USE OF NON-STANDARD BASES AND PROXIMITY EFFECTS FOR GENE ASSEMBLY AND CONVERSION OF NON-STANDARD BASES TO STANDARD BASES DURING DNA SYNTHESIS
The present methods relate to generating nucleic acid molecules using non-natural nucleotides. In some methods, the nucleic acid molecules may be generated by hybridizing a plurality of oligonucleotides comprising one or more non-natural nucleotides and using a polymerase and/or a coupling agent to link the hybridized oligonucleotides. The methods also relate to the use of proximity effects to generate nucleic acid molecules using non-natural nucleotides. Furthermore, the methods relate to the use of at least one non-natural base in a DNA template in order to generate a replicate of the DNA template in which the non-natural base has been replaced with a natural base.
1 . A method of generating a DNA molecule, the method comprising:
(a) hybridizing a plurality of oligonucleotides to a complementary portion of a nucleotide template, wherein the oligonucleotides comprise at least one non-natural nucleotide;
(b) coupling the plurality of hybridized oligonucleotides to form a first nucleotide strand;
(c) synthesizing a first complement to the first nucleotide strand, thereby generating a DNA molecule.
2 . The method of claim 1 , wherein synthesizing comprises reacting a mixture that comprises:
(a) the first nucleotide strand;
(b) at least one oligonucleotide primer that hybridizes to the first nucleotide strand;
(c) a polymerase; and
(d) a nucleotide mixture that comprises dATP, dCTP, dGTP, and dTTP.
3 . The method of claim 2 , wherein the nucleotide mixture does not comprise a non-natural nucleotide triphosphate.
4 . The method of claim 1 , wherein synthesizing comprises:
(a) hybridizing a second plurality of oligonucleotides to the first nucleotide strand; and
(b) coupling the second plurality of oligonucleotides to generate the first complement to the first nucleotide strand.
5 . The method of claim 4 , wherein the second plurality of oligonucleotides comprises at least one non-natural nucleotide that base-pairs with a corresponding non-natural nucleotide present in the first nucleotide strand.
6 . The method claim 1 , wherein coupling comprises enzymatic coupling.
7 . The method of claim 7 , wherein enzymatic coupling comprises ligation.
8 . The method of claim 1 , further comprising amplifying the DNA molecule.
9 . The method of claim 8 , wherein the DNA molecule is amplified in a reaction mixture that does not comprise a non-natural nucleotide triphosphate.
10 . The method of claim 1 , further comprising transforming or transfecting the DNA molecule into a cell and subjecting the cell to conditions suitable for replicating the DNA.
11 . The method of claim 1 , further comprising transforming or transfecting the DNA molecule into a cell and subjecting the cell to conditions suitable to select for expression of at least one gene product encoded by the DNA.
12 . The method of claim 1 , further comprising sequencing the double-stranded DNA.
13 . The method of claim 1 , wherein the at least one non-natural base is selected from isoguanine and isocytosine.
14 . The method of claim 1 , wherein synthesizing is performed in the presence of at least one non-natural nucleotide.
15 . The method of claim 1 , wherein the DNA molecule is no less than about 1000 nucleotides in length.
16 . The method of claim 1 , wherein hybridizing comprises base-pairing between at least one non-natural nucleotide in a first oligonucleotide and at least one non-natural nucleotide in a second oligonucleotide and base-pairing between at least one non-natural nucleotide in the first oligonucleotide and at least one non-natural nucleotide in a third oligonucleotide, and wherein the coupling occurs between the second and third oligonucleotides.
17 . The method of claim 16 , wherein the first oligonucleotide or the second oligonucleotide includes at least one non-natural base within 5 nucleotides of the 5′ or 3′ terminus of the oligonucleotide.
18 . The method of claim 16 , wherein the third oligonucleotide includes at least one non-natural base within 5 nucleotides of the 5′ or 3′ terminus of the oligonucleotide.
19 . The method of claim 16 , wherein:
(a) at least one of the first oligonucleotide and the second oligonucleotide are located at a first position on a solid substrate;
(b) the third oligonucleotide is located at a second position on the solid substrate adjacent to the first position; and
(c) the first position and second position are proximal and at a distance of no more than about 10 microns.
20 . The method of claim 19 , wherein at least one of the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide is reversibly immobilized on the substrate.
21 . The method of claim 19 , wherein at least one of the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide is irreversibly immobilized on the substrate.
22 . The method of claim 19 , wherein at least one of the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide is covalently conjugated to the substrate.
23 . A method for synthesizing a DNA molecule, the method comprising:
(a) replicating a DNA template that includes a first base pair at a selected position, wherein at least one base of the first base pair is a non-natural base;
(b) converting the at least one non-natural nucleotide of the first base pair to a selected natural nucleotide to generate a second base pair.
24 . The method of claim 23 , wherein the converting is by replicating the DNA template using a polymerase.
25 . The method of claim 23 , wherein the first base pair is selected from the group consisting of A:iC, iC:T, iG:iC, and iG:T, and the second base pair is A:T.
26 . The method of claim 23 , wherein the first base pair is selected from the group consisting of iC:iG, iC:iC, iG:iG, and A:iG and the second base pair is T:A.
27 . The method of claim 23 , wherein the first base pair is selected from the group consisting of iC:C, G:iC, and G:iG and the second base pair is G:C or T:A.
28 . The method of claim 23 , wherein the first base pair is iG:C and the second base pair is G:C.
29 . The method of claim 23 , wherein the replicating and converting occurs in a cell.
30 . A method for synthesizing a DNA molecule comprising replicating a DNA template that includes at least one non-natural base in a cell.