Methods for high speed, high throughput analysis of polynucleotide sequences, and apparatuses with which to carry out the methods are provided in the invention.

Methods and apparatuses for analyzing polynucleotide sequences

The invention provides methods for sequencing a polynucleotide comprising stopping an extension cycle in a sequence by synthesis reaction before the reaction has run to near or full completion.

Short cycle methods for sequencing polynucleotides

Purpose: To prospectively compare clinical breast examination (CBE), mammography, ultrasonography (US), and contrast material–enhanced magnetic resonance (MR) imaging for screening women at genetic-familial high risk for breast cancer and report interim results, with pathologic findings as standard. Materials and Methods: Institutional review board of each center approved the research; informed written consent was obtained. CBE, mammography, US, and MR imaging were performed for yearly screening of BRCA1 or BRCA2 mutation carriers, first-degree relatives of BRCA1 or BRCA2 mutation carriers, or women enrolled because of a strong family history of breast or ovarian cancer (three or more events in first- or second-degree relatives in either maternal or paternal line; these included breast cancer in women younger than 60 years, ovarian cancer at any age, and male breast cancer at any age). Results: Two hundred seventy-eight women (mean age, 46 years ± 12 [standard deviation]) were enrolled. Breast cancer was ...

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Multicenter Comparative Multimodality Surveillance of Women at Genetic-Familial High Risk for Breast Cancer (HIBCRIT Study): Interim Results

The white light emitting diode comprising a light emitting component using a semiconductor as a light emitting layer and a phosphor which absorbs a part of light emitted by the light emitting component and emits light of wavelength different from that of the absorbed light, wherein the light emitting layer of the light emitting component is a nitride compound semiconductor and the phosphor contains garnet fluorescent material activated with cerium which contains at least one element selected from the group consisting of Y, Lu, Sc, La, Gd and Sm, and at least one element selected from the group consisting of Al, Ga and In and, and is subject to less deterioration of emission characteristic even when used with high luminance for a long period of time.

Light emitting device and display

The invention provides methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension. Some applications of the invention utilize total internal reflection fluorescence microscopy to image polynucleotide molecules at single molecule resolution.

Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension

Improved detection methods and apparatus which may be used individually or in various combinations enhance the ability of the electrophoresis apparatus to detect fluorophore-labeled materials in short periods of time. One such apparatus comprises a housing adapted to receive an electrophoresis gel holder; an excitation source of electromagnetic radiation having a frequency effective to induce emission of electromagnetic radiation from the fluorophore; a plurality of optical fibers for delivering electromagnetic radiation from the excitation source to a linear array of excitation/detection sites on the gel holder, optical switching means for sequentially directing electromagnetic radiation into one of several pre-defined groups of the optical fibers; detection means such as a photomultiplier tube, or an array of photomultiplier tubes for detecting emission from the fluorophore induced by a radiation from the excitation source; and means for correlating a detected emission with the switching of the excitation electromagnetic radiation such that a given emission may be linked with the excitation/detection site being irradiated. For example, the optical switching means may alternate between directing radiation from the source into every other optical fiber, or may provide radiation in rotation to every third or fourth fiber. Alternatively, a spot array generation grating can be used for dividing an incident beam of coherent radiation into an array of excitation beamlets and directing each excitation beamlet to an excitation/detection site on the electrophoresis gel. Light emitting diode disposed to deliver excitation energy to an array of excitation/detection sites may also be used. This latter form of the apparatus is particularly advantageous due to the low costs of light emitting diodes (LEDs) compared to coherent light sources (e.g. lasers).

Automated electrophoresis and fluorescence detection apparatus and method

An apparatus and method for thermally cycling a reaction mixture in a reaction vessel to expose the mixture to the varying temperatures necessary to, for example, achieve PCR amplification or the preparation of sequencing fragments using a cycle sequencing operation makes use of flow-through reaction vessels, such as capillary tubes, for the preparation and thermal cycling of reaction mixtures. In order to prevent loss of the reaction mixture from the vessels during heating, the thermal cycling apparatus of the invention provides means for reversibly sealing the proximal and distal ends of each reaction vessel. The proximal ends can be sealed by coupling to a pump which permits movement of the samples within the reaction vessels. As to the distal ends, the reaction vessels can be sealed by pressing the distal end of each vessel against a sealing element with a conformable surface, or by immersing the distal end of each vessel in the reservoir of liquid, preferably of an oil, that is not miscible with the reaction mixture.

Method and apparatus for thermal cycling and for automated sample preparation with thermal cycling

The Visible Genetics Clipper sequencer is a new platform for automated DNA sequencing which employs disposable MicroCel cassettes and 50 microm thick polyacrylamide gels. Two DNA ladders can be analyzed simultaneously in each of 16 lanes on a gel, after labeling with far-red absorbing dyes such as Cy5 and Cy5.5. This allows a simultaneous bidirectional sequencing of four templates. We have evaluated the Clipper sequencer, by cycle-sequencing of an M13 single-stranded DNA standard, and by coupled amplification and sequencing (CLIP) of reverse-transcribed human immunodeficiency virus (HIV-1) RNA standards and clinical patient samples. (i) Limitations of instrument. We have examined basic instrument parameters such as detector stability, background, digital sampling rate, and gain. With proper usage, the optical and electronic subsystems of the Clipper sequencer do not limit the data collection or sequence-determination processes. (ii) Limitations of gel performance. We have also examined the physics of DNA band separation on 50 microm thick MicroCel gels. We routinely obtain well-resolved sequence which can be base-called with 98.5% accuracy to position approximately 450 on an 11 cm gel, and to position approximately 900 on a 25 cm gel. Resolution on 5 and 11 cm gels ultimately is limited by a sharp decrease in spacing between adjacent bands, in the biased reptation separation regime. Fick's (thermal) diffusion appears to be of minor importance on 6 cm or 11 cm gels, but becomes an additional resolution-limiting factor on 25 cm gels. (iii) Limitations of enzymology. Template quality, primer nesting, choice of DNA polymerase, and choice between dye primers and dye terminators are key determinants of the ability to detect mutations and polymorphisms on the Clipper sequencer, as on other DNA sequencers. When CLIP is used with dye-labeled primers and a DNA polymerase of the F667Y, delta(5'--> 3' exo) class, we can routinely detect single-nucleotide mutations and polymorphisms over the 0.35-0.65 heterozygosity range. We present an example of detecting therapeutically relevant mutations in a clinical HIV-1 RNA isolate.

High performance DNA sequencing, and the detection of mutations and polymorphisms, on the Clipper sequencer.

An electrophoresis microgel is formed in a gel holder. The gel holder comprises a top substrate, a bottom substrate and a spacer disposed between the top substrate and the bottom substrate. The spacer establishes a separation of from 25 to 250 microns between the top substrate and the bottom substrate. A gel compartment is formed by partially sealing the top substrate to the bottom substrate, while leaving an opening for the introduction of unpolymerized gel. The gel compartment is then filled with an unpolymerized gel, which is polymerized in the gel compartment. Electrodes may be printed on the substrates, may be contacted to an exposed edge of gel, or may be applied through windows cut into one of the substrates. One type of gel holder makes use of graded beads having a diameter of 25 to 250 microns slurried in an adhesive such as an acrylate adhesive as the spacer. The slurry is printed onto the surface of one or both substrates to form a spacer of the desired shape, and then hardened using heat or light. If desired, the spacer can establish lanes within the gel.

Microgels for use in medical diagnosis and methods of making and using same

An electrophoresis microgel is formed in a gel holder. The gel holder comprises a top substrate, a bottom substrate and a spacer disposed between the top substrate and the bottom substrate. The spacer establishes a separation of from 25 to 250 microns between the top substrate and the bottom substrate. A gel compartment is formed by partially sealing the top substrate to the bottom substrate, while leaving an opening for the introduction of unpolymerized gel. The gel compartment is then filled with an unpolymerized gel, which is polymerized in the gel compartment. Electrodes may be printed on the substrates, may be contacts to an exposed edge of gel, or may be applied through windows cut into one of the substrates. One type of gel holder makes use of graded beads having a diameter of 25 to 250 microns slurried in an adhesive such as an acrylate adhesive as the spacer. The slurry is printed onto the surface of one or both substrates to form a spacer of the desired shape, and then hardened using heat or light. If desired, the spacer can establish lanes within the gel.

John A. Renfrew

Papers

Automated electrophoresis and fluorescence detection apparatus and method

Method and apparatus for thermal cycling and for automated sample preparation with thermal cycling

High performance DNA sequencing, and the detection of mutations and polymorphisms, on the Clipper sequencer.

Microgels for use in medical diagnosis and methods of making and using same

Microgels for use in medical diagnosis and holders useful in fabricating same