DNA sequencing using multiple fluorescent labels being distinguishable by their decay times

• US 6,716,394 B2
• Filed: 06/24/2002
• Issued: 04/06/2004
• Est. Priority Date: 08/11/1998
• Status: Expired due to Term

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Holdings Incorporated
• Inventors: Jensen, Morten, Parce, J. Wallace
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/179,686
• Publication Number:
  US 20030036080A1
• Time in Patent Office: 652 Days
• Field of Search: 422/68.1, 435/6, 55/58, 55/820.5, 55/99
• US Class Current: 422/68.1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2561/12 Fluorescence lifetime measurement
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

A method is provided for identifying components of a mixture by labeling the individual components with fluorescent agents having different fluorescence lifetimes. The components are subsequently separated, fluorescent labels detected and their lifetimes measured. Based on the measured fluorescent lifetimes, the components of mixtures of small organic molecules, polymers, peptides, saccharides and nucleic acids can be identified.

Claims

1. A method of distinguishing between a plurality of fluorescent species, wherein each of said fluorescent species has a fluorescence emission, said emission having a characteristic fluorescence lifetime, said method comprising:

(a) transporting each of said fluorescent species through a microfluidic channel;

(b) detecting each of said fluorescent species in said channel; and

,

(c) identifying each of said fluorescent species by measuring said characteristic flourescence lifetime,

wherein at least a first fluorescent species and a second fluorescent species have characteristic fluorescence lifetime that differ by at least about 5 nanoseconds.

2. A method of distinguishing between a plurality of fluorescent species;

wherein each of said fluorescent species has a fluorescence emission, said emission having characteristic fluorescence lifetime, said method comprising

(a) transporting each of said fluorescent species through a microfluidic channel;

(b) detecting each of said fluorescent species in said channel; and

(c) identifying each of said fluorescent species by measuring said characteristic fluorescence lifetime, and

(d) contacting said plurality of fluorescent species with one or more sequencing reagents in said microfluidic channel, thereby performing a sequencing reaction.

3. The method of claim 2, wherein the one or wore sequencing reagent is selected from one or more of;

a nucleic acid polymer, a nucleic acid template, nucleic add primer, a fluorescently labeled nucleic acid, a polymerase, an exonuclease, an endonuclease, a metal ion, a chelating agent, surfactant, an acid, a base, a solvent, a buffer and a catalyst.

4. The method of claim 2, wherein performing the sequencing reaction comprises producing a plurality of nucleic acid polymers complementary to a region of a nucleic acid template, the method further comprising electrokinetically separating the plurality of nucleic acid polymers prior to stop (b).

5. A method of sequencing a nucleic acid, the method comprising:

(a) introducing the nucleic acid into a microfluidic device;

(b) contacting the nucleic acid with a plurality of sequencing reagents, thereby performing a sequencing reaction resulting in a plurality of fluorescently labeled nucleic acid sequence fragments, wherein each of said fragments has a fluorescence emission, said emission having a characteristic fluorescence lifetime;

(c) electrokinetically separating said fragments;

(d) detecting each of said fragments in said microfluidic device; and

,

(e) identifying each of said fragments by measuring said characteristic fluorescence lifetime, thereby sequencing the nucleic acid.

6. The method of claim 5, wherein the plurality of sequencing reagents comprises a nucleic acid primer, a polymerize, and one or more fluorescent species selected from:

a nucleotide, a nucleoside, a dideoxynucleoside, dideoxynucleotide, a deoxynucleoside, a deoxynucleotide, a nucleotide analog, and a nucleoside analog.