HIGH SPEED PARALLEL MOLECULAR NUCLEIC ACID SEQUENCING

• US 20110008794A1
• Filed: 09/21/2010
• Published: 01/13/2011
• Est. Priority Date: 08/30/1999
• Status: Active Grant

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: Government of The USA Represented By The Secretary
• Inventors: Schneider, Thomas D.
• Granted Patent: US 8,535,881 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• 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 2563/107 fluorescence

Abstract

A method and device is disclosed for high speed, automated sequencing of nucleic acid molecules. A nucleic acid molecule to be sequenced is exposed to a polymerase in the presence of nucleotides which are to be incorporated into a complementary nucleic acid strand. The polymerase carries a donor fluorophore, and each type of nucleotide (e.g. A, T/U, C and G) carries a distinguishable acceptor fluorophore characteristic of the particular type of nucleotide. As the polymerase incorporates individual nucleic acid molecules into a complementary strand, a laser continuously irradiates the donor fluorophore, at a wavelength that causes it to emit an emission signal (but the laser wavelength does not stimulate the acceptor fluorophore). In particular embodiments, no laser is needed if the donor fluorophore is a luminescent molecule or is stimulated by one. The emission signal from the polymerase is capable of stimulating any of the donor fluorophores (but not acceptor fluorophores), so that as a nucleotide is added by the polymerase, the acceptor fluorophore emits a signal associated with the type of nucleotide added to the complementary strand. The series of emission signals from the acceptor fluorophores is detected, and correlated with a sequence of nucleotides that correspond to the sequence of emission signals.

Claims

1. A method for high speed parallel molecular nucleic acid sequencing, comprising:

(a) attaching a nucleic acid molecule to be sequenced to a substrate;

(b) adding an oligonucleotide primer under conditions where the primer anneals to the nucleic acid molecule;

(c) adding a green fluorescent protein (GFP)-polymerase and nucleotides, wherein the nucleotides are labeled with a fluorophore, and wherein each type of nucleotide comprises a different fluorophore that emits a unique emission signal corresponding to addition of that type of nucleotide as the nucleotide is incorporated into a complementary nucleic acid molecule by the GFP-polymerase; and

(d) detecting the unique emission signal.

2. The method of claim 1, wherein the unique emission signal is converted into a signal for a specific nucleotide in a nucleic acid sequence.

3. The method of claim 1, wherein the nucleic acid molecule is DNA and the polymerase is a DNA or RNA polymerase.

4. The method of claim 1, wherein the nucleic acid molecule is RNA and the polymerase is reverse transcriptase.

5. The method of claim 1, wherein the polymerase is a Klenow fragment of DNA polymerase I.

6. The method of claim 1, wherein the unique emission signal is generated by fluorescent resonance energy transfer (FRET) or luminescence resonance energy transfer (LRET).

7. The method of claim 1, wherein the fluorophore of each nucleotide is an acceptor fluorophore.

8. The method of claim 1, wherein the GFP is a GFP mutant protein.

9. The method of claim 1, wherein the GFP mutant protein is RSGFP4, BFP5, or H9-40.

10. The method of claim 7, wherein the acceptor fluorophores are BODIPY, fluorescein, rhodamine green, and Oregon green or derivatives thereof, or tetramethylrhodamine, Lissamine™

, Texas Red and naphthofluorescein.

11. The method of claim 1, wherein the GFP-polymerase is an aequorin-GFP-polymerase.

12. The method of claim 1, further comprising performing a plurality of sequencing reactions substantially simultaneously, and detecting the signals from the plurality of sequencing reactions.

13. The method of claim 12, wherein a plurality of nucleic acid molecules are fixed directly or indirectly to the substrate in a predetermined pattern, and detecting the signal further comprises correlating the signal with a single nucleic acid molecule corresponding to a predetermined position within that pattern.

14. The method of claim 1, wherein the substrate comprises a biocompatible material that is transparent to light.

15. The method of claim 1, further comprising adding four deoxyribonucleoside monophosphates (dNMPs) or deoxyribonucleoside diphosphates (dNDPs), wherein the dNMPs or dNDPs comprise a fluorophore distinct from the fluorophores on the nucleotides.

16. The method of claim 1, further comprising adding an oxygen scavenger system.

17. A method for high speed parallel molecular nucleic acid sequencing, comprising:

(a) attaching a green fluorescent protein (GFP)-polymerase to a substrate;

(b) adding a nucleic acid molecule to be sequenced;

(c) adding a oligonucleotide primer under conditions where the primer anneals to the nucleic acid molecule to be sequenced;

(d) adding nucleotides, wherein the nucleotides are labeled with a fluorophore, and wherein each type of nucleotide comprises a different fluorophore that emits a unique emission signal corresponding to addition of that type of nucleotide as the nucleotide is incorporated into a complementary nucleic acid molecule by the GFP-polymerase; and

(e) detecting the unique emission signal.

18. The method of claim 17, wherein the GFP-polymerase is attached to the substrate via a histidine, S-tag, or glutathione-S-transferase (GST) linker.

19. The method of claim 17, wherein the GFP-polymerase is a GFP-aequorin-polymerase.

20. The method of claim 17, further comprising adding an oxygen scavenger system.