Method for sequencing nucleic acid molecules

• US 7,033,764 B2
• Filed: 12/15/2004
• Issued: 04/25/2006
• Est. Priority Date: 05/19/1999
• Status: Expired due to Term

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Craighead, Harold G., Turner, Stephen, Foquet, Mathieu, Korlach, Jonas, Levene, Michael, Webb, Watt W.
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US11/014,015
• Publication Number:
  US 20050158761A1
• Time in Patent Office: 496 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/287.2, 536/23.1, 536/24.31, 536/24.33, 536/25.72, 436/94, 436/164
• 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, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Claims

1. A single-step method for sequencing a plurality of target nucleic acid molecules, said method comprising subjecting the plurality of target nucleic acid molecules to a polymerization reaction with a plurality of types of nucleotides or nucleotide analogs to yield a population of growing nucleic acid strands that are complementary to their respective target nucleic acid molecules, wherein the target nucleic acid molecule and/or the nucleic acid polymerization enzyme is attached to a support;

and optically identifying a time sequence of incorporation of said plurality of types of nucleotides or nucleotide analogs into at least two growing nucleic acid strands in said population, wherein the time sequence for each of said at least two growing nucleic acid strands is different.

3. A method according to claims 1 or 2, wherein the polymerization reaction is performed by a nucleic acid polymerizing enzyme selected from the group consisting of a DNA polymerase, an RNA polymerase, reverse transcriptase, and mixtures thereof.

4. A method according to claim 3, wherein the nucleic acid polymerizing enzyme is a thermostable polymerase or a thermodegradable polymerase.

5. A method according to claim 1, wherein the target nucleic acid molecules are selected from the group consisting of double-stranded DNA, single-stranded DNA, single stranded DNA hairpins, DNA/RNA hybrids, RNA with a recognition site for binding of the polymerase, and RNA hairpins.

6. A method according to claim 1, wherein the nucleotide analogs are selected from the group consisting of a ribonucleotide, a deoxyribonucleotide, a modified ribonulcleotide, a modified deoxyribonucleotide, a peptide nucleotide, a modified peptide nucleotide, and a modified phosphate-sugar backbone nucleotide.

7. A method according to claim 1, wherein the nucleotides or nucleotide analogs further comprise a label.

8. A method according to claim 7, wherein the label is selected from the group consisting of chromophores, fluorescent moieties, enzymes, antigens, dyes, phosphorescent groups, chemiluminescent moieties, scattering or fluorescent nanoparticles and Raman signal generating moieties.

9. A method according to claim 7, wherein the label is attached to the nucleotides or nucleotide analogs at a base, sugar moiety, alpha phosphate, beta phosphate, or gamma phosphate of the nucleotides or nucleotide analogs.

10. A method according to claim 7, wherein each of the plurality of types of nucleotides or nucleotide analogs have different labels which are distinguished from one another during said identifying.

11. A method according to claim 7, wherein three or less of the plurality of types of nucleotides or nucleotide analogs have a different label.

12. A method according to claim 7, wherein the different types of nucleotides or nucleotide analogs have the same label but are distinguished by different properties due a presence of base fluorophores, quenched fluorophores, or fluorogenic nucleotides or nucleotide analogs.

13. A method according to claims 3, wherein the nucleic acid polymerizing enzyme carries a label and said identifying is carried out by detecting interaction between the label and the nucleotide analog.

14. A method according to claim 13, wherein the label is a fluorescence resonance energy transfer donor or acceptor.

15. A method according to claims 1 or 2, wherein said identifying is carried out by optical procedures selected from the group consisting of far-field microspectroscopy, near-field microspectroscopy, evanescent wave or wave guided illumination, nanostructure enhancement, and combinations thereof.

16. A method according to claims 1 or 2, wherein said identifying is carried out by single photon excitation, multiphoton excitation, fluorescence resonance energy transfer, or photoconversion or a combination thereof.

17. A method according to claims 1 or 2, wherein said identifying is performed by spectral wavelength discrimination, measurement and separation of fluorescence lifetimes, fluorophore identification, background suppression or a combination thereof.

18. A method according to claim 17, wherein fluorophore identification or background suppression utilizes fast switching between excitation modes and illumination sources, and combinations thereof.

19. A method according to claims 1 or 2, wherein said plurality of target nucleic acids are attached to a support.

20. The method of claims 1 or 2, wherein each target nucleic acid is hybridized to a separate oligonucleotide which is attached to a support.

21. The method of claim 3 wherein said DNA polymerizing enzyme is attached to a support.

22. A method according to claims 1 or 2, wherein said identifying is carried out by reducing background noise resulting from free nucleotides or nucleotide analogs.

23. A method according to claim 1, wherein the nucleotides or nucleotide analogs comprise labels attached to the 5'"'"' termini of the nucleotides or analogs.

24. A method according to claim 23, wherein said identifying is effected by detecting the labels attached to the nucleotides or nucleotide analogs.

2. A method for sequencing a plurality of target nucleic acid molecules, said method comprising:

subjecting the plurality of target nucleic acid molecules to a polymerization reaction in the presence of a plurality of types of nucleotides or nucleotide analogs to yield a population of growing nucleic acid strands that are complementary to their respective target nucleic acid molecules, wherein the target nucleic acid molecule and/or the nucleic acid polymerization enzyme is attached to a support; and

optically identifying in real-time a time sequence of incorporation of a plurality of types of nucleotides or nucleotide analogs into at least two growing nucleic acid strands in said population.