Methods and devices for de novo oligonucleic acid assembly

Abstract

Methods and devices are provided herein for surfaces for de novo nucleic acid synthesis which provide for low error rates. In addition, methods and devices are provided herein for increased nucleic acid mass yield resulting from de novo nucleic acid synthesis.

What is claimed is: 
     
         1 . A method for preparing a surface for oligonucleic acid synthesis, the method comprising:
 (a) providing a structure comprising a surface, wherein the structure comprises silicon dioxide;   (b) depositing a first molecule on the surface at a first region, wherein the first molecule binds to the surface and lacks a reactive group that binds to a nucleoside phosphoramidite;   (c) depositing a second molecule on the surface at a second region, wherein the second region comprises a plurality of loci surrounded by the first region, wherein the second molecule binds to the surface and lacks a reactive group that binds to the nucleoside phosphoramidite; and   (d) depositing a mixture on the surface at the second region, wherein the mixture comprises the second molecule and a third molecule, wherein the third molecule binds to the surface and nucleoside phosphoramidite, and wherein the mixture comprises a greater amount of the second molecule than the third molecule.   
     
     
         2 . The method of  claim 1 , wherein the second molecule and the third molecule both have a higher surface energy than a surface energy of the first molecule, and wherein surface energy is a measurement of water contact angle on a smooth planar surface. 
     
     
         3 . The method of  claim 2 , wherein a difference in water contact angle between the first region and the second region is at least 10, 20, 50, or 75 degrees. 
     
     
         4 . The method of any one of  claims 1  to  3 , wherein the third molecule is a silane. 
     
     
         5 . The method of any one of  claims 1  to  4 , wherein the third molecule is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane. 
     
     
         6 . The method of any one of  claims 1  to  5 , wherein the third molecule is 3-glycidoxypropyltrimethoxysilane. 
     
     
         7 . The method of  claim 4 , wherein the silane is an aminosilane. 
     
     
         8 . The method of any one of  claims 1  to  7 , wherein the second molecule is propyltrimethoxysilane. 
     
     
         9 . The method of any one of  claims 1  to  8 , wherein the first molecule is a fluorosilane. 
     
     
         10 . The method of  claim 9 , wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. 
     
     
         11 . The method of any one of  claims 1  to  10 , wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 100:1 to about 2500:1. 
     
     
         12 . The method of  claim 11 , wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 2000:1. 
     
     
         13 . The method of  claim 12 , wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of 2000:1. 
     
     
         14 . The method of any one of  claims 1  to  13 , wherein the first molecule lacks a free hydroxyl, amino, or carboxyl group. 
     
     
         15 . The method of any one of  claims 1  to  14 , wherein the second molecule lacks a free hydroxyl, amino, or carboxyl group. 
     
     
         16 . The method of any one of  claims 1  to  15 , wherein the mixture or the first molecule is in a gaseous state when deposited on the surface. 
     
     
         17 . The method of any one of  claims 1  to  16 , wherein the surface comprises a layer of silicon oxide. 
     
     
         18 . A method for preparing a surface for oligonucleic acid synthesis, the method comprising:
 (a) providing a structure comprising a surface, wherein the structure comprises silicon dioxide; wherein the surface comprises a layer of silicon oxide;   (b) coating the surface with a light-sensitive material that binds silicon oxide;   (c) exposing predetermined regions of the surface to a light source to remove a portion of the light-sensitive material coated on the surface;   (d) depositing a first molecule on the surface, wherein the first molecule binds the surface at the predetermined regions and lacks a reactive group that binds to a nucleoside phosphoramidite;   (e) removing a remaining portion of the light-sensitive material coated on the surface to expose loci, wherein each of the loci are surrounded by the predetermined regions comprising the first molecule;   (f) depositing a second molecule on the surface at the loci, wherein the second molecule binds to the loci and lacks a reactive group that binds to the nucleoside phosphoramidite; and   (g) depositing a mixture on the surface at the loci, the mixture comprises the second molecule and a third molecule, wherein the third molecule binds to the surface and nucleoside phosphoramidite.   
     
     
         19 . The method of  claim 18 , wherein the second molecule and the third molecule both have a higher surface energy than a surface energy of the first molecule, and wherein surface energy is a measurement of water contact angle on a smooth planar surface. 
     
     
         20 . The method of  claim 19 , wherein a difference in water contact angle between the first region and the second region is at least 10, 20, 50, or 75 degrees. 
     
     
         21 . The method of any one of  claims 18  to  20 , wherein the third molecule is a silane. 
     
     
         22 . The method of any one of  claims 18  to  21 , wherein the third molecule is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane. 
     
     
         23 . The method of any one of  claims 18  to  22 , wherein the third molecule is 3-glycidoxypropyltrimethoxysilane. 
     
     
         24 . The method of  claim 21 , wherein the silane is an aminosilane. 
     
     
         25 . The method of any one of  claims 18  to  24 , wherein the second molecule is propyltrimethoxysilane. 
     
     
         26 . The method of any one of  claims 18  to  25 , wherein the first molecule is a fluorosilane. 
     
     
         27 . The method of  claim 26 , wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. 
     
     
         28 . The method of any one of  claims 18  to  27 , wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 100:1 to about 2500:1. 
     
     
         29 . The method of  claim 28 , wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 2000:1. 
     
     
         30 . The method of  claim 29 , wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of 2000:1. 
     
     
         31 . The method of any one of  claims 18  to  30 , wherein the first molecule lacks a free hydroxyl, amino, or carboxyl group. 
     
     
         32 . The method of any one of  claims 18  to  31 , wherein the second molecule lacks a free hydroxyl, amino, or carboxyl group. 
     
     
         33 . The method of any one of  claims 18  to  32 , wherein the first molecule is in a gaseous state when deposited on the surface. 
     
     
         34 . The method of any one of  claims 18  to  33 , wherein the mixture is in a gaseous state when deposited on the surface. 
     
     
         35 . The method of any one of  claims 18  to  34 , further comprising applying oxygen plasma to the surface prior to coating the surface with the light-sensitive material that binds silicon oxide. 
     
     
         36 . The method of any one of  claims 18  to  35 , further comprising applying oxygen plasma to the surface after exposing predetermined regions of the surface to light. 
     
     
         37 . A method for oligonucleic acid synthesis, the method comprising:
 (a) providing predetermined sequences for at least 30,000 non-identical oligonucleic acids;   (b) providing a structure comprising a patterned surface, wherein the structure comprises silicon dioxide; wherein the patterned surface is generated by:
 (i) depositing a first molecule on the surface at a first region, wherein the first molecule binds to the surface and lacks a reactive group that binds to a nucleoside phosphoramidite; and 
 (ii) depositing a second molecule on the surface at a second region, wherein the second region comprises a plurality of loci surrounded by the first region, wherein the second molecule binds to the surface and lacks a reactive group that binds to the nucleoside phosphoramidite; and 
 (iii) depositing a mixture on the surface at the second region, wherein the mixture comprises the second molecule and a third molecule, wherein the third molecule binds to the surface and nucleoside phosphoramidite, wherein the mixture comprises a greater amount of the second molecule than the third molecule; and 
   (c) synthesizing the at least 30,000 non-identical oligonucleic acids each at least 10 bases in length, wherein the at least 30,000 non-identical oligonucleic acids encode sequences with an aggregate deletion error rate of less than 1 in 1500 bases compared to the predetermined sequences, and wherein each of the at least 30,000 non-identical oligonucleic acids extends from a different locus.   
     
     
         38 . The method of  claim 37 , wherein the second molecule and the third molecule both have a higher surface energy than a surface energy of the first molecule, and wherein surface energy is a measurement of water contact angle on a smooth planar surface. 
     
     
         39 . The method of  claim 38 , wherein a difference in water contact angle between the first region and the second region is at least 10, 20, 50, or 75 degrees. 
     
     
         40 . The method of any one of  claims 37  to  39 , wherein the third molecule is a silane. 
     
     
         41 . The method of any one of  claims 37  to  40 , wherein the third molecule is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane. 
     
     
         42 . The method of  claim 40 , wherein the silane is an aminosilane. 
     
     
         43 . The method of  claim 41  wherein the third molecule is 3-glycidoxypropyltrimethoxysilane. 
     
     
         44 . The method of any one of  claims 37  to  43 , wherein the second molecule is propyltrimethoxysilane. 
     
     
         45 . The method of any one of  claims 37  to  44 , wherein the first molecule is a fluorosilane. 
     
     
         46 . The method of  claim 45 , wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. 
     
     
         47 . The method of any one of  claims 37  to  46 , wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 100:1 to about 2500:1. 
     
     
         48 . The method of  claim 47 , wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 2000:1. 
     
     
         49 . The method of  claim 48 , wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of 2000:1. 
     
     
         50 . The method of any one of  claims 37  to  49 , wherein the first molecule lacks a free hydroxyl, amino, or carboxyl group. 
     
     
         51 . The method of any one of  claims 37  to  50 , wherein the second molecule lacks a free hydroxyl, amino, or carboxyl group. 
     
     
         52 . The method of any one of  claims 37  to  51 , wherein the mixture is in a gaseous state when deposited on the surface. 
     
     
         53 . The method of any one of  claims 37  to  52 , wherein the first molecule is in a gaseous state when deposited on the surface. 
     
     
         54 . The method of any one of  claims 37  to  53 , wherein each of the at least 30,000 non-identical oligonucleic acids is at least 30 bases in length. 
     
     
         55 . The method of any one of  claims 37  to  53 , wherein each of the at least 30,000 non-identical oligonucleic acids is 10 bases to 1 kb in length. 
     
     
         56 . The method of  claim 55 , wherein each of the at least 30,000 non-identical oligonucleic acids is about 50 to about 120 bases in length. 
     
     
         57 . The method of any one of  claims 37  to  56 , wherein the aggregate deletion error rate is less than about 1 in 1700 bases compared to the predetermined sequences. 
     
     
         58 . The method of any one of  claims 37  to  57 , wherein the aggregate deletion error rate is achieved without correcting errors. 
     
     
         59 . The method of any one of  claims 37  to  58 , wherein the at least 30,000 non-identical oligonucleic acids synthesized encode sequences with an aggregate error rate of less than 1 in 1500 bases compared to the predetermined sequences without correcting errors. 
     
     
         60 . The method of  claim 59 , wherein the aggregate error rate is less than 1 in 2000 bases compared to the predetermined sequences. 
     
     
         61 . The method of  claim 60 , wherein the aggregate error rate is less than 1 in 3000 bases compared to the predetermined sequences. 
     
     
         62 . A method for nucleic acid synthesis, the method comprising
 (a) providing predetermined sequences for at least 200 preselected nucleic acids;   (b) providing a structure comprising a patterned surface, wherein the structure comprises silicon dioxide; wherein the patterned surface is generated by:
 (i) depositing a first molecule on the surface at a first region, wherein the first molecule binds to the surface and lacks a reactive group that binds to a nucleoside phosphoramidite; and 
 (ii) depositing a second molecule on the surface at a second region, wherein the second region comprises a plurality of loci surrounded by the first region, wherein the second molecule binds to the surface and lacks a reactive group that binds to the nucleoside phosphoramidite; and 
 (iii) depositing a mixture on the surface at the second region, wherein the mixture comprises the second molecule and a third molecule, wherein the third molecule binds to the surface and nucleoside phosphoramidite, wherein the mixture comprises a greater amount of the second molecule than the third molecule; 
   (c) synthesizing at least 20,000 non-identical oligonucleic acids each at least 50 bases in length, wherein each of the at least 20,000 non-identical oligonucleic acids extends from a different locus of the patterned surface;   (d) releasing the at least 20,000 non-identical oligonucleic acids from the patterned surface;   (e) suspending the at least 20,000 non-identical oligonucleic acids in a solution; and   (f) subjecting the solution comprising at least 20,000 non-identical oligonucleic acids to a polymerase chain assembly reaction to assemble at least 200 genes, wherein the assembled at least 200 preselected nucleic acids encode sequences with an aggregate deletion error rate of less than 1 in 1500 bases compared to the predetermined sequences.   
     
     
         63 . The method of  claim 62 , wherein the second molecule and the third molecule both have a higher surface energy than a surface energy of the first molecule, and wherein surface energy is a measurement of water contact angle on a smooth planar surface. 
     
     
         64 . The method of  claim 63 , wherein a difference in water contact angle between the first region and the second region is at least 10, 20, 50, or 75 degrees. 
     
     
         65 . The method of any one of  claims 62  to  64 , wherein the third molecule is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane. 
     
     
         66 . The method of any one of  claims 62  to  64 , wherein the third molecule is a silane. 
     
     
         67 . The method of  claim 66 , wherein the third molecule is 3-glycidoxypropyltrimethoxysilane. 
     
     
         68 . The method of  claim 66 , wherein the silane is an aminosilane. 
     
     
         69 . The method of any one of  claims 62  to  68 , wherein the second molecule is propyltrimethoxysilane. 
     
     
         70 . The method of any one of  claims 62  to  69 , wherein the first molecule is a fluorosilane. 
     
     
         71 . The method of  claim 70 , wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. 
     
     
         72 . The method of any one of  claims 62  to  71 , wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 100:1 to about 2500:1. 
     
     
         73 . The method of  claim 72 , wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 2000:1. 
     
     
         74 . The method of  claim 72 , wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of 2000:1. 
     
     
         75 . The method of any one of  claims 62  to  74 , wherein the first molecule lacks a free hydroxyl, amino, or carboxyl group. 
     
     
         76 . The method of any one of  claims 62  to  75 , wherein the second molecule lacks a free hydroxyl, amino, or carboxyl group. 
     
     
         77 . The method of any one of  claims 62  to  76 , wherein each of the at least 20,000 non-identical oligonucleic acids is about 50 to about 120 bases in length. 
     
     
         78 . The method of any one of  claims 62  to  77 , wherein the aggregate deletion error rate is less than about 1 in 1700 bases compared to the predetermined sequences. 
     
     
         79 . The method of any one of  claims 62  to  78 , wherein the aggregate deletion error rate is achieved without correcting errors. 
     
     
         80 . The method of any one of  claims 62  to  79 , wherein the assembled at least 200 preselected nucleic acids encode sequences with an aggregate error rate of less than 1 in 1500 bases compared to the predetermined sequences without correcting errors. 
     
     
         81 . The method of  claim 80 , wherein the aggregate error rate is less than 1 in 2000 bases compared to the predetermined sequences. 
     
     
         82 . A device for oligonucleic acid synthesis prepared by any one of methods 1 to 36. 
     
     
         83 . A device for oligonucleic acid synthesis, the device comprising:
 (a) a structure having a surface, wherein the structure comprises silicon dioxide;   (b) a plurality of recesses or posts on the surface, wherein each recess or post comprises:
 (i) a width length that is 6.8 nm to 500 nm, 
 (ii) a pitch length that is about twice the width length, and 
 (iii) a depth length that is about 60% to about 125% of the pitch length; 
   (c) a plurality of loci on the surface, wherein each locus has a diameter of 0.5 to 100 μm, wherein each locus comprises at least two of the plurality of recesses or posts; and   (d) a plurality of clusters on the surface, wherein each of the clusters comprise 50 to 500 loci and has a cross-section of 0.5 to 2 mm.   
     
     
         84 . The device of  claim 83 , wherein each of the clusters comprise 100 to 150 loci. 
     
     
         85 . The device of  claim 83 , wherein the structure comprises at least 30,000 loci. 
     
     
         86 . The device of any one of  claims 83  to  85 , wherein the pitch length is 1 μm or less. 
     
     
         87 . The device of any one of  claims 83  to  86 , wherein the depth length is 1 μm or less. 
     
     
         88 . The device of any one of  claims 83  to  87 , wherein each of the loci has a diameter of 0.5 μm. 
     
     
         89 . The device of any one of  claims 83  to  87 , wherein each of the loci has a diameter of 10 μm. 
     
     
         90 . The device of any one of  claims 83  to  87 , wherein each of the loci has a diameter of 50 μm. 
     
     
         91 . The device of any one of  claims 83  to  90 , wherein the cross-section of each of the clusters is about 1.125 mm. 
     
     
         92 . The device of any one of  claims 83  to  91 , wherein each of the clusters has a pitch of about 1.125 mm. 
     
     
         93 . The device of any one of  claims 83  to  92 , wherein each locus comprises a molecule that binds to the surface and a nucleoside phosphoramidite. 
     
     
         94 . The device of  claim 93 , wherein the molecule that binds to the surface and the nucleoside phosphoramidite is a silane. 
     
     
         95 . The device of any one of  claims 83  to  94 , wherein the molecule that binds to the surface and the nucleoside phosphoramidite is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane. 
     
     
         96 . The device of  claim 95 , wherein the silane is 3-glycidoxypropyltrimethoxysilane. 
     
     
         97 . The device of  claim 94 , wherein the silane is an aminosilane. 
     
     
         98 . The device of any one of  claims 83  to  97 , wherein a region surrounding the plurality of loci comprises a molecule that binds to the surface and lacks a nucleoside phosphoramidite. 
     
     
         99 . The device of  claim 98 , wherein the molecule that binds to the surface and lacks the nucleoside phosphoramidite is a fluorosilane. 
     
     
         100 . The device of  claim 99 , wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. 
     
     
         101 . The device of  claim 83 , wherein the surface comprises a layer of silicon oxide. 
     
     
         102 . A method for oligonucleic acid synthesis, comprising:
 a) providing predetermined sequences;   b) providing the device of any one of  claims 83  to  101 ;   c) synthesizing a plurality of non-identical oligonucleic acids at least 10 bases in length, wherein each of the non-identical oligonucleic acids extends from a different locus.   
     
     
         103 . The method of  claim 102 , wherein the aggregate deletion error rate is less than about 1 in 1700 bases compared to the predetermined sequences. 
     
     
         104 . The method of any one of  claim 102  or  103 , wherein the aggregate deletion error rate is achieved without correcting errors. 
     
     
         105 . The method of any one of  claims 102  to  104 , wherein the plurality of non-identical oligonucleic acids synthesized encode sequences with an aggregate error rate of less than 1 in 1500 bases compared to the predetermined sequences without correcting errors. 
     
     
         106 . The method of  claim 105 , wherein the aggregate error rate is less than 1 in 2000 bases compared to the predetermined sequences. 
     
     
         107 . The method of  claim 106 , wherein the aggregate error rate is less than 1 in 3000 bases compared to the predetermined sequences. 
     
     
         108 . A method for nucleic acid synthesis, the method comprising
 (a) providing predetermined sequences for at least 200 preselected nucleic acids;   (b) providing the device of any one of  claims 83  to  101 ;   (c) synthesizing at least 20,000 non-identical oligonucleic acids each at least 50 bases in length, wherein each of the at least 20,000 non-identical oligonucleic acids extends from a different locus;   (d) releasing the at least 20,000 non-identical oligonucleic acids from the surface;   (e) suspending the at least 20,000 non-identical oligonucleic acids in a solution; and   (f) subjecting the solution comprising at least 20,000 non-identical oligonucleic acids to a polymerase chain assembly reaction to assemble at least 200 genes, wherein the assembled at least 200 preselected nucleic acids encode sequences with an aggregate deletion error rate of less than 1 in 1500 bases compared to the predetermined sequences.   
     
     
         109 . The method of  claim 108 , wherein the aggregate deletion error rate is less than about 1 in 1700 bases compared to the predetermined sequences. 
     
     
         110 . The method of any one of  claim 108  or  109 , wherein the aggregate deletion error rate is achieved without correcting errors. 
     
     
         111 . The method of any one of  claims 108  to  110 , wherein the assembled at least 200 preselected nucleic acids encode sequences with an aggregate error rate of less than 1 in 1500 bases compared to the predetermined sequences without correcting errors. 
     
     
         112 . The method of  claim 111 , wherein the aggregate error rate is less than 1 in 2000 bases compared to the predetermined sequences.