Compounds for protecting hydroxyls and methods for their use
First Claim
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1. A method for sequencing a nucleic acid comprising the steps of:
- a) contacting a target nucleic acid with a primer under conditions wherein said primer anneals to said target nucleic acid in a sequence specific manner and wherein at least a portion of said primer is complementary to a portion of said target nucleic acid;
b) incorporating a hydrocarbyldithiomethyl-modified nucleotide into said primer; and
c) detecting incorporation of said hydrocarbyldithiomethyl-modified nucleotide, wherein said hydrocarbyldithiomethyl-modified nucleotide is complementary to said target nucleic acid at said hydrocarbyldithiomethyl-modified nucleotide'"'"'s site of incorporation thereby identifying the sequence of one nucleobase of said target nucleic acid.
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Abstract
A hydrocarbyldithiomethyl-modified compound of the Formula:
or a salt thereof wherein R1 is an organic molecule and R2 is a hydrocarbyl is useful for protecting and/or blocking hydroxyl groups in organic molecules such as nucleotides. The hydrocarbyldithiomethyl-modified compounds can also be used for chemically synthesizing oligonucleotides and for sequencing nucleic acid compounds.
159 Citations
14 Claims
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1. A method for sequencing a nucleic acid comprising the steps of:
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a) contacting a target nucleic acid with a primer under conditions wherein said primer anneals to said target nucleic acid in a sequence specific manner and wherein at least a portion of said primer is complementary to a portion of said target nucleic acid;
b) incorporating a hydrocarbyldithiomethyl-modified nucleotide into said primer; and
c) detecting incorporation of said hydrocarbyldithiomethyl-modified nucleotide, wherein said hydrocarbyldithiomethyl-modified nucleotide is complementary to said target nucleic acid at said hydrocarbyldithiomethyl-modified nucleotide'"'"'s site of incorporation thereby identifying the sequence of one nucleobase of said target nucleic acid. - View Dependent Claims (2, 3, 4)
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5. A method for sequencing a nucleic acid comprising the steps of:
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a) contacting a target nucleic acid with a primer under conditions wherein said primer anneals to said target nucleic acid in a sequence specific manner and wherein at least a portion of said primer is complementary to a portion of said target nucleic acid;
b) incorporating a first 3′
-hydrocarbyldithiomethyl-modified nucleotide into said primer;
c) detecting said incorporation of said first 3′
-hydrocarbyldithiomethyl-modified nucleotide thereby identifying the sequence of a nucleobase of said target nucleic acid;
d) removing said hydrocarbyldithiomethyl group from said first incorporated hydrocarbyldithiomethyl-modified nucleotide to form a first elongated primer having a free hydroxyl group;
e) incorporating a second 3′
-hydrocarbyldithiomethyl-modified nucleotide into said first elongated primer; and
f) detecting said second hydrocarbyldithiomethyl-modified nucleotide thereby identifying the sequence of another nucleobase of said target nucleic acid, wherein said first 3′
-hydrocarbyldithiomethyl-modified nucleotide and said second 3′
-hydrocarbyldithiomethyl-modified nucleotide are complementary to said target nucleic acid at each said nucleotide'"'"'s site of incorporation.- View Dependent Claims (6, 7, 8, 9)
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10. A method for sequencing a nucleic acid comprising the steps of:
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a) providing a primer array comprising a plurality of sequencing primers;
b) contacting a target nucleic acid with said primer array under conditions wherein said sequencing primers anneal to said target nucleic acid in a sequence specific manner thereby forming target-primer complexes between complementary portions of said sequencing primers and said target nucleic acid;
c) incorporating a first 3′
-hydrocarbyldithiomethyl-modified nucleotide into at least one sequencing primer portion of said target-primer complexes, said first 3′
-hydrocarbyldithiomethyl-modified nucleotide being complementary to said target nucleic acid; and
d) detecting said incorporation of said first 3′
-hydrocarbyldithiomethyl-modified nucleotide, wherein said first 3′
-hydrocarbyldithiomethyl-modified nucleotide is complementary to said target sequence at said first 3′
-hydrocarbyldithiomethyl-modified nucleotide'"'"'s site of incorporation.- View Dependent Claims (11, 12, 13, 14)
e) removing said hydrocarbyldithiomethyl group from said first incorporated 3′
-hydrocarbyldithiomethyl-modified nucleotide to form a first elongated target-primer complex having a free 3′
hydroxyl group;
f) incorporating a second hydrocarbyldithiomethyl-modified nucleotide into said first elongated target-primer complex; and
g) detecting said second 3′
-hydrocarbyldithiomethyl-modified nucleotide, wherein said second 3′
-hydrocarbyldithiomethyl-modified nucleotide is complementary to said target sequence at said second 3′
-hydrocarbyldithiomethyl-modified nucleotide'"'"'s site of incorporation.
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12. The method of claim 10 wherein said detecting said incorporation step is performed before removing a hydrocarbyldithiomethyl moiety.
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13. The method of claim 10 wherein said method is effective for producing a plurality of nucleotide sequences, said nucleotide sequences corresponding to overlapping nucleotide sequences of said target nucleic acid.
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14. The method of claim 11 wherein said step e) is performed under conditions that do not disrupt said target-primer complexes.
Specification