Tunneling junctions for sequencing

Patent metadata

Field	Value
Publication number	US-11718870-B2
Application number	US-201916445635-A
Country	US
Kind code	B2
Filing date	Jun 19, 2019
Priority date	Jun 21, 2018
Publication date	Aug 8, 2023
Grant date	Aug 8, 2023

Abstract

Molecules may be analyzed (e.g., sequencing of nucleic acid molecules) by tunneling recognition at a tunneling junction. Embodiments of the present invention may allow detecting individual nucleotides and the sequencing of a nucleic acid molecule using a tunneling junction. By labeling a specific nucleotide with a moiety, tunneling junctions may generate a signal with a suitable signal-to-noise ratio. The tunneling recognition uses a tunneling current that is mostly through the moiety rather than mostly through the nucleotide or a portion of the molecule of interest. Because a single nucleotide can be detected with a signal with a suitable signal-to-noise ratio resulting from the tunneling current passing through the moiety, embodiments of the present invention may allow for fast detection of nucleotides using a tunneling current.

First claim

What is claimed is:

1. A method of determining a sequence of a nucleic acid using a sequencing device, the method comprising:
adding a set of nucleotides to the sequencing device, wherein:
each nucleotide of the set of nucleotides is attached to a label compound comprising a moiety,
the sequencing device comprises a tunneling junction, and
the tunneling junction comprises a first conductor and a second conductor separated by an insulating layer, wherein:
the thickness of the insulating layer separating the first conductor and the second conductor is greater than the size of each moiety of each nucleotide of the set of nucleotides,
the first conductor comprises a first ferromagnetic material, and
the second conductor comprises a second ferromagnetic material;

elongating a nascent strand using a polymerase attached to the tunneling junction and connected to a template parent strand to be sequenced, wherein the elongating includes the polymerase incorporating a first nucleotide of the set of nucleotides into the nascent strand via hybridization to the template parent strand;
measuring a value of an electrical characteristic or a magnetic characteristic through the first conductor, a first moiety of a first label compound attached to the first nucleotide, and the second conductor; and
detecting the first nucleotide as being hybridized to the template parent strand using the value of the electrical characteristic or the magnetic characteristic.

2. The method of claim 1 , wherein:
the first conductor is a first electrode,
the second conductor is a second electrode,
measuring the value of the electrical characteristic or the magnetic characteristic comprises measuring the value of the electrical characteristic, and
detecting the first nucleotide as being hybridized to the template parent strand comprises using the value of the electrical characteristic, and
the method further comprising:
applying a voltage across the first conductor and the second conductor.

3. The method of claim 2 , wherein detecting the first nucleotide as being hybridized to the template parent strand based on the value of the electrical characteristic comprises:
comparing the value of the electrical characteristic to a reference value of the electrical characteristic, and
determining the value exceeds the reference value.

4. The method of claim 3 , wherein the reference value is of a background tunneling current that is through the first electrode and the second electrode and that does not pass through the moiety.

5. The method of claim 2 , wherein the electrical characteristic is current, and the value is greater than 10 nA.

6. The method of claim 2 , wherein the moiety is selected from the group consisting of an organometallic compound, a nanoparticle, a conjugated aromatic, and a conductive organic molecule.

7. The method of claim 2 , wherein the label compound comprises a terminator configured to prevent further elongation of the nascent strand.

8. The method of claim 2 , wherein:
each nucleotide of the set of nucleotides is a same type of nucleotide, and
each moiety of each label compound attached to each nucleotide of the set of nucleotides is a same type of moiety.

9. The method of claim 2 , wherein:
the set of nucleotides comprises a second nucleotide attached to a second label compound comprising a second moiety,
detecting the first nucleotide as being hybridized to the template parent strand based on the value of the electrical characteristic comprises:
comparing the value of the electrical characteristic to a first reference value of the electrical characteristic,
the method further comprising:
determining the second nucleotide is not being hybridized to the template parent strand based on the value of the electrical characteristic by comparing the value of the electrical characteristic to a second reference value of the electrical characteristic.

10. The method of claim 2 , wherein:
the sequencing device comprises a plurality of tunneling junctions,
each tunneling junction comprises a respective first electrode, a respective second electrode, and a respective insulating layer, and
each respective tunneling junction is attached to a respective polymerase,
the method further comprising:
for each tunneling junction of the plurality of tunneling junctions:
elongating a respective nascent strand using the respective polymerase attached to the respective tunneling junction and connected to a respective template parent strand to be sequenced, wherein the elongating includes the respective polymerase incorporating a respective nucleotide of the set of nucleotides into the respective nascent strand via hybridization to the respective template parent strand,
applying a respective voltage across the respective first electrode and the respective second electrode of the respective tunneling junction,
measuring a respective value of the electrical characteristic through the respective first electrode, the respective moiety of a respective label compound attached to the respective nucleotide, and the respective second electrode, and
detecting the respective nucleotide as being hybridized to the respective template parent strand using the respective value of the electrical characteristic.

11. The method of claim 10 , further comprising:
removing remaining nucleotides of the set of nucleotides that have not been incorporated into a nascent strand, wherein removing the set of nucleotides occurs before measuring the value of the electrical characteristic.

12. The method of claim 11 , wherein removing the set of nucleotides except the first nucleotide from contacting the tunneling junction comprises rinsing the tunneling junction with water.

13. The method of claim 11 , wherein:
the set of nucleotides is a first set of nucleotides, and
the value of the electrical characteristic is a first value of the electrical characteristic,

the method further comprising:
cleaving the first label compound from the first nucleotide,
adding a second set of nucleotides to the sequencing device, each nucleotide of the second set of nucleotides attached to a second label compound comprising a second moiety, each nucleotide of the set of nucleotides being a different type of nucleotide than the first nucleotide, each second moiety the same as the first moiety,
elongating the nascent strand by the polymerase, wherein the elongating includes the polymerase incorporating a second nucleotide of the second set of nucleotides into the nascent strand via hybridization to the template parent strand,
measuring a second value of the electrical characteristic through the first electrode, the second moiety of the second label compound attached to the second nucleotide, and the second electrode, and
detecting the second nucleotide as being hybridized to the template parent strand based on the second value of the electrical characteristic.

14. The method of claim 1 , wherein the first ferromagnetic material is a permanent magnet with a first polarity, the method further comprising:
applying a magnetic field to set a second polarity of the second ferromagnetic material to be anti-parallel to the first polarity.

15. The method of claim 1 , wherein:
measuring the value of the electrical characteristic or the magnetic characteristic comprises measuring the value of the electrical characteristic,
detecting the first nucleotide as being hybridized to the template parent strand comprises using the value of the electrical characteristic, and
the electrical characteristic is a current.

16. The method o

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Astier Yann
• Topolancik Juraj
• Kuchelmeister Hannes
• Bergmann Frank
• Heindl Dieter
• Stengele Nikolaus Klaus-Peter

Classifications

• G01N15/1023

  Microstructural devices for non-optical measurement · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• G01N15/1031

  by measuring electrical or magnetic effects · CPC title

• G01N15/1056

  Physics · mapped topic