High speed nucleic acid sequencing

• US 7,943,305 B2
• Filed: 04/13/2006
• Issued: 05/17/2011
• Est. Priority Date: 05/19/1999
• Status: Expired due to Fees

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Turner, Stephen, Foquet, Mathieu, Korlach, Jonas, Levene, Michael, Webb, Watt W., Craighead, Harold G.
• Primary Examiner(s): Sisson; Bradley L
• Application Number:
  US11/279,711
• Publication Number:
  US 20060188900A1
• Time in Patent Office: 1,860 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2561/12 Fluorescence lifetime measurement
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid. Each type of labeled nucleotide comprises an acceptor fluorophore attached to a phosphate portion of the nucleotide such that the fluorophore is removed upon incorporation into a growing strand. Fluorescent signal is emitted via fluorescent resonance energy transfer between the donor fluorophore and the acceptor fluorophore as each nucleotide is incorporated into the growing strand. The sequence is deduced by identifying which base is being incorporated into the growing strand.

Claims

1. A method of sequencing a nucleic acid molecule, comprising:

forming on a support a complex comprising the nucleic acid molecule, an oligonucleotide primer, and a polymerase comprising a donor fluorophore;

exposing the complex to a plurality of types of labeled nucleotide, each type of labeled nucleotide comprises an acceptor fluorophore that is distinguishable from acceptor fluorophores of other types of labeled nucleotides, and the fluorophore of each type of labeled nucleotide is attached to a phosphate portion of the nucleotide such that the fluorophore is removed upon incorporation into a growing strand, wherein at least one of the donor fluorophore of the polymerase and the acceptor fluorophore of the nucleotide comprises a fluorescent nanoparticle, and wherein a fluorescent signal is emitted via fluorescent resonance energy transfer between the donor fluorophore of the polymerase and the acceptor fluorophore of the nucleotide as each nucleotide is incorporated into the growing strand which is complementary to the sample nucleic acid molecule; and

detecting the signal as each nucleotide is incorporated into the growing strand.

2. The method of claim 1, wherein the method comprises sequencing a plurality of DNA molecules simultaneously by monitoring individual complexes on said support with an optical detector.

3. The method of claim 1, wherein the substrate comprises a nanostructure comprising a hole in an opaque layer entrapping the nucleic acid molecule, wherein said hole is sized to permit distinguishing an incorporated nucleotide from an unincorporated nucleotide.

4. The method of claim 1 wherein the nucleic acid molecule is bound to the support, and further comprising replacing the polymerase with a second polymerase comprising a donor-fluorophore.

5. The method of claim 1, wherein the fluorophore of the nucleotide is linked to terminal phosphate group of the nucleotide.

6. The method of claim 1, wherein the fluorophore of the nucleotide is removed via enzymatic activity of the polymerase.