Methods of producing and sequencing modified polynucleotides
View Patent ↗The present invention encompasses methods for producing a modified polynucleotide sequence that comprises a (e.g., one or more) phosphorothiolate linkage, methods for determining a polynucleotide sequence comprising a (e.g., one or more) phosphorothiolate linkage, and methods for separating forward and reverse extension products that comprise a (e.g., one or more) phosphorothiolate linkage. The invention also encompasses kits for producing and/or determining the sequence of a modified polynucleotide that comprises a (e.g., one or more) phosphorothiolate linkage.
1. A method for determining a polynucleotide sequence comprising:
a) annealing a plurality of primers to a plurality of template polynucleotide sequences;
b) extending the plurality of primers in the presence of one or more nucleoside triphosphates wherein at least one of the nucleoside triphosphates is modified, thereby producing a plurality of extension products that comprise a modified nucleotide sequence having one or more phosphorothiolate linkages;
c) cleaving the phosphorothiolate linkages in the extension products, under conditions in which a plurality of fragments are produced;
d) identifying from among the fragments produced in c), the fragments which comprise the primer; and
e) identifying the nucleotide at the 3′ end of the fragments identified in d), thereby determining a polynucleotide sequence.
2. The method of claim 1 , wherein each primer in the plurality of primers comprises a first tag.
3. The method of claim 1 , wherein the one or more nucleoside triphosphates comprises a tag.
4. The method of claim 1 , wherein the phosphorothiolate linkage is cleaved by Ag + , Hg 2+ or Cu 2+ .
5. The method of claim 4 , wherein the phosphorothiolate linkage is cleaved at a pH of about 7 and at a temperature of about 22° C. to about 37° C.
6. A method for determining a polynucleotide sequence comprising:
a) annealing a plurality of primers to a plurality of template polynucleotide sequences;
b) extending the plurality of primers in the presence of one or more nucleoside triphosphates, wherein at least one of the nucleoside triphosphates is a modified nucleoside triphosphate comprising a general structure [I]:
wherein R 1 is hydrogen, a substituted or non-substituted: alkyl, alkenyl, alkynyl or aryl group, or R 2 ;
wherein R 2 is —SH, or —SR 3 ; and
wherein R 3 is a substituted or non-substituted: alkyl, akenyl, alkynyl or aryl group;
thereby producing a plurality of extension products that comprise a modified polynucleotide sequence, wherein said modified polynucleotide sequence comprises a general structure [II]:
wherein the general structure [II] comprises at least one 3′ phosphorothiolate linkage;
c) cleaving the at least one 3′ phosphorothiolate linkage in the extension products under conditions in which a plurality of fragments are produced; and
d) identifying from among the fragments produced in c), the fragments that comprise the primer; and
e) identifying the nucleotide at the 3′ end of the fragments identified in d), thereby determining a polynucleotide sequence.
7. The method of claim 6 , wherein R 1 is hydrogen (—H) or —SCH 3 .
8. The method of claim 6 , wherein each template polynucleotide sequence in the plurality of template polynucleotide sequences comprises a sense strand and an antisense strand and wherein at least one primer is annealed to each strand prior to step (b).
9. The method of claim 6 , wherein each primer in the plurality of primers comprises a tag.
10. A method for determining a polynucleotide sequence comprising:
a) annealing a plurality of primers to a plurality of template polynucleotide sequences, wherein each primer in the plurality of primers comprises a first tag;
b) extending the plurality of primers in the presence of one or more nucleoside triphosphates, wherein at least one of the nucleoside triphosphates is a modified nucleoside triphosphate comprising a general structure [III]:
thereby producing a plurality of extension products that comprise a modified polynucleotide sequence having a general structure [IV]:
wherein the general structure [IV] comprises at least one 5′ phosphorothiolate linkage;
c) cleaving the at least one 5′ phosphorothiolate linkage in the extension products under conditions in which a plurality of fragments are produced; and
d) identifying from among the fragments produced in c), the fragments that comprise a primer; and
e) identifying the nucleotide at the 3′ end of the fragments identified in d), thereby determining a polynucleotide sequence.
11. The method of claim 10 , wherein each template polynucleotide sequence in the plurality of template polynucleotide sequences comprises a sense strand and an antisense strand and wherein at least one primer is annealed to each strand prior to step (b).