Real time scanning electrophoresis apparatus for DNA sequencing

• US 4,811,218 A
• Filed: 06/02/1986
• Issued: 03/07/1989
• Est. Priority Date: 06/02/1986
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Lytle, John D., Mordan, William J., Connell, Charles R., Hunkapiller, Michael W., Bridgham, John A.
• Primary Examiner(s): Smith, Jerry
• Assistant Examiner(s): Jablon, Clark A.
• Application Number:
  US06/869,421
• Time in Patent Office: 1,009 Days
• Field of Search: 435/6, 204/299 R, 935/87, 935/77, 364/413
• US Class Current: 204/461
• CPC Class Codes: G01N 27/44721 by optical means

Abstract

A real-time, automated, nucleic acid sequencing apparatus that offers high speed, definitive sequencing on many samples at the same time. The apparatus permits more than one clone to be sequenced at a time, thus vastly decreasing the time required to sequence longer fragments and reducing sequencing costs accordingly. The apparatus detects electromagnetic radiation from a plurality of lanes in an electrophoresis system wherein the plurality of lanes are arranged in a planar array. The apparatus includes an optical system for detecting the radiation at a plurality of wavelengths and is made up of a collection element, a filter for selectively transmitting the plurality of wavelengths received from the collection element, and a detection system for measuring intensity of the radiation received from the filter means. A translational stage is used for mounting the optical system and for moving the optical system parallel to the planar array in order to move the collection element back and forth across the lanes in order to receive radiation from the lanes, one lane at a time during electrophoresis. Also included is a computer system for controlling the filter and the stage, and for receiving intensity data from the detector and correlating that data with the corresponding lane and corresponding wavelengths transmitted by the filter in substantially real time.

Claims

1. An apparatus for detecting electromagnetic radiation from a plurality of lanes in an electrophoresis system wherein said plurality of lanes are arranged in a planar array comprising:

optical means for detecting said radiation at a plurality of wavelengths emanating from said plurality of lanes, comprising;

collecting means for collecting and focussing said radiation;

filter means for receiving said radiation from said collecting means and for selectively transmitting said plurality of wavelengths of said radiation received from said collecting means;

detection means for measuring intensity of radiation received from said filter means;

stage means for mounting said optical means, and for translating said optical means in a direction parallel to said planar array in order to move said collecting means to receive radiation from said lanes, one lane at a time;

computer means coupled to said filter means and said stage means for controlling said filter means and said stage means, for receiving intensity data from said detection means, and for correlating said intensity data with corresponding lanes of said electrophoresis system and corresponding wavelengths transmitted by said filter means.

2. An apparatus as in claim 1 further comprising:

source means for providing said electromagnetic radiation and for directing said radiation in a direction parallel to said planar array;

and where said optical means further comprises deflection means fixed relative to said stage means for deflecting said radiation away from said direction parallel to said planar array and towards said planar array.

3. An apparatus for determining the base sequences of nucleic acids, wherein said nucleic acids have been fragmented and the fragments labelled with four different dyes that fluoresce at four different wavelengths, each dye being attached to fragments terminating at a different one of A, G, T, or C, comprising:

source means for providing electromagnetic radiation in a first direction;

electrophoresis means constructed to have a plurality of lanes, said lanes arranged in a line parallel to said first direction, with each lane having a side constrained by a material transparent to radiation from said source means, for electrophoresing said nucleic acids simultaneously in said plurality of lanes;

a radiation detection system comprising;

deflection means for deflecting said radiation from said source means toward said line of lanes in a direction incident on said transparent material;

collection means for collecting light fluorescing from said lanes in response to receiving radiation from said source means;

filter means for selectively transmitting four bands of wavelengths of radiation received from said collection means, one band at a time, each band corresponding to a different dye in the set of four dyes;

detector means for receiving radiation transmitted by said filter means and for providing a signal functionally related to intensity of the radiation received;

stage means for holding said radiation detection system and for translating said radiation detection system back and forth across said plurality of lanes in a direction parallel to said line of lanes while said nucleic acids are being electrophoresed; and

computer means for controlling the radiation detection system and for analyzing signals from the detector means to detemine a time ordered sequence of said dyes traversing each of said lanes during electrophoresis.

4. A method of detecting fluorescence from multiple lanes of an electrophoresis apparatus comprising:

illuminating molecules in said lanes with electromagnetic radiation, said molecules having fluorescent dyes attached thereto, said molecules moving through said lanes by electrophoresis;

collecting light resulting from fluorescence of said dyes, by moving a collecting lens across said lanes during electrophoresis;

focussing said collected light on an intensity sensitive apparatus moving with said collecting lens to provide an intensity profile as said collecting lens is moved across said lanes; and

sorting said intensity profile in time to identify a time ordered sequence of dyes traversing each of said lanes.

5. An electrophoresis apparatus comprising:

means for providing a plurality of substantially parallel electrophoretic lanes along which fluorescently labeled samples migrate, said electrophoretic lanes being substantially disposed in a plane;

electrodes disposed at opposite ends of said electrophoretic lanes;

a power source coupled to said electrodes for driving said samples along said electrophoretic lanes;

source means for providing a beam of electromagnetic radiation;

deflection means for deflecting said beam of electromagnetic radiation toward said lanes, one lane at a time, said deflected beam having a directional component orthogonal to said plane as said deflected beam traverses each lane;

detector means for detecting radiation fluorescing from said lanes in response to receiving radiation from said source means, and for providing a signal functionally related to the intensity of the radiation received;

computer means coupled to said detector means and to said deflection means for analyzing said signal from said detector means to determine a time ordered sequence of said samples traversing each of said lanes during electrophoresis.

6. Apparatus as in claim 5 further comprising collection means for increasing the proportion of fluorescent radiation that impinges on said detector means.

7. Apparatus as in claim 5 wherein said deflection means comprises a mirror means for deflecting said beam toward said plane and a stage means for mounting said mirror, said stage means for translating said mirror means in a direction parallel to said planar array.

8. Apparatus as in claim 7 further comprising collection means for increasing the proportion of fluorescent radiation that impinges on said detector means.

9. Apparatus as in claim 8 wherein said stage means is coupled to said collection means and to said detector means for translating said collection means and said detector means as said stage means translates said mirror means.

10. An electrophoretic fluorescence detection apparatus comprising:

source means for providing electromagnetic radiation;

electrophoresis means constructed to have a plurality of lanes, said lanes arranged in a line parallel to a first direction, with each lane having a side constrained by a material transparent to radiation from said source means, for electrophoresing fluorescently labeled samples simultaneously in said plurality of lanes;

deflection means for directing said electromagnetic radiation onto said lanes, a single lane at a time;

a radiation detection system comprising;

filter means for selectively transmitting a plurality of bands of wavelenghts of radiation of fluorescing from said lanes in response to receiving radiation from said source means;

detector means for receiving said radiation transmitted by said filter means and for providing a signal functionally related to intensity of the radiation received;

computer means coupled to said deflection means and to said radiation detection system for controlling said detection system and for analyzing said signal from the detector means to determine a time ordered sequence of the samples traversing each of said lanes during electrophoresis.

11. Apparatus as in claim 10 wherein said radiation detection system further comprises collection means for increasing the proportion of fluorescent radiation that impinges on said detector means.

12. Apparatus as in claim 11 wherein said deflection means comprises translation means coupled to said computer means for moving said deflection means parallel to said first direction lanes, said translation means also including mounting means for mounting said radiation detection system thereto, so that said radiation detection system is also translated parallel to said lanes.

13. An apparatus for determining the base sequences of nucleic acids, wherein said nucleic acids have been fragmented and the fragments labelled with four different dyes that fluoresce at four different wavelengths, each dye being attached to fragments terminating in a different one of A, G, T, or C, comprising:

source means for providing electromagnetic radiation;

electrophoresis means constructed to have a plurality of lanes, said lanes arranged in a line parallel to a first direction, with each lane having a side constrained by a material transparent to radiation from said source means, for electrophoresing the fluorescently labeled fragments simultaneously in said plurality of lanes;

deflection means for directing said elecromagnetic radiation onto said lanes, a single lane at a time;

a radiation detection system comprising;

filter means for selectively transmitting four bands of wavelengths of radiation fluorescing from the fragments being electrophoresed in said lanes in response to receiving radiation from said source means, said bands corresponding to the four wavelengths of said dyes;

detector means for receiving radiation transmitted by said filter means and for providing a signal functionally related to intensity of the radiation received;

computer means coupled to said deflection means and to said detection system for controlling said deflection means and for analyzing signals from the detector means to determine a time ordered sequence of the fragments traversing each of said lanes during electrophoresis.

14. An apparatus as in claim 13 wherein said radiation detection system further comprises collection means for increasing the proportion of fluorescent radiation that impinges on said detector means.