Methods and compositions for sequencing a nucleic acid
View Patent ↗The invention provides a family of tethered nucleotide analogs useful in sequencing nucleic acids containing a homopolymer region comprising, for example, two or more base repeats, and to sequencing methods using such tethered nucleotide analogs.
1. A nucleoside triphosphate analog comprising a chemical structure within the following formula:
wherein:
NTP is a nucleoside triphosphate or analog thereof capable of incorporating onto the 3′ end of a polynucleotide strand hybridized to a template presenting the complement of said NTP;
R1 is an alkyl, alkenyl, alkynyl amide, or alkyl amide;
R2 is a cleavable bond or group;
R3 is an alkyl, alkenyl, alkyl amide, alkynyl amide, aryl, ether, or ester;
R4 is selected from the group consisting of: OH, phosphoryl , sulfate, NH 2 , SH, an amino acid, a peptide comprising 1 to 12 amino acids,
Y, at each occurrence, independently is O or S;
Z represents H or a halogen;
R5 and R6 are independently selected from the group consisting of: H, OH, phosphoryl, P 2 O 7 −3 , sulfate, NH 2 , SH, or a halogen; and
X is selected from the group consisting of: O, S, or CH 2 .
2. The analog of claim 1 wherein said cleavable bond is selected from: a disulfide bond, an ester, an azo bond, and an amido bond.
3. The analog of claim 1 wherein said Base and the nitrogenous base of said NTP are different bases.
4. The analog of claim 1 , wherein said Base and the nitrogenous base of said NTP are each independently selected from the group consisting of: cytosine, uracil, thymine, adenine, guanine, and analogs thereof.
5. The analog of claim 1 wherein R6 is selected from the group consisting of PO 4 −2 and SO 4 − .
6. The analog of claim 1 wherein R4 is selected from the group consisting of PO 4 −2 , SO 4 − , P 2 O 7 −3 , PO 3 S −2 , and P 2 O 6 S 2 3− .
7. The analog of claim 1 wherein Z is fluorine.
8. The analog of claim 1 wherein R5 is H or OH, and R4 and R6 are each independently PO 4 −2 or SO 4 − .
9. The analog of claim 1 wherein R3 comprises ethylene glycol or propylene glycol.
10. The analog of claim 1 wherein R3 comprises a diethylene glycol or dipropylene glycol.
11. The analog of claim 10 wherein R3 is polyethylene glycol.
12. The analog of claim 1 wherein R3 comprises at least one of: ester, amide, ether, divalent alkyl, divalent alkenyl, or divalent alkynyl.
13. The analog of claim 1 wherein said Base is deaza adenine or deaza guanine and R3 is linked to the C-7 or C-8 position of said deaza adenine or deaza guanine.
14. The analog of claim 1 wherein said base is thymine, cytosine, or uracil and R3 is linked to the N-3 or C-5 positions of said thymine, cytosine, or uracil.
15. The analog of claim 1 , wherein said Label is an optically-detectable label.
16. The analog of claim 15 , wherein said optically-detectable label is a fluorophore.
17. The analog of claim 16 , wherein said fluorophore is selected from the group consisting of: Cy5 and Cy3.
18. A molecule having a structure selected from the group consisting of:
Wherein L is a detectable label and n is from 1 to 10;
Wherein L is a detectable label, Base 1 and Base 2 are independently selected from adenine, thymine, guanine, cytosine, uracil and derivatives of the foregoing, and n is from 1 to 10;
Wherein L is a detectable label, Base 1 and Base 2 are independently selected from adenine, thymine, guanine, cytosine, uracil and derivatives of the foregoing, and n is from 1 to 10.
19. A molecule having the following structure:
Wherein, R 1 is a dectable label, R 2 is an alkyl, alkenyl, amide, or ether, and Base 1 and Base 2 are independently selected from adenine, thymine, guanine, cytosine, uracil and derivatives of the foregoing.
20. A molecule having the following structure:
Wherein Base 1 and Base 2 are independently is selected from adenine, thymine, guanine, cytosine, uracil and derivatives of the foregoing; R1 is a detectable label; and R2 is an alkyl, alkenyl, amide, or ether.
21. A molecule having the following structure:
Wherein Base 1 and Base 2 are independently is selected from adenine, thymine, guanine, cytosine, uracil and derivatives of the foregoing; and R1 is a detectable label.
22. A molecule having the following structure:
Wherein Base is selected from adenine, thymine, guanine, cytosine, uracil and derivatives of the foregoing and R1 is a detectable label.
23. A molecule having the following structure:
Wherein Base 1 and Base 2 are independently is selected from adenine, thymine, guanine, cytosine, uracil and derivatives of any of the foregoing; R1 is a detectable label, R2 is selected from OH and PO 3 − , R3 is selected from a monophosphate, a diphosphate, a triphosphate, and OH, and n is from about 1 to about 10 atoms in length.
24. A method of sequencing a nucleic acid , the method comprising the steps of:
a) contacting a field of template molecules and primers annealed thereto with an analog of any of claims 1 - 23 in the presence of a polymerase to extend said primers by covalent attachment of one analog in a template-dependent manner;
b) washing the field to remove non-covalently attached analog;
c) detecting signal from label bound to said attached analog;
d) cleaving said cleavable bond thereby separating said label from said NTP; and
e) repeating steps a) through d).