Methods for sequencing individual nucleic acids under tension
View Patent ↗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.
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.