DNA sequencing by synthesis using Raman and infrared spectroscopy detection
View Patent ↗This invention provides a process of labeling a polynucleotide analogue to be detected by Raman and/or infrared spectroscopy detection.
1. A nucleoside triphosphate analogue having the structure:
wherein B is a base and is adenine, guanine, cytosine, uracil or thymine,
wherein R″ is OH or H, and
wherein R′:
(i) comprises an azidomethyl moiety with chemical side groups, wherein said chemical side groups generate a unique or multiple Raman band shifts;
(ii) has a Raman spectroscopy peak with wavenumber from 2000 cm −1 to 2300 cm −1 or a Fourier transform-infrared spectroscopy peak with wavenumber from 2000 cm −1 to 2300 cm −1 ; and
(iii) is cleaved by treatment with a reducing agent, which destroys the N 3 group of the azidomethyl moiety with chemical side groups, thereby resulting in a 3′-OH.
2. The nucleoside triphosphate analogue of claim 1 having a Raman spectroscopy peak with wavenumber from 2100 cm −1 to 2260 cm −1 .
3. The nucleoside triphosphate analogue of claim 1 , wherein R′ has the structure:
wherein the wavy line indicates the point of attachment of R′ to the 3′-O, m is C 1 -C 5 , n is C 1 -C 5 , q is C 1 -C 5 , and i is C 0 -C 4 , or
wherein the wavy line represents the point of attachment to the 3′ oxygen atom, and R x is a C 1 -C 5 cyanoalkyl, a C 1 -C 5 alkyl, a C 2 -C 5 alkenyl, or a C 2 -C 5 alkynyl, which is substituted or unsubstituted,
wherein a substituted C 1 -C 5 cyanoalkyl, C 1 -C 5 alkyl, C 2 -C 5 alkenyl, or a C 2 -C 5 alkynyl is a C 1 -C 5 cyanoalkyl, C 1 -C 5 alkyl, C 2 -C 5 alkenyl, or C 2 -C 5 alkynyl in which at least one bond to a hydrogen atom contained therein is replaced by one or more bonds to a non-hydrogen or non-carbon atom with the proviso that normal valencies are maintained.
4. The nucleoside triphosphate analogue of claim 3 , wherein R′ has the structure:
wherein the wavy line indicates the point of attachment of R′ to the 3′-O, m is C 1 -C 5 , n is C 1 -C 5 , q is C 1 -C 5 , and i is C 0 -C 4 .
5. The nucleoside triphosphate analogue of claim 3 , wherein R′ has the structure:
wherein the wavy line indicates the point of attachment to the 3′ oxygen atom.
6. The nucleoside triphosphate analogue of claim 3 , wherein R′ has the structure:
wherein the wavy line represents the point of attachment to the 3′ oxygen atom, and R x is a C 1 -C 5 cyanoalkyl, a C 1 -C 5 alkyl, a C 2 -C 5 alkenyl, or a C 2 -C 5 alkynyl, which is substituted or unsubstituted,
wherein a substituted C 1 -C 5 cyanoalkyl, C 1 -C 5 alkyl, C 2 -C 5 alkenyl, or a C 2 -C 5 alkynyl is a C 1 -C 5 cyanoalkyl, C 1 -C 5 alkyl, C 2 -C 5 alkenyl, or C 2 -C 5 alkynyl in which at least one bond to a hydrogen atom contained therein is replaced by one or more bonds to a non-hydrogen or non-carbon atom with the proviso that normal valencies are maintained.
7. The nucleoside triphosphate analogue of claim 3 , wherein R″ is —H.
8. The nucleoside triphosphate analogue of claim 3 , wherein R″ is —OH.
9. The nucleoside triphosphate analogue of claim 3 , wherein R′ has a Raman spectroscopy peak with wavenumber at ˜2105 cm−1.
10. The nucleoside triphosphate analogue of claim 3 , wherein R′ has a Raman spectroscopy peak with wavenumber at ˜2138 cm−1.
11. The nucleoside triphosphate analogue of claim 3 , wherein R′ has a Raman spectroscopy peak with wavenumber at ˜2249 cm−1.
12. The nucleoside triphosphate analogue of claim 3 , wherein R′ has a Raman spectroscopy peak with wavenumber at ˜2259 cm−1.
13. The nucleoside triphosphate analogue of claim 3 , wherein the reducing agent is aqueous Tris-(2-carboxyethyl)phosphine.