Assembly of high fidelity polynucleotides

Abstract

Methods and apparatus relate to the synthesis of high fidelity polynucleotides and to the reduction of sequence errors generated during synthesis of nucleic acids on a solid support. Specifically, design of support-bound template oligonucleotides is disclosed. Assembly methods include cycles of annealing, stringent wash and extension of polynucleotides comprising a sequence region complementary to immobilized template oligonucleotides. The error free synthetic nucleic acids generated therefrom can be used for a variety of applications, including synthesis of biofuels and value-added pharmaceutical products.

What is claimed is: 
     
         1 . A method for producing at least one polynucleotide having a predefined sequence, the method comprising:
 a) providing at least a first and a second plurality of support-bound single-stranded oligonucleotides, wherein each first and second plurality of oligonucleotides has a predefined sequence and is bound to a discrete feature of the support, each first plurality of oligonucleotides comprising a sequence region at its 5′ end that is the same as a sequence region of a 3′ end of the second plurality of oligonucleotides;   b) providing a plurality of first input single-stranded oligonucleotides, wherein the 3′ end of the plurality of the first input oligonucleotide is complementary to the 3′ end of the first plurality of oligonucleotides;   c) hybridizing the plurality of first input oligonucleotides to the first plurality of support-bound oligonucleotides at a first feature;   d) generating a first plurality of complementary oligonucleotides in a chain extension reaction, thereby forming an extension product duplex;   e) dissociating the extension product duplex, thereby producing a first plurality of complementary oligonucleotides;   f) transferring the first plurality of complementary oligonucleotides from the first feature to a second feature, thereby bringing into contact the first plurality of complementary oligonucleotides to the second plurality of support-bound oligonucleotides; and   g) annealing the first plurality of complementary oligonucleotides to the second plurality of support-bound single-stranded oligonucleotides at the second feature, wherein the annealing of the first plurality of complementary oligonucleotides to the second plurality of support-bound oligonucleotides serves as a primer for extension of the first plurality of complementary oligonucleotides, thereby producing the polynucleotide.   
     
     
         2 . The method of  claim 1  further providing a third plurality of support-bound single-stranded oligonucleotides wherein each third plurality of oligonucleotides has a predefined sequence and is bound to a third discrete feature of the support, each third plurality of oligonucleotides comprising a sequence region at its 3′ end that is the same as a sequence region of a 5′ end of the second plurality of oligonucleotides, and repeating steps c) through g) thereby producing a longer polynucleotide. 
     
     
         3 . The method of  claim 1  further sequentially repeating steps c) through g) using at least a third plurality of oligonucleotides, wherein each plurality of oligonucleotides comprises a sequence region at its 3′ end that is the same as a sequence region of a 5′ end of a next plurality of oligonucleotides, thereby producing a longer polynucleotide. 
     
     
         4 . The method of  claim 1  wherein the first input oligonucleotide is a primer. 
     
     
         5 . The method of  claim 4  wherein the primer is a universal primer or a unique primer. 
     
     
         6 . The method of  claim 1  wherein the first input oligonucleotide is a synthetic oligonucleotide. 
     
     
         7 . The method of  claim 1  wherein the first input oligonucleotide is a single-stranded nucleic acid fragment. 
     
     
         8 . The method of  claim 1  further amplifying the polynucleotide. 
     
     
         9 . The method of  claim 1  wherein the plurality of support-bound oligonucleotides is synthesized on the solid support. 
     
     
         10 . The method of  claim 1  wherein the plurality of support-bound oligonucleotides is immobilized on the solid support. 
     
     
         11 . The method of  claim 1  wherein the solid support is a microarray device. 
     
     
         12 . The method of  claim 1  wherein steps b) through e) are performed within a first droplet volume at the first feature thereby releasing the first plurality of complementary oligonucleotides in the first droplet volume and transferring the first droplet volume to the second feature comprising the second plurality of support-bound oligonucleotides. 
     
     
         13 . The method of  claim 12  wherein the droplet volumes are transferred by electrowetting, temperature gradients, wettability gradients, mechanical force or any combination thereof. 
     
     
         14 . The method of  claim 1  wherein at least one features is subjected to conditions promoting primer extension. 
     
     
         15 . The method of  claim 14  wherein at least one features is subjected to thermocycling conditions. 
     
     
         16 . The method of  claim 1  comprising N pluralities of support-bound single-stranded oligonucleotides wherein the first plurality of oligonucleotides comprises at its 5′ end a sequence region that is the same as a sequence region at the 3′ end of a second oligonucleotide and wherein a N plurality of oligonucleotides comprises at its 3′ end a sequence region that is the same as a sequence region of the (N−1) oligonucleotide. 
     
     
         17 . A method for producing at least one high fidelity target polynucleotide having a predefined sequence, the method comprising:
 a) providing at least a first and a second plurality of support-bound single-stranded oligonucleotides, each first and second plurality of oligonucleotides having a predefined sequence and each first and second plurality of oligonucleotides being bound to a discrete feature of the support,
 wherein each first plurality of support-bound oligonucleotides a sequence region at its 5′ end that is the same as a sequence region of the 3′ end of the second plurality of oligonucleotides, 
 wherein each first plurality of oligonucleotides has a 3′ end that is complementary to a 3′ end of an input single-stranded polynucleotide; 
   b) hybridizing a plurality of first input polynucleotides with the first plurality of support-bound oligonucleotides at a first feature under hybridizing conditions thereby forming duplexes;   c) subjecting the duplexes to stringent melt conditions to denature duplexes having at least one mismatch in a complementary region without denaturing the duplexes that do not comprise a mismatch in the complementary region, thereby releasing a population of error-containing input polynucleotides;   d) removing error-containing polynucleotides;   e) generating a first plurality of complementary polynucleotides in a chain extension reaction under conditions promoting extension of the input polynucleotides, thereby forming extension product duplexes;   f) dissociating the extension product duplexes, thereby releasing a plurality of second input polynucleotides;   g) annealing the plurality of second input polynucleotides to a second plurality of support-bound single-stranded oligonucleotides at a second feature of the support wherein each second plurality of oligonucleotides has a 3′ end that is complementary to a 3′ end of the second input single-stranded polynucleotide; and   h) optionally repeating the cycles of stringent melt, extension, dissociation and annealing until the target polynucleotide is synthesized.   
     
     
         18 . The method of  claim 17  wherein the plurality of first input polynucleotides is generated by a previous cycle of chain extension reaction using a plurality of support-bound oligonucleotides as a template. 
     
     
         19 . The method of  claim 17  further amplifying the target polynucleotide. 
     
     
         20 . The method of  claim 17  wherein the pluralities of support-bound oligonucleotides are synthesized on the solid support. 
     
     
         21 . The method of  claim 17  wherein the pluralities of support-bound oligonucleotides are immobilized on the solid support. 
     
     
         22 . The method of  claim 17  wherein the solid support is a microarray device. 
     
     
         23 . The method of  claim 17   wherein steps b) through d) are performed within a first droplet volume at the first feature,   wherein step d) is performed by removing the first droplet volume,   wherein steps e) and f) are performed within a second droplet volume at the first feature, thereby releasing the plurality of second input polynucleotides in the second droplet volume, and   wherein step h) comprises transferring the second droplet volume to the second feature comprising the second plurality of support-bound oligonucleotides.   
     
     
         24 . The method of  claim 23  wherein the droplet volumes are transferred by electrowetting, temperature gradients, wettability gradients, mechanical force or any combination thereof. 
     
     
         25 . The method of  claim 23  wherein the first droplet is subjected first to annealing conditions and second to stringent melt conditions and wherein the second droplet is subjected to conditions promoting primer extension. 
     
     
         26 . The method of  claim 17  wherein each plurality of support bound oligonucleotides comprises a spacer sequence region at the 3′ end of the oligonucleotide. 
     
     
         27 . A method for producing at least one high fidelity target polynucleotide having a predefined sequence, the method comprising:
 a) providing at least a first and a second plurality of support-bound single-stranded oligonucleotides, each plurality of oligonucleotides having a predefined sequence and each plurality of oligonucleotides being bound to a discrete feature of a solid support,
 wherein each first plurality of oligonucleotides has at least three sequence regions, a 5′ end sequence region N, at least two sequence regions (N−1) and (N−2) that are complementary to the 3′ end of an input polynucleotide, and a 3′ end sequence region, 
 wherein each first plurality of oligonucleotides has a 3′ end that is complementary to a 3′ end of a plurality of an input single-stranded polynucleotide; 
   b) providing a plurality of first input polynucleotides at a first feature comprising the first plurality of support-bound oligonucleotides, wherein the plurality of first input polynucleotides comprises sequences regions complementary at least in part to the two sequences regions (N−1) and (N−2);   c) hybridizing the plurality of first input polynucleotides with the first plurality of support-bound oligonucleotides under hybridizing conditions wherein the 3′ end of the plurality of first input polynucleotides hybridize to the at least two sequence regions (N−1) and (N−2) of the first plurality of oligonucleotides, thereby forming duplexes;   d) subjecting the duplexes to stringent melt conditions sufficient to denature duplexes having at least one mismatch in a complementary region without denaturing the duplexes that do not comprise a mismatch in the complementary region, thereby releasing a population of error-containing input polynucleotides;   e) removing error-containing polynucleotides;   f) generating a first plurality of complementary oligonucleotides by template-dependent synthesis under condition promoting extension of the input polynucleotides, thereby forming extension product duplexes;   g) dissociating the extension product duplexes, thereby releasing a plurality of second input polynucleotides;   h) annealing the plurality of second input polynucleotides to the second plurality of support-bound single-stranded oligonucleotides; and   i) optionally repeating the cycles of stringent melt, extension, dissociation and annealing until the target polynucleotide is synthesized.   
     
     
         28 . A method of removing error-containing polynucleotides synthesized on a solid support, the method comprising:
 a) providing a plurality of support-bound single stranded oligonucleotides on a solid support, each plurality of oligonucleotides having a predefined sequence, wherein the plurality of oligonucleotides comprise a 5′ end sequence region, a 3′ end sequence region and at least two different sequences regions (N−1) and (N−2) between the 5′ end and the 3′ end sequence regions;   b) providing a plurality of input polynucleotides wherein the plurality of input polynucleotides has at its 3′ end a region that is complementary at least in part to the (N−1) and (N−2) sequences regions of the plurality of support-bound oligonucleotides;   c) hybridizing the plurality of input polynucleotides to the plurality of support-bound oligonucleotides, thereby generating duplexes;   d) subjecting the duplexes to stringent melt conditions sufficient to denature duplexes having at least one mismatch in a complementary region without denaturing the duplexes that do not comprise a mismatch in the complementary region, thereby releasing a population of error-containing input polynucleotides; and   e) removing error-containing input polynucleotides.   
     
     
         29 . The method of  claim 27  or  28  wherein the (N−1) sequence region is adjacent to the 5′ end sequence region and the (N−2) sequence region is adjacent to the (N−1) sequence region. 
     
     
         30 . The method of  claim 27  or  28  wherein the support-bound oligonucleotides comprise at least three different sequences regions (N−1), (N−2) and (N−3) between the 5′ end and the 3′ end sequence regions, and wherein the input polynucleotide hybridize to the (N−1), (N−2) and (N−3) sequences regions of the support-bound oligonucleotides. 
     
     
         31 . The method of  claim 27  or  28  wherein the input polynucleotide is a product of at least two consecutive extension chain reactions using the sequences (N−2) and (N−1) as templates. 
     
     
         32 . The method of  claim 27  or  28  wherein each extension cycle is performed at a different feature of the solid support and wherein each extension cycle uses a different plurality of oligonucleotides as template. 
     
     
         33 . The method of  claim 1 ,  17 ,  27  or  28  wherein the extension duplexes are subjected to a shuffling process before undergoing a next cycle of extension. 
     
     
         35 . The method of  claim 33  wherein the shuffling process comprises:
 a) denaturing extension duplexes, thereby releasing single-stranded extension products in solution; 
 b) re-annealing single-stranded extension products to the support-bound oligonucleotides, thereby producing re-annealed duplexes; 
 c) subjecting the re-annealed duplexes to stringent melt conditions sufficient to dissociate error-containing duplexes; 
 d) removing error-containing single-stranded extension products; and 
 f) dissociating error-free duplexes, thereby releasing error-free extension products in solution. 
 
     
     
         36 . The method of  claim 27  or  28  wherein the 3′ end sequence is a spacer sequence. 
     
     
         37 . The method of  claim 36  wherein the spacer sequence comprises a primer binding site. 
     
     
         38 . The method of  claim 27  or  28  wherein each plurality of oligonucleotides is designed to serve as a template to a different polymerase extension reaction, thereby forming pluralities of extension duplexes, wherein each plurality of extension duplexes has a substantially identical melting temperature. 
     
     
         39 . The method of  claim 38  wherein the difference of melting temperature between the plurality of duplexes is less than 10° C. 
     
     
         40 . The method of  claim 35  wherein the difference of melting temperature between the plurality of duplexes is less than 1° C. 
     
     
         41 . A method for producing at least one double-stranded polynucleotide having a predefined sequence, the method comprising:
 a) synthesizing a polynucleotide on a discrete feature of a support according to the method of  claim 1 ;   b) providing at least a first plurality of support-bound oligonucleotides, wherein the at least first plurality of oligonucleotides has a predefined sequence and is bound to a first discrete feature of the support, each first plurality of oligonucleotides comprising a primer binding sequence at its 3′ end and a sequence region at its 5′ end substantially identical to a 5′ end of the polynucleotide;   c) annealing the primer to the first plurality of oligonucleotides at the first discrete feature, wherein the annealing of the primer to the first plurality of support-bound oligonucleotides serves as a primer for extension of the first plurality of complementary oligonucleotides, thereby generating a first extension product duplex;   d) removing the primer from the first extension product duplex;   e) dissociating the first extension product duplex thereby producing a first plurality of complementary oligonucleotides;   f) transferring the first plurality of complementary oligonucleotides to a the discrete feature comprising the polynucleotide thereby bringing into contact the first plurality of oligonucleotides with the polynucleotide, wherein the first plurality of oligonucleotides is complementary to the 5′ end of the polynucleotide;   g) annealing the first plurality of complementary oligonucleotides to the polynucleotide, wherein the annealing of the oligonucleotides serves as a primer for extension of the polynucleotide, thereby producing a double stranded polynucleotide.   
     
     
         42 . The method of  claim 41  wherein the polynucleotide comprises a 3′ terminal sequence region complementary to a 5′ region of an oligonucleotide at a discrete feature and a 5′ terminal region that is not complementary to the oligonucleotide. 
     
     
         43 . The method of  claim 42  wherein the primer sequence comprises at least one Uracil. 
     
     
         44 . The method of  claim 42  wherein the primer is removed using a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII. 
     
     
         45 . The method of  claim 41  further comprising a wash step after the removal of the primer. 
     
     
         46 . A method for producing at least one double-stranded polynucleotide having a predefined sequence, the method comprising:
 a) providing at least a first, a second and a third plurality of support-bound single-stranded oligonucleotides, each first, second and third plurality of oligonucleotides having a predefined sequence and being bound to a discrete feature of the support,   each first and second plurality of oligonucleotides comprising a primer binding site at its 3′ end that is complementary to a primer sequence,   wherein the first plurality of oligonucleotide has a sequence 5′ sequence region that is complementary to the 5′ sequence region of the second plurality of oligonucleotides and a sequence region between the primer binding site and the 5′ sequence region that is identical to a 5′ end of the third plurality of oligonucleotides;   b) annealing the primers to the primer binding sites of the first and the second plurality of oligonucleotides, wherein the annealing of the primer to the first and second plurality of support-bound oligonucleotides serves as a primer for extension of the first and second plurality of complementary oligonucleotides, thereby producing a first and second plurality of extension product duplexes;   c) removing the primer sequences from the extension product duplexes;   d) dissociating the extension product duplexes, thereby producing a first and second plurality of complementary oligonucleotides;   e) hybridizing the first plurality of complementary oligonucleotides to the third plurality of oligonucleotides; and   f) hybridizing the second plurality of complementary oligonucleotides to the first plurality of oligonucleotides, thereby producing the polynucleotide.   
     
     
         47 . The method of  claim 46  further providing a fourth plurality of support-bound single-stranded oligonucleotides wherein each fourth plurality of oligonucleotides has a predefined sequence and is bound to a fourth discrete feature of the support, each fourth plurality of oligonucleotides comprising a primer binding site at its 3′ end that is complementary to a primer sequence and a sequence region that is complementary to a 5′ end of the polynucleotide, and repeating steps b) through f) thereby producing a longer polynucleotide. 
     
     
         48 . The method of  claim 46  wherein the primers hybridizing to the first and second plurality of oligonucleotides are the same. 
     
     
         49 . The method of  claim 46  wherein the primer sequence comprises at least one Uracil. 
     
     
         50 . The method of  claim 46  wherein the primer is removed using a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII. 
     
     
         51 . A method for producing at least one double-stranded polynucleotide having a predefined sequence, the method comprising:
 a) providing at least a first and a second plurality of support-bound single-stranded oligonucleotides, each first and second plurality of oligonucleotides having a predefined sequence and being bound to a first and second discrete feature of the support,   each first plurality of oligonucleotides comprising a primer binding site at its 3′ end which is complementary to a primer sequence, a first sequence region at the 5′ end of the primer binding site and a second 3′ end sequence region,   wherein the second plurality of oligonucleotides comprises a sequence region at its 5′ end that is identical to the first sequence region of the first plurality of oligonucleotides;   b) annealing the primer to the primer binding sites of the first plurality of oligonucleotides at the first feature, wherein the annealing of the primer to the first plurality of support-bound oligonucleotides serves as a primer for extension of the first plurality of complementary oligonucleotides, thereby producing a first plurality of extension product duplexes;   c) removing the primer sequences from the extension product duplexes;   d) dissociating the extension product duplexes, thereby producing a first plurality of complementary oligonucleotides;   e) hybridizing the first plurality of complementary oligonucleotides to the second plurality of oligonucleotides at the second feature;   f) providing a stem-loop oligonucleotide, wherein the 3′ end of the stem structure is complementary to the 3′ end of the extension product;   g) hybridizing the stem-loop oligonucleotide to the first plurality of oligonucleotides at the second feature; and   h) ligating the stem-loop oligonucleotide to the first extension product, thereby generating the double-stranded stem and loop polynucleotide.   
     
     
         52 . The method of  claim 51  further comprising:
 a) providing at least a third and a fourth plurality of support-bound single-stranded oligonucleotides, each third and fourth plurality of oligonucleotides having a predefined sequence and being bound to a third and fourth discrete feature of the support, 
 each third plurality of oligonucleotides comprising a primer binding site at its 3′ end which is complementary to a primer sequence, a first sequence region at the 5′ end of the primer binding site, the first region sequence being substantially identical to the 5′ end of the double-stranded stem-loop polynucleotide and a second 3′ end sequence region, 
 wherein the fourth plurality of oligonucleotides comprises a sequence region at its 5′ end which is substantially identical to the first sequence region of the third plurality of oligonucleotides; 
 b) annealing the primer to the primer binding sites of the third plurality of oligonucleotides at the third feature, wherein the annealing of the primer to the third plurality of support-bound oligonucleotides serves as a primer for extension of the third plurality of complementary oligonucleotides, thereby producing a third plurality of extension product duplexes; 
 c) removing the primer sequences from the extension product duplexes; 
 d) dissociating the extension product duplexes, thereby producing a third plurality of complementary oligonucleotides; 
 e) hybridizing the third plurality of complementary oligonucleotides to the fourth plurality of oligonucleotides at the fourth feature; 
 f) dissociating the double-stranded stem-loop polynucleotide from the second feature; 
 g) transferring the stem-loop polynucleotide to the fourth feature; 
 h) hybridizing the stem-loop polynucleotide to the fourth plurality of oligonucleotides at the fourth feature, thereby extending the stem-loop polynucleotide; and 
 i) ligating the 3′ end of the stem-loop polynucleotide with the 5′ end of the third plurality of oligonucleotides, thereby forming a longer double-stranded polynucleotide. 
 
     
     
         53 . The method of  claim 52  further repeating steps a) through h) thereby producing a longer polynucleotide. 
     
     
         54 . The method of  claim 51  wherein the primers hybridizing to the first and second plurality of oligonucleotides are the same. 
     
     
         55 . The method of  claim 51  wherein the primer sequence comprises at least one Uracil. 
     
     
         56 . The method of  claim 51  wherein the primer is removed using a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII. 
     
     
         57 . A method for producing at least one double-stranded polynucleotide having a predefined sequence, the method comprising
 a) synthesizing a polynucleotide at a first discrete feature according to  claim 1 ;   b) synthesizing a complementary oligonucleotide at a second discrete feature, wherein the 3′ terminal region of the complementary oligonucleotide is complementary to the 5′ terminal region of the polynucleotide;   c) transferring the complementary oligonucleotide to the first feature; and   d) hybridizing the complementary oligonucleotide to the polynucleotide.   
     
     
         58 . A method of producing at least one double-stranded polynucleotide having a predefined sequence, the method comprising
 a) synthesizing a plurality of construction oligonucleotides at discrete feature of a support by primer extension, wherein the 3′ end of each construction oligonucleotide are complementary to one another and wherein each construction oligonucleotides is synthesized at a different feature of the support;   b) releasing the construction oligonucleotides from the discrete features;   c) transferring the construction oligonucleotides to a discrete feature comprising an anchor oligonucleotide, wherein the anchor oligonucleotide comprises a 5′ end complementary to the 5′ end of a first construction oligonucleotide; and   d) hybridizing the construction oligonucleotides to each other and to the anchor oligonucleotide thereby producing a double-stranded polynucleotide.   
     
     
         59 . The method of  claim 59  wherein the plurality of construction oligonucleotides are synthesized on discrete features of the support, each discrete feature comprising a different plurality of oligonucleotides, each plurality of oligonucleotides comprising a primer binding site at a 3′ end. 
     
     
         60 . The method of  claim 58  further comprising providing at least one construction oligonucleotide has a stem-loop structure