DNA fragment sizing and sorting by laser-induced fluorescence

• US 5,558,998 A
• Filed: 06/05/1995
• Issued: 09/24/1996
• Est. Priority Date: 02/25/1992
• Status: Expired due to Fees

• Current Assignee: Los Alamos National Security LLC (Government of the United States of America)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Marrone, Babetta L., Hammond, Mark L., Martin, John C., Keller, Richard A., Jett, James H.
• Primary Examiner(s): Fleisher, Mindy
• Assistant Examiner(s): CARTER, PHILIP
• Application Number:
  US08/486,400
• Time in Patent Office: 477 Days
• Field of Search: 435/6, 436/52, 436/63
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/626 being a flow cytometer
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: Y10T 436/117497 with a continuously flowing sample or carrier stream

Abstract

A method is provided for sizing DNA fragments using high speed detection systems, such as flow cytometry to determine unique characteristics of DNA pieces from a sample. In one characterization the DNA piece is fragmented at preselected sites to produce a plurality of DNA fragments. The DNA piece or the resulting DNA fragments are treated with a dye effective to stain stoichiometrically the DNA piece or the DNA fragments. The fluorescence from the dye in the stained fragments is then examined to generate an output functionally related to the number of nucleotides in each one of the DNA fragments. In one embodiment, the intensity of the fluorescence emissions from each fragment is linearly related to the fragment length. The distribution of DNA fragment sizes forms a characterization of the DNA piece for use in forensic and research applications.

Claims

1. A method for quantitatively determining the number of nucleotides in DNA fragments of about 50 kbp or less, naturally occurring or obtained by fragmenting a larger piece of DNA at known sites within said piece of DNA by induced fluorescence, comprising the steps of:

staining said piece of DNA or said DNA fragments with a single dye effective to form stoichiometrically stained DNA or DNA fragments;

forming a flow stream of said stained DNA fragments serially spaced apart in said flow stream;

illuminating said flow stream with a light effective to cause said dye to fluoresce;

measuring the intensity of the fluorescence from each one of said stained fragments of DNA in said flow stream, wherein said intensity of said fluorescence is linearly related to said number of nucleotides in said stained DNA fragments Of about 50 kbp or less.

2. A method according to claim 1, wherein the step of staining said DNA piece or said DNA fragments with a dye includes the step of adding said DNA piece or said DNA fragments to a solution containing a dye selected from the group consisting of ethidium homodimer, asymmetric cyanine homo- and heterodimers, ethidium bromide, acridine orange, propidium iodide, 9 amino acridine, and ethidium acridine heterodimer.

3. A method according to claim 1, wherein said step of measuring said fluorescence intensity further includes the step of differentiating dye molecules bound to said DNA fragments from unbound dye molecules.

4. A method according to claim 1, wherein said step of illuminating said flow stream with a light includes the step of illuminating said flow stream with a pulsed laser.

5. A method according to claim 1, wherein said step of illuminating said flow stream with a light includes the step of illuminating said flow stream with a cw laser.

6. A method according to claim 1, wherein the step of measuring the intensity of said fluorescence includes the step of gating detection electronics to measure fluorescence at a predetermined time after said pulsed laser is initiated.

7. A method according to claim 1, wherein the step of staining said piece of DNA or said DNA fragments includes the step of adding said piece of DNA or said DNA fragments to a solution containing an intercalating dye.

8. A method for quantitatively determining the number of nucleotides in DNA fragments containing 50 kbp or less of nucleotides, naturally occurring or obtained by fragmenting a larger piece of DNA at known sites within said piece of DNA by induced fluorescence, comprising the steps of:

staining said piece of DNA or said DNA fragments with a single dye effective to form stoichiometrically stained DNA or DNA fragments;

forming a flow stream of said stained DNA fragments serially spaced apart in said flow stream;

passing said flow stream through an excitation and detection volume effective to contain only a single DNA fragment at a time;

illuminating said flow stream with a light effective to cause said dye to fluoresce;

counting the number of photons emitted by said dye to output a fluorescence signal from each one of said stained fragments of DNA in said flow stream; and

determining a centroid value for each peak of said fluorescence signal, wherein said centroid value is linearly related to said number of nucleotides in each one of said stained DNA fragments having 50 kbp or less of nucleotides.

9. A method according to claim 8, wherein the step of passing only a single DNA fragment at a time through a flow volume includes the steps of:

providing a small excitation volume;

forming a flow stream with a selected velocity effective to retain said single DNA fragment in said excitation volume for a time effective to optimize said fluorescence from said DNA fragment; and

providing said DNA fragments at a concentration wherein only a single DNA fragment is within said excitation volume at said selected velocity.

10. A method according to claim 8 wherein said step of illuminating said flow stream with a light includes the step of illuminating said flow stream with a pulsed laser.

11. A method according to claim 10, wherein the step of counting said number of photons includes the step of gating detection electronics to count said photons at a predetermined time after said pulsed laser is initiated.

12. A method according to claim 8, wherein said step of illuminating said flow stream with a light includes the step of illuminating said flow stream with a cw laser.