Methods for sequencing individual nucleic acids under tension

• US 8,906,617 B2
• Filed: 03/09/2011
• Issued: 12/09/2014
• Est. Priority Date: 08/08/2008
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Rothberg, Jonathan M., Leamon, John H., Davidson, John F., Van Oijen, Antoine M., Hinz, Wolfgang, Davey, Melville, Hann, Bradley, Schultz, Jonathan
• Primary Examiner(s): Crow, Robert T
• Assistant Examiner(s): Dauner, Joseph G
• Application Number:
  US13/044,361
• Publication Number:
  US 20110250700A1
• Time in Patent Office: 1,371 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2523/303 Applying a physical force on a nucleic acid
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/131 Single/double strand conformational analysis, i.e. SSCP/DSCP

Abstract

The invention provides apparatuses and methods of use thereof for sequencing nucleic acids subjected to a force, and thus considered under tension. The methods may employ but are not dependent upon incorporation of extrinsically detectably labeled nucleotides.

Claims

1. A method for sequencing comprising:

immobilizing a single stranded nucleic acid target by its 3′

end or 5′

end to a surface of a substrate, the single-stranded nucleic acid target including a plurality of tandem repeat nucleic acid sequences, the single-stranded nucleic acid target including a detectable moiety at its free end;

applying at least one oligonucleotide complementary to at least a portion of a tandem repeat single stranded nucleic acid sequence of the nucleic acid target, the at least one oligonucleotide having a known sequence and hybridizing to the at least a portion of the tandem repeat nucleic acid sequence of the single stranded nucleic acid target;

applying a force to the single stranded nucleic acid target;

determining with an ion sensitive field effect transistor array and when the force is applied, a change in a length of the single stranded nucleic acid target arising from hybridizing the at least one oligonucleotide, the determining based on a change in a position of the detectable moiety caused by the conversion of single stranded bases to double stranded bases; and

associating the change in the length of the single stranded nucleic acid target with a nucleotide sequence for the nucleic acid target.

2. The method of claim 1, wherein immobilizing the single stranded nucleic acid target includes coupling the nucleic acid target to a linker or primer secured to the surface of the substrate.

3. The method of claim 1, wherein the force is in a range of approximately 1 pN to approximately 12 pN.

4. The method of claim 1, wherein the force is a flow-based force inducing tension in the nucleic acid target.

5. The method of claim 1, wherein the detectable moiety is responsive to a magnetic force and wherein the force is a magnetic force that induces tension in the nucleic acid target.

6. The method of claim 1, wherein the force is an electrical force that induces tension in the nucleic acid target.

7. The method of claim 1, wherein at least two different forces induce tension in the nucleic acid target.

8. The method of claim 1, wherein the detectable moiety includes a particle or bead.

9. The method of claim 8, wherein the particle or bead is magnetic.

10. The method of claim 1, wherein the detectable moiety includes a magnetic bead, the method further comprising applying a magnetic field proximal to the nucleic acid target.

11. The method of claim 10, wherein the magnetic field provides tension to the nucleic acid target substantially perpendicular to a flow field.

12. The method of claim 11, wherein the tension is substantially parallel to a substrate to which the nucleic acid target is immobilized.

13. The method of claim 10, wherein the magnetic field urges the magnetic bead toward an approximate central locality of a flow field.

14. A method for sequencing comprising:

immobilizing a single stranded nucleic acid target by its 3′

end or 5′

end to a surface of a substrate, the single stranded nucleic acid target including a plurality of tandem repeat nucleic acid sequences, the single stranded nucleic acid target including a detectable moiety at its free end;

applying a plurality of oligonucleotides complementary to at least a portion of the tandem repeat nucleic acid sequences, the plurality of oligonucleotides having a known sequencing and hybridizing to the plurality of tandem repeat nucleic acid sequences;

applying a force to the single stranded nucleic acid target;

determining with an ion sensitive field effect transistor array and when the force is applied, a change in a position of the detectable moiety arising from the hybridizing of the plurality of oligonucleotides and determining a change in a length of the single stranded nucleic acid target caused by the conversion of single stranded bases to double stranded bases; and

associating the change in the length of the single stranded nucleic acid target with a nucleotide sequence for the single stranded nucleic acid target.

15. The method of claim 14, further comprising applying tension to the single stranded nucleic acid target prior to determining a change in position.

16. The method of claim 15, wherein the detectable moiety includes a magnetic bead and wherein the tension is applied using a magnetic field.