ENZYMATIC SYNTHESIS OF POLYNUCLEOTIDES USING 3'-O-AMINO-2'-DEOXYRIBONUCLEOSIDE TRIPHOSPHATE MONOMERS
The invention is directed to improvements to methods of enzymatic synthesis of polynucleotides employing 3′-O-amino-nucleoside triphosphate monomers in which aldehyde scavenging agents which are O-substituted hydroxy lamines or polyhydroxylamines reduce or prevent spurious capping of growing polynucleotide chains. thereby increasing yields of full length product.
1 . A method of synthesizing a polynucleotide, the method comprising the steps of:
(a) providing initiators each with a free 3′-hydroxyl;
(b) repeating in a reaction mixture until the polynucleotide is formed, cycles of (i) contacting under elongation conditions the initiators or elongated fragments having free 3′-O-hydroxyls with a 3′-O-amino nucleoside triphosphate and a template-independent polymerase so that the initiators or elongated fragments are elongated by incorporation of a 3′-O-amino nucleoside triphosphate to form 3′-O-amino elongated fragments, and (ii) deprotecting the elongated fragments to form elongated fragments having free 3′-hydroxyls,
wherein an effective amount of at least one aldehyde scavenger is present in the reaction mixture.
2 . The method of claim 1 , wherein said effective amount of the at least one aldehyde scavenger is delivered to said reaction mixture by at least one synthesis reagent.
3 . The method of claim 2 , wherein said effective amount of said aldehyde scavenger is delivered to the reaction mixture by said synthesis reagent comprising a 3′-O-amino nucleoside triphosphate or a template-independent polymerase, or a mixture of both.
4 . The method of claim 1 , wherein said polynucleotide is a DNA and wherein said template-independent polymerase is a terminal deoxynucleotidyltransferase (TdT).
5 . The method of claim 1 , wherein said aldehyde scavenger is an O-substituted mono- or polyhydroxylamine.
6 . The method of claim 5 , wherein said O-substituted mono- or polyhydroxylamine is selected from the group consisting of compounds defined by the formula:
7 . The method of claim 1 , wherein said effective amount is provided by a concentration of said aldehyde scavenger in the range of from 1 to 100 mM in said reaction mixture.
8 . A kit for synthesizing a polynucleotide using a template-free polymerase comprising one or more vials of synthesis reagents each containing an effective amount of at least one aldehyde scavenger.
9 . The kit of claim 8 wherein said aldehyde scavenger is an O-substituted mono- or polyhydroxylamine
10 . The kit of claim 9 , wherein said O-substituted mono- or polyhydroxylamine is selected from the group consisting of compounds defined by the formula:
11 . The method of claim 1 , wherein said aldehyde scavenger is of formula (1)
12 . The kit of claim 8 , wherein said aldehyde scavenger is of formula (1)
13 . The method of claim 1 , wherein the total concentration of the at least one aldehyde scavenger in the reaction mixture is about 1 mM to about 500 mM.
14 . The method of claim 1 , wherein the total concentration of the at least one aldehyde scavenger in the reaction mixture is about 1 mM to about 25 mM.
15 . The method of claim 11 , wherein the total concentration of the at least one aldehyde scavenger in the reaction mixture is about 1 mM to about 25 mM.