Digital DNA typing

• US 5,863,403 A
• Filed: 08/26/1996
• Issued: 01/26/1999
• Est. Priority Date: 03/29/1984
• Status: Expired due to Fees

• Current Assignee: Board of Regents of the University of Nebraska
• Original Assignee: Board of Regents of the University of Nebraska
• Inventors: Jang, Gi Young, Brumbaugh, John Albert, Middendorf, Lyle Richard
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US08/703,106
• Time in Patent Office: 883 Days
• Field of Search: 204/612, 204/461
• US Class Current: 204/612
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules

Abstract

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.

Claims

1. DNA sequencing apparatus comprising:

electrophoresis means;

said electrophoresis means including channels of separating medium;

one end of said electrophoresis means communicating with a first buffer solution;

at least one other side of said electrophoresis means communicating with at least a second buffer solution;

means for applying opposite polarity electrical potentials to said first buffer solution and to said at least second buffer solution;

means for receiving a plurality of fluorescently marked DNA bases from an individual'"'"'s DNA in a plurality of the channels of said separating medium, whereby said fluorescently marked DNA bases are electrophoresed along said separating medium so that bands of more mobile strands in at least one channel are fully resolved while some of the less mobile strands to be later formed into bands are unresolved in a continuous process;

a diode laser;

means for moving the diode laser and said channels with respect to each other wherein said channels may be scanned with light emitted from the diode laser as said diode laser and channels are moved with respect to each other;

means for detecting fluorescent light emitted by said fluorescently marked DNA bases, whereby a time sequence of separated bands may be obtained to provide a sensed pattern indicative of an individual; and

means for comparing the sensed patterns of DNA bases with other information to identify the individual.

2. DNA sequencing apparatus according to claim 1 in which said means for receiving comprises means for receiving fluorescently marked alleles in a plurality of the channels of said separating medium, whereby said fluorescently marked DNA bands are electrophoresed along said separating medium;

said means for scanning including means for scanning with light from said diode laser in a band of wavelengths of light incorporating at least the near infrared and infrared regions;

said means for detecting being responsive to light in said band of wavelengths including at least said near infrared and infrared regions; and

means for recording sensed patterns of bands, wherein alleles are detected from the patterns so that individuals are identified.

3. DNA sequencing apparatus according to claim 2 in which said means for moving a diode laser includes means for moving a microscope and the diode laser together across said channels to perform said scanning.

4. DNA sequencing apparatus according to claim 3 wherein the light from the diode laser is scanned at an angle chosen to impinge on the bands with full intensity after refraction but not be reflected into the microscope.

5. DNA sequencing apparatus according to claim 4 wherein the light from the diode laser is scanned at an angle to the surface being scanned equal to the Brewster'"'"'s angle.

6. DNA sequencing apparatus according to claim 1 in which said channels of separating medium include capillary tubes containing said medium.