Methods for real-time single molecule sequence determination

• US 7,329,492 B2
• Filed: 12/08/2004
• Issued: 02/12/2008
• Est. Priority Date: 07/07/2000
• Status: Expired due to Term

• Current Assignee: VisiGen Biotechnologies, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: VisiGen Biotechnologies, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Willson, Richard, Hardin, Susan, Briggs, James, Tu, Shiao-Chun, Gao, Xiaolian
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US11/007,797
• Publication Number:
  US 20050260614A1
• Time in Patent Office: 1,161 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/133 conformational analysis
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

A sequencing methodology is disclosed that allows a single DNA or RNA molecule or portion thereof to be sequenced directly and in substantially real time. The methodology involves engineering a polymerase and/or dNTPs with atomic and/or molecular tags that have a detectable property that is monitored by a detection system.

Claims

1. A method for sequencing a plurality of target nucleic acid molecules, the method comprising:

subjecting the plurality of target nucleic acid molecules to a polymerization reaction including 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 a nucleic acid polymerase is attached to a support, where at least one of the types of nucleotides or nucleotide analogs includes a first atomic or molecular tag bonded to a nucleotide or nucleotide analog part released during incorporation; and

optically identifying a time sequence of incorporation events, at least one of the incorporation events involving the incorporation of a tagged nucleotide or nucleotide analog into at least one growing nucleic acid strand in the population, where each event involving a tagged nucleotide or nucleotide analog is characterized by a change in a detectable property of the first tag.

2. A method for sequencing a plurality of target nucleic acid molecules, the 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 a nucleic acid polymerase is attached to a support, where at least one of the types of nucleotides or nucleotide analogs includes a first atomic or molecular tag bonded to a nucleotide or nucleotide analog part released during incorporation; and

optically identifying in real-time or near real-time a time sequence of incorporation events, where at least one of the incorporation events involves the incorporation of a tagged nucleotide or nucleotide analog into at least one growing nucleic acid strand in the population, where each event involving a tagged nucleotide or nucleotide analog is characterized by a change in a detectable property of the first tag.

3. The method of claims 1 or 2, wherein the polymerase is selected from the group consisting of a DNA polymerase, reverse transcriptase, and mixtures thereof.

4. The method of claim 3, wherein the polymerase is a thermostable polymerase or a thermodegradable polymerase.

5. The method of 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, and DNA/RNA.

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

7. The method of claim 1, wherein the tagged nucleotides or nucleotide analogs types further include one additional tag or a plurality of additional tags.

8. The method of claim 1, wherein the first tags are selected from the group consisting of chromophores, fluorescent moieties, dyes, and fluorescent nanoparticles.

9. The method of claim 7, wherein the additional tag is or the additional tags are attached to the nucleotides or nucleotide analogs at a base, sugar moiety, or alpha phosphate.

10. The method of claim 1, wherein each nucleotide and nucleotide analog type includes a first tag and where the first tags make the nucleotide or nucleotide analog types distinguishable during the identifying step.

11. The method of claim 7, wherein three or less of the plurality of types of nucleotides or nucleotide analogs have a different tag.

12. The method according to claim 7, wherein the different types of nucleotides or nucleotide analogs have the same tag but are distinguished by different properties of the nucleotides or nucleotide analogs, and/or emission properties of the tags.

13. The method of claim 3, wherein the polymerase includes an atomic or molecular tag and the identifying is carried out by detecting interaction between the polymerase tag and the nucleotide, the nucleotide analog, or the tags on the nucleotide or nucleotide analog.

14. The method of claim 13, wherein the polymerase tag and at least one tag on at least one or the types of nucleotides or nucleotide analogs form a donor and acceptor pair and the interaction is fluorescence resonance energy transfer.

15. The method of claims 1 or 2, wherein the identifying is carried out by fluorescence resonance energy transfer.

16. The method of claims 1 or 2, wherein the identifying is performed by spectral wavelength discrimination, measurement and separation of fluorescence lifetimes, fluorophore identification, background suppression or a combination thereof.

17. The method of claims 1 or 2, wherein the plurality of target nucleic acids are attached to the support.

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

19. The method of claim 3, wherein the DNA polymerase is attached to the support.

20. The method of claims 1 or 2, wherein the identifying is carried out by reducing background noise resulting from free nucleotides or nucleotide analogs.

21. The method of claim 1, wherein the tag is attached to the terminal phosphate of the nucleotides or nucleotide analogs.

22. The method of claim 21, wherein the identifying is effected by detecting the tags attached to the nucleotides or nucleotide analogs.

23. The method of claim 3, wherein the polymerase lack 3′

to 5′

exonuclease activity.