DNA SEQUENCING BY SYNTHESIS USING RAMAN AND INFRARED SPECTROSCOPY DETECTION

• US 20150080232A1
• Filed: 05/23/2012
• Published: 03/19/2015
• Est. Priority Date: 05/23/2011
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Ju, Jingyue, Wu, Jian, Li, Zengmin
• Granted Patent: US 9,624,539 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2565/632 being a surface enhanced, e.g. resonance, Raman spectrometer

Abstract

This invention provides nucleoside triphosphate analogues having the structure:

• • wherein B is a base and is adenine, guanine, cytosine, uracil or thymine, wherein R″ is an OH or an H, and wherein R′ is azidomethyl, a hydrocarbyl, or a substituted hydrocarbyl, and which has a Raman spectroscopy peak with wavenumber from 2000 cm⁻¹ to 2300 cm⁻¹ or a Fourier transform-infrared spectroscopy spectroscopy peak with wavenumber from 2000 cm⁻¹ to 2300 cm⁻¹, and also to methods of DNA sequencing and SNP detection.

Claims

1. A nucleoside triphosphate analogue having the structure:

6. The nucleoside triphosphate analogue of claim 1, having a Raman spectroscopy peak with wavenumber from 2100 cm⁻

1 to 2260 cm<sup>−

1</sup>.

8. A composition comprising four different nucleoside triphosphate (NTP) analogues of claim 1, wherein R″

is H; and

wherein R′

has the structure;

9. A method for determining the sequence of consecutive nucleotide residues present in a single-stranded DNA comprising:

(a) contacting the single-stranded DNA, having a primer hybridized to a portion thereof, with a DNA polymerase and four different nucleoside triphosphate (NTP) analogues of claim 1 under conditions permitting the DNA polymerase to catalyze incorporation onto the primer of a NTP analogue complementary to a nucleotide residue of the single-stranded DNA which is immediately 5′

to a nucleotide residue of the single-stranded DNA hybridized to the 3′

terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein (i) each of the four NTP analogues has the structure;

10. A method for determining the sequence of consecutive nucleotide residues of a single-stranded DNA comprising:

(a) contacting the single-stranded DNA, having a primer hybridized to a portion thereof, with a DNA polymerase and four different nucleoside triphosphate (NTP) analogues of claim 1 under conditions permitting the DNA polymerase to catalyze incorporation into the primer of a NTP analogue complementary to a nucleotide residue of the single-stranded DNA which is immediately 5′

to a nucleotide residue of the single-stranded DNA hybridized to the 3′

terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein (i) each of the four NTP analogues has the structure;

12. The method of claim 10, wherein the wavenumber of the Fourier transform-infrared spectroscopy peak is determined by irradiating the incorporated dNTP analogue with grazing angle infra-red light.

13. The method of claim 9, wherein the wavenumber of the Raman spectroscopy peak is determined by irradiating the incorporated dNTP analogue with 532 nm light.

15. The method of claim 9, wherein the Raman spectroscopy is surface-enhanced Raman spectroscopy.

20. A composition comprising four different nucleoside triphosphate (NTP) analogues of claim 1, wherein R″

is OH,

wherein B is a base and is adenine, guanine, cytosine, or uracil, and wherein R′

has the structure;

41. A method for determining the identity of a nucleotide residue within a stretch of consecutive nucleic acid residues in a single-stranded DNA comprising:

(a) contacting the single-stranded DNA, having a primer hybridized to a portion thereof such that the 3 terminal nucleotide residue of the primer is hybridized to a nucleotide residue of the single-stranded DNA immediately 3′

to the nucleotide residue being identified, with a DNA polymerase and at least four nucleoside triphosphate (NTP) analogues of claim 1 under conditions permitting the DNA polymerase to catalyze incorporation into the primer of an NTP analogue complementary to the nucleotide residue of the single-stranded DNA being identified, so as to form a DNA extension product, wherein (i) each of the four NTP analogues has the structure;

42. A method for determining the identity of a nucleotide residue within a stretch of consecutive nucleic acid residues in a single-stranded DNA comprising:

(a) contacting the single-stranded DNA, having a primer hybridized to a portion thereof such that the 3 terminal nucleotide residue of the primer is hybridized to a nucleotide residue of the single-stranded DNA immediately 3′

to the nucleotide residue identified, with a DNA polymerase and a nucleoside triphosphate (NTP) analogue of claim 1 under conditions permitting the DNA polymerase to catalyze incorporation into the primer of the NTP analogue if it is complementary to the nucleotide residue of the single-stranded DNA being identified, so as to form a DNA extension product, wherein (i) the NTP analogue has the structure;

47. The method of claim 41, wherein the Raman spectroscopy is surface-enhanced Raman spectroscopy.

77. The method of claim 42, wherein the Raman spectroscopy is surface-enhanced Raman spectroscopy.

2-5. -5. (canceled)

7. A polynucleotide analogue, wherein the polynucleotide analogue differs from a polynucleotide by comprising at its 3′

terminus one of the following structures in place of the H atom of the 3′

OH group normally present in the polynucleotide;

26. A process for preparing a polynucleotide analogue of claim 7, comprising:

contacting the polynucleotide with a deoxyribonucleoside triphosphate analogue having the structure;

29. The process of claim 26, wherein the DNA polymerase is 9°

N polymerase or a variant thereof, E. Coli DNA polymerase I, Bacteriophage T4 DNA polymerase, Sequenase, Tag DNA polymerase or 9°

N polymerase (exo-)A485L/Y409V.

30. A process for preparing a polynucleotide analogue of claim 7 comprising:

contacting the polynucleotide with a ribonucleoside triphosphate analogue having the structure;

11. (canceled)

14. (canceled)

16-19. -19. (canceled)

21. A nucleoside triphosphate analogue having the structure:

22-25. -25. (canceled)

27-28. -28. (canceled)

31-40. -40. (canceled)

43. A method for determining the identity of a nucleotide residue within a stretch of consecutive nucleic acid residues in a single-stranded DNA comprising:

(a) contacting the single-stranded DNA with four different oligonucleotide probes, (1) wherein each of the oligonucleotide probes comprises (i) a portion that is complementary to a portion of consecutive nucleotides of the single stranded DNA immediately 3′

to the nucleotide residue being identified, and (ii) a 3′

terminal nucleotide residue analogue comprising on its sugar a 3′

-O—

R′

group wherein R′

is (a) is azidomethyl, or a substituted or unsubstituted hydrocarbyl group and (b) has a predetermined Raman spectroscopy peak with wavenumber which is from 2000 cm<sup>−

1</sup> to 2300 cm<sup>−

1</sup>, and which is different from the wavenumber of the Raman spectroscopy peak of the R′

of the 3′

terminal nucleotide residue analogue of the other three oligonucleotide probes, or has a predetermined Fourier-transform infra red spectroscopy peak with wavenumber which is from 2000 cm<sup>−

1</sup> to 2300 cm<sup>−

1</sup>, and is different from the wavenumber of the Fourier-transform infra red spectra peak of the R′

of the 3′

terminal nucleotide residue analogue of the other three oligonucleotide probes, and (iii) each of the four terminal nucleotide residue analogue comprises a base which is different from the base of the terminal nucleotide residue analogue of the other three oligonucleotide probes, and (2) under conditions permitting hybridization of the primer which is fully complementary to the portion of consecutive nucleotides of the single stranded DNA immediately 3′

to the nucleotide residue being identified;

(b) removing oligonucleotide primers not hybridized to the single-stranded DNA; and

(c) determining the wavenumber of the Raman spectroscopy peak or wavenumber of the Fourier-transform infra red peak of the dNTP analogue of the oligonucleotide probe hybridized in step (a) so as to thereby determine the identity of the dNTP analogue of the hybridized oligonucleotide probe and thus determine the identity of the complementary nucleotide residue in the single-stranded DNA,

thereby identifying the nucleotide residue within the stretch of consecutive nucleic acid residues in the single-stranded DNA.

78. The method of claim 43, wherein the Raman spectroscopy is surface-enhanced Raman spectroscopy.

44-46. -46. (canceled)

48-76. -76. (canceled)