allows for the quick and accurate detection of a point mutation in the DNA strand and, in some applications, the determination of the approximate location of the mutation within the sequence.

Probe for detecting point mutations in DNA utilizing fluorescence energy transfer

A method for detecting point mutations in DNA using a fluorescently labeled oligomeric probe and Forster resonance energy transfer (FRET) is disclosed. The selected probe is initially labeled at each end with a fluorescence dye, which act together as a donor/acceptor pair for FRET. The fluorescence emission from the dyes changes dramatically from the duplex stage, wherein the probe is hybridized to the complementary strand of DNA, to the single strand stage, when the probe is melted to become detached from the DNA. The change in fluorescence is caused by the dyes coming into closer proximity after melting occurs and the probe becomes detached from the DNA strand. The change in fluorescence emission as a function of temperature is used to calculate the melting temperature of the complex or T,. In the case where there is a base mismatch between the probe and the DNA strand, indicating a point mutation, the T,has been found to be significantly lower than the T,for a perfectly match probelstand duplex. The present invention allows for the detection of the existence and magnitude of T,, which allows for the quick and accurate detection of a point mutation in the DNA strand and, in some applications, the determination of the approximate location of the mutation within the sequence.

Method for detecting point mutations in DNA utilizing fluorescence energy transfer

A universal terminator includes a heterocycle other than naturally occuring DNA heterocycles such as adenine, cytosine, guanine and thymine. In terminating strand synthesis, the hybridized primer-template is split into four aliquots. In the &ldquo;A&rdquo; vial is added DNA polymerase and normal amounts of &ldquo;C&rdquo;, &ldquo;G&rdquo;, and &ldquo;T&rdquo; deoxynucleotides, along with a reduced amount of &ldquo;A&rdquo; deoxynucleotide and an amount of the universal terminator such that statistically the universal terminator has less than a one percent chance of being incorporated at sites where a &ldquo;C&rdquo;, &ldquo;G&rdquo;, or &ldquo;T&rdquo; deoxynucleotide should be incorporated and about a one percent chance of being incorporated at sites where an &ldquo;A&rdquo; deoxynucleotide should be incorporated. Similar strategies are followed for the &ldquo;C&rdquo;, &ldquo;G&rdquo;, and &ldquo;T&rdquo; vials, except that the amount of &ldquo;C&rdquo;, &ldquo;G&rdquo; and &ldquo;T&rdquo; deoxynucleotides are reduced for their respective vial.

DNA sequencing and DNA terminators

To sequence long strands of DNA, clone strands having lengths longer than 100 bases are, in one embodiment, marked on one end with biotin. These strands are divided into 4 aliquots and each aliquot: (1) is uniquely chemically treated to randomly terminate the strands at the non-biotinylated end at a selected type of base; and (2) is moved continuously by electrophoresis through a different one of four identical channels. In the one embodiment, the strands are randomly terminated at a selected base type and they are moved into avidin, which due to high affinity, combines with the biotin marked ends of shorter strands before the longer strands are fully resolved in the gel. The avidin is marked with fluorescein, the strands are scanned and the signals are decoded. In another embodiment, the strands are synthesized, with termination at a selected base type and marked either by the above method of by ethidium bromide.

DNA sequencing

To sequence long strands of DNA, clone strands having lengths longer than 100 bases are, in one embodiment, marked on one end with biotin. These strands are divided into 4 aliquots and each aliquot: (1) is uniquely chemically treated to randomly terminate the strands at the nonbiotinylated end at a selected type of base; and (2) is moved continuously by electrophoresis through a different one of four identical channels. In the one embodiment, the strands are randomly terminated at a selected base type and they are moved into avidin, which due to high affinity, combines with the biotin marked ends of shorter strands before the longer strands are fully resolved in the gel. The avidin is marked with fluorescein, the strands are scanned and the signals are decoded. In another embodiment, the strands are synthesized, with termination at a selected base type and marked either by the above method of by ethidium bromide.

To sequence DNA automatically, DNA marked with far infrared, near infrared, or infrared fluorescent dyes are electrophoresed in a plurality of channels through a gel electrophoresis slab or capillary tubes wherein the DNA samples are resolved in accordance with the size of DNA fragments in the gel electrophoresis slab or capillary tubes into fluorescently marked DNA bands. The separated samples are scanned photoelectrically with a laser diode and a sensor, wherein the laser scans with scanning light at a wavelength within the absorbance spectrum of said fluorescently marked DNA samples and light is sensed at the emission wavelength of the marked DNA.

Sequencing near infrared and infrared fluorescence labeled DNA for

To sequence DNA, DNA samples marked with flourscent infrared dye are applied at a plurality of locations for electrophoresing in a plurality of channels through a gel electrophoresis slab. The channels are scanned with a laser and a sensor, that include a microscope focused on the gel slab. The focal point and slab are adjusted with respect to each other so that the focal point of the microscope remains on the gel slab during a scan. The data from the scan is directly used to amplitude modulate density readings on a display, and the scan is displayed in a horizontal sweep of a cathode ray tube, whereby said cathode ray tube provides intensity displays of bands representing DNA. Different sizes of glass gel sandwiches may be mounted to the same console for different sequencing tasks.

DNA Sequencing

To sequence DNA automatically, fluorescently marked DNA are electrophoresed in a plurality of channels through a gel electrophoresis slab; wherein the DNA samples are resolved in accordance with the size of DNA fragments in the gel electrophoresis slab into fluorescently marked DNA bands. The separated samples are scanned photoelectrically with a laser and a sensor, wherein the laser scans with scanning light at a scanning light frequency within the absorbance spectrum of said fluorescently marked DNA samples and light is sensed at the emission frequency of the marked DNA. The light is modulated from said laser at a predetermined modulation frequency and fluorescent light emitted by said DNA bands at said modulation frequency is detected, whereby background noise from the medium through which the light is transmitted is discriminated against.

Growing community of inventors

Lincoln, NE, United States of America

Lyle R Middendorf