Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides
First Claim
1. A nucleoside 5′
- -triphosphate having a removable blocking moiety protecting the 3′
position which is a hydrocarbyl wherein said removable blocking moiety is linked to the 3′
carbon of said nucleoside 5′
-triphosphate.
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Abstract
A method for the stepwise creation of phosphodiester bonds between desired nucleosides resulting in the synthesis of polynucleotides having a predetermined nucleotide sequence by preparing an initiation substrate containing a free and unmodified 3′-hydroxyl group; attaching a mononucleotide selected according to the order of the predetermined nucleotide sequence to the 3′-hydroxyl of the initiating substrate in a solution containing a catalytic amount of an enzyme capable of catalyzing the 5′ to 3′ phosphodiester linkage of the 5′-phosphate of the mononucleotide to the 3′-hydroxyl of the initiating substrate, wherein the mononucleotide contains a protected 3′-hydroxyl group, whereby the protected mononucleotide is covalently linked to the initiating substrate and further additions are hindered by the 3′-hydroxyl protecting group. Methods in which a mononucleotide immobilized on a solid support is added to a free polynucleotide chain are also disclosed.
199 Citations
6 Claims
-
1. A nucleoside 5′
- -triphosphate having a removable blocking moiety protecting the 3′
position which is a hydrocarbyl wherein said removable blocking moiety is linked to the 3′
carbon of said nucleoside 5′
-triphosphate. - View Dependent Claims (3, 4)
- -triphosphate having a removable blocking moiety protecting the 3′
-
2. A nucleoside 5′
- -triphosphate having a removable blocking moiety protecting the 3′
position which is selected from the group consisting of an ester, an ether and a phoshate, wherein said removable blocking moiety is linked to a 3′
carbon of said nucleoside 5′
-triphosphate.
- -triphosphate having a removable blocking moiety protecting the 3′
-
5. A composition of matter, comprising:
-
(a) a nuleoside 5′
-triphosphate having a removable hydrocarbyl blocking moiety linked to a 3′
carbon of said nucleoside 5′
-triphosphate; and
(b) a solid support linked to said removable bloclcing moiety.
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6. A composition of matter, comprising:
-
(a) a nucleoside 5′
-triphosphate having a removable blocking moiety selected from the group consisting of an ester, an other and a phosphate linked to a 3′
carbon of said nucleoside 5′
-tiphosphate; and
(b) a solid support linked to said removable blocking moiety.
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Specification