APPARATUSES FOR REAL-TIME, SINGLE MOLECULE SEQUENCE DETERMINATION

• US 20110021383A1
• Filed: 03/26/2009
• Published: 01/27/2011
• Est. Priority Date: 07/07/2000
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Willson, Richard, Hardin, Susan, Briggs, James, Tu, Shiao-Chun, Gao, Xiaolian
• Application Number:
  US12/411,997
• Publication Number:
  US 20110021383A1
• Time in Patent Office: Days
• US Class Current: 506/39
• 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 single molecule sequencing apparatus comprising:

a support comprising at least one complex, wherein the complex comprises one or more tagged polymerases and one or more target nucleic acid templates immobilized on the support through at least one polymerase and/or at least one template;

at least one tagged nucleotide or nucleotide analog for the polymerase, where the tag of the at least one nucleotide or nucleotide analog is covalently bonded to a part of the nucleotide or nucleotide analog that is released upon incorporation due to action of the polymerase;

a light source; and

a detector;

where, during one or more incorporations of a tagged nucleotide or nucleotide analog by the polymerase, a detectable property of the tag of a nucleotide or nucleotide analog and/or the polymerase tag undergoes one or more changes that is detected by the detector, where the one or more changes detected by the detector can be further analyzed to determine the identity of one or more incorporated nucleotides or nucleotide analogs, thereby determining a sequence of one or more templates.

2. The apparatus of claim 1, wherein the tag of at least one nucleotide or nucleotide analog comprises a Forster resonance energy transfer (FRET) acceptor, and the tag of the polymerase comprises a FRET donor located at a position capable of supporting FRET with the acceptor tag of a nucleotide or nucleotide analog that is incorporated by the polymerase.

3. The apparatus of claim 1, wherein the detector simultaneously detects changes in detectable properties relating to two or more different complexes, which can be further analyzed to determine a sequence of two or more templates in parallel.

4. The apparatus of claim 1, wherein the support comprises regions, areas, wells, grooves, channels or patterned arrays, where at least one complex is immobilized within the regions, areas, wells, grooves, channels or patterned arrays.

5. A single molecule sequencing apparatus comprising:

a support comprising a polymerase immobilized on the support, where the polymerase is detectably tagged with a Forster resonance energy transfer (FRET) donor, and

one or more target nucleic acid templates;

at least one tagged nucleotide or nucleotide analog comprising a tag, wherein the tag comprises at least one FRET acceptor covalently bonded to a part of the nucleotide or nucleotide analog that is released upon incorporation due to action of the polymerase;

an excitation light source; and

a fluorescent detector;

where the light source irradiates the polymerase with light of a wavelength sufficient to excite the donor tag on the polymerase and the detector detects FRET events indicative of one or more incorporations of a tagged nucleotide or nucleotide analog and analyzes the events to determine the identity of one or more tagged nucleotides or nucleotide analogs, thereby generating sequence information relating to one or more templates.

6. The apparatus of claim 5, wherein each type of nucleotide or nucleotide analog comprises an acceptor tag that is different and detectably discernible from the acceptor tag of any other type of nucleotide or nucleotide analog.

7. The apparatus of claim 5, wherein at least one tag of a nucleotide or nucleotide analog is bonded to a beta (β

) phosphate, a gamma (γ

) phosphate, or any other terminal phosphate of the nucleotide or nucleotide analog.

8. The apparatus of claim 5, wherein the detector detects events and generates sequence information relating to two or more templates in parallel.

9. The apparatus of claim 5, wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, SEQUENASE®

, and the Klenow fragment from E. coli DNA polymerase I, Superscript™

reverse transcriptase, HIV reverse transcriptase and mixtures or combinations thereof.

10. The apparatus of claim 5, wherein the polymerase naturally lacks or has been genetically modified to lack 3′

to 5′

exonuclease activity.

11. The apparatus of claim 5, wherein the support comprises regions, areas, wells, grooves, channels or patterned arrays, where the polymerase is immobilized within the regions, areas, wells, grooves, channels or patterned arrays.

12. The apparatus of claim 5, wherein the tag of the polymerase is covalently attached to the —

SH group of a cysteine residue of the polymerase.

13. A single molecule sequencing apparatus, comprising:

a support;

a tagged polymerase, wherein the tag comprises a Forster resonance energy transfer (FRET) donor;

at least one target nucleic acid template, where the polymerase and the template form a complex and where the complex is immobilized on the support through the polymerase or the template;

at least one tagged nucleotide or nucleotide analog, wherein the tag of one or more nucleotides or nucleotide analogs comprises a FRET acceptor bonded to a part of the nucleotide or nucleotide analog that is released due to action of the polymerase;

an excitation light source;

a fluorescent detector; and

an analyzer;

where the light source irradiates the support with light of a wavelength sufficient to excite the donor tag on the polymerase, the detector detects one or more FRET events between the donor tag on the polymerase and the acceptor tag of a tagged incorporating nucleotide or nucleotide analog, and the analyzer analyzes the events and determines a sequence of at least one template.

14. The apparatus of claim 13, wherein each type of nucleotide or nucleotide analog comprises an acceptor tag that is different and detectably discernible from the acceptor tag of any other type of nucleotide or nucleotide analog.

15. The apparatus of claim 13, wherein at least one tag of a nucleotide or nucleotide analog is bonded to a beta (β

) phosphate, a gamma (γ

) phosphate, or any other terminal phosphate of the nucleotide or nucleotide analog.

16. The apparatus of claim 13, wherein the analyzer determines the sequence of two or more templates in parallel.

17. The apparatus of claim 13, wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, SEQUENASE®

, and the Klenow fragment from E. coli DNA polymerase I, Superscript™

reverse transcriptase, HIV reverse transcriptase and mixtures or combinations thereof.

18. The apparatus of claim 13, wherein the polymerase naturally lacks or has been genetically modified to lack 3′

to 5′

exonuclease activity.

19. The apparatus of claim 13, wherein the support comprises regions, areas, wells, grooves, channels or patterned arrays, where the polymerase is immobilized within the regions, areas, wells, grooves, channels or patterned arrays.

20. The apparatus of claim 13, wherein the tag of the polymerase is covalently attached to the —

SH group of a cysteine residue of the polymerase.