NUCLEIC ACID TERMINATORS INCORPORATING A CATIONIC MOIETY AND METHODS FOR THEIR USE
Disclosed are methods and kits applicable to sequencing methods, such as Sanger dideoxy sequencing methods. The methods and kits disclosed utilize a cationically charged nucleic acid terminator in combination with a discriminatory polymerase.
1 . A method of sequencing a target polynucleotide sequence comprising:
(a) providing a cationically charged nucleic acid terminator wherein the cationically charged nucleic acid terminator comprises a labeled compound of structure (I):
Z-X—S—B-L (I)
wherein Z is selected from the group consisting of a mono-phosphate, a di-phosphate, a tri-phosphate, a thiophosphate, and a boranophosphate;
X is selected from the group consisting of O, CH 2 , S, and NH;
S is selected from the group consisting of a sugar and a sugar analogue;
B is selected from the group consisting of a naturally occurring base, a synthetic base, and a nucleobase;
L is a linker that is selected from the group consisting of alkyl, alkenyl, and alkynyl;
wherein at least one of L, B, S, X or Z is substituted with a moiety that imparts a positive charge to structure (I), and wherein at least one of L, B, S, X, or Z is substituted with a reporter moiety;
(b) reacting the cationically charged nucleic acid terminator with a discriminatory polymerase that is exonuclease minus;
(c) separating the reacted cationically charged nucleic acid terminator on the basis of size; and
(d) determining the sequence of the target polynucleotide sequence.
2 . The method of claim 1 , wherein the discriminatory polymerase is a Thermus aquaticus DNA polymerase.
3 . The method of claim 1 , wherein the discriminatory polymerase is an Escherichia coli DNA polymerase.
4 . The method of claim 1 , wherein the discriminatory polymerase is a Pfu DNA polymerase from Pyrococcus furiosus.
5 . The method of claim 1 , wherein the discriminatory polymerase is a DNA polymerase from Bacillus stearothermophilus.
6 . The method of claim 1 , wherein the moiety which imparts a positive charge to structure (I) is selected from the group consisting of amines, higher alkyl amines, aryl amines and imidazoles.
7 . The method of claim 6 , wherein the moiety is a primary, secondary, tertiary, or quaternary amine.
8 . The method of claim 1 wherein the moiety which imparts a positive charge to structure (I) is selected from the group consisting of tetraalkyl ammonium moieties, trialkyl ammonium moieties, imidazolium moieties, aryl ammonium moieties, iminium moieties, amidinium moieties, guanadinium moieties, thiazolium moieties, pyrazolylium moieties, pyrazinium moieties, pyridinium moieties, and phosphonium moieties.
9 . The method of claim 1 , wherein L contains up to about 1000 atoms.
10 . The method of claim 1 , wherein L contains from about 2 to about 500 atoms.
11 . The method of claim 1 , wherein L contains from about 11 to about 250 atoms.
12 . The method of claim 1 , wherein L contains from about 18 to about 25 atoms.
13 . The method of claim 1 , wherein L further comprises the reporter moiety.
14 . The method of claim 13 , wherein the reporter moiety is selected from the group consisting of a radioisotope label, an electrochemical label, a fluorescent label, an energy transfer label, a mass spectrometry label, a Raman label, a hapten, a chemilluminescent group label, an enzyme, a chromophore label, and combinations thereof.
15 . A kit for sequencing a polynucleotide sequence, the kit comprising:
(a) a cationically charged nucleic acid terminator, the cationically charged nucleic acid terminator comprising a labeled compound of structure (I):
Z-X—S—B-L (I)
wherein Z is selected from the group consisting of a mono-phosphate, a di-phosphate, a tri-phosphate, a thiophosphate, and a boranophosphate;
X is selected from the group consisting of O, CH 2 , S, and NH;
S is selected from the group consisting of a sugar and a sugar analogue;
B is selected from the group consisting of a naturally occurring base, a synthetic base, and a nucleobase;
L is a linker that is selected from the group consisting of alkyl, alkenyl, and alkynyl;
wherein at least one of L, B, S, X or Z is substituted with a moiety which imparts a positive charge to structure (I), and wherein at least one of L, B, S, X, or Z is substituted with a reporter moiety; and
(b) a discriminatory polymerase that is exonuclease minus.
16 . The kit of claim 15 , wherein the discriminatory polymerase is a Thermus aquaticus DNA polymerase.
17 . The kit of claim 15 , wherein the discriminatory polymerase is an Escherichia coli DNA polymerase.
18 . The kit of claim 15 , wherein the discriminatory polymerase is a Pfu DNA polymerase from Pyrococcus furiosus.
19 . The kit of claim 15 , wherein the discriminatory polymerase is a DNA polymerase from Bacillus stearothermophilus.
20 . The kit of claim 15 , wherein the moiety which imparts a positive charge to structure (I) is selected from the group consisting of amines, higher alkyl amines, aryl amines, imidazoles, and combinations thereof.
21 . The kit of claim 20 , wherein the moiety is a primary, secondary, tertiary, or quaternary amine.
22 . The kit of claim 15 wherein the moiety which imparts a positive charge to structure (I) is selected from the group consisting of tetraalkyl ammonium moieties, trialkyl ammonium moieties, imidazolium moieties, aryl ammonium moieties, iminium moieties, amidinium moieties, guanadinium moieties, thiazolium moieties, pyrazolylium moieties, pyrazinium moieties, pyridinium moieties, and phosphonium moieties.
23 . The kit of claim 15 , wherein L contains up to about 1000 atoms.
24 . The kit of claim 15 , wherein L contains from about 2 to about 500 atoms.
25 . The kit of claim 15 , wherein L contains from about 11 to about 250 atoms.
26 . The kit of claim 15 , wherein L contains from about 18 to about 25 atoms.
27 . The kit of claim 15 , wherein L further comprises the reporter moiety.
28 . The kit of claim 27 , wherein the reporter moiety is selected from the group consisting of a radioisotope label, an electrochemical label, a fluorescent label, an energy transfer label, a mass spectrometry label, a Raman label, a hapten, a chemilluminescent group label, an enzyme, a chromophore label, and combinations thereof.