Methods and devices for high fidelity polynucleotide synthesis

Abstract

Disclosed are methods for synthesizing and/or assembling at least one polynucleotide product having a predefined sequence from a plurality of different oligonucleotides. In exemplary embodiments, the methods involve synthesis and/or amplification of different oligonucleotides immobilized on a solid support, release of synthesized/amplified oligonucleotides in solution to form droplets, recognition and removal of error-containing oligonucleotides, moving or combining two droplets to allow hybridization and/or ligation between two different oligonucleotides, and further chain extension reaction following hybridization and/or ligation to hierarchically generate desired length of polynucleotide products.

What is claimed is: 
     
         1 . A method for assembling a polynucleotide having a predefined sequence from a plurality of different oligonucleotides, the method comprising:
 a) providing a plurality of single-stranded template oligonucleotides on a support, wherein each of the plurality of template oligonucleotides comprises a predefined sequence and includes a primer binding site;   b) generating a complementary oligonucleotide for each of the plurality of template oligonucleotides by enzyme-catalyzed synthesis within a primary droplet, thereby producing a plurality of double-stranded oligonucleotides;   c) releasing the complementary oligonucleotide from the double-stranded oligonucleotides into the primary droplet;   d) combining at least a first and second primary droplets, thereby forming a secondary droplet, wherein the first primary droplet includes a released oligonucleotide that comprises a portion that is complementary to a portion of a released or template oligonucleotide from the second primary droplet; and   e) exposing the secondary droplet to conditions suitable for hybridization and ligation, polymerase extension, or polymerase extension and ligation to assemble a double-stranded polynucleotide having a predefined sequence.   
     
     
         2 . The method of  claim 1  wherein the plurality of single-stranded template oligonucleotides are synthesized onto the solid support. 
     
     
         3 . The method of  claim 1  wherein the plurality of template oligonucleotides are immobilized onto the solid support. 
     
     
         4 . The method of  claim 1  wherein the plurality of template oligonucleotides are spotted onto the solid support. 
     
     
         5 . The method of  claim 1  wherein each of the plurality of template oligonucleotides includes a universal primer binding site. 
     
     
         6 . The method of  claim 1  wherein at least one of the pluralities of template oligonucleotides includes a primer binding site that is different than at least one other plurality of template oligonucleotides. 
     
     
         7 . The method of  claim 1  wherein each of the plurality of single-stranded oligonucleotides is provided on a different feature of the support. 
     
     
         8 . The method of  claim 1  wherein a plurality of primary droplets are separately defined by surface tension. 
     
     
         9 . The method of  claim 1  wherein at least a one feature is spotted with a solution suitable for primer extension. 
     
     
         10 . The method of  claim 9  wherein the solution comprises a polymerase, a primer and dNTPs. 
     
     
         11 . The method of  claim 9  wherein at least one feature is subjected to thermocycling promoting primer extension. 
     
     
         12 . The method of  claim 11  wherein the thermocycling is performed using a scanning laser. 
     
     
         13 . The method of  claim 11  wherein the thermocycling is modulated at individual features. 
     
     
         14 . The method of  claim 9  wherein spotting is performed using a mechanical wave liquid handler. 
     
     
         15 . The method of  claim 1  wherein the step of combining is performed by spotting a solution in between two first or two primary droplets. 
     
     
         16 . The method of  claim 1  wherein primary or secondary droplets are combined in parallel sets or two or more. 
     
     
         17 . The method of  claim 1  further comprising confirming the sequence of the assembled polynucleotide. 
     
     
         18 . The method of  claim 1  further comprising for each assembled polynucleotide, consecutively interrogating the identity of each of a plurality of contiguous nucleotides of the polynucleotide thereby determining a sequence for each polynucleotide. 
     
     
         19 . The method of  claim 18  further comprising selectively isolating a polynucleotide determined to have a desired sequence from one or more polynucleotides determined not to have the desired sequence. 
     
     
         20 . The method of  claim 19  wherein the step of isolating comprises selectively inactivating one or more polynucleotides determined not to have the desired sequence. 
     
     
         21 . The method of  claim 19  wherein the step of isolating comprises selectively irradiating one or more polynucleotides determined not to have the desired sequence. 
     
     
         22 . The method of  claim 19  wherein the polynucleotides determined to have the correct sequence are selectively captured using a laser tweezer. 
     
     
         23 . The method of  claim 1  wherein the secondary droplet comprises at least two different oligonucleotides, wherein the oligonucleotides are at least about 20, at least about 50, at least about 100, at least about 200, at least about 300 nucleotides in length. 
     
     
         24 . The method of  claim 1  wherein the assembled polynucleotide is at least about 1,000, at least about 1,500, at least about 2,000, at least about 5,000, at least about 10,000, at least about 50,000 nucleotides in length. 
     
     
         25 . A method for assembling at least one polynucleotide having a predefined sequence, the method comprising:
 a) providing a plurality of different oligonucleotides segregated in separate primary droplets, wherein the plurality of oligonucleotides together comprise the polynucleotide sequence, and wherein each of said primary droplets comprises multiple copies of at least one of said plurality of different oligonucleotides;   b) combining at least two primary droplets, thereby forming a secondary droplet;   c) exposing the secondary droplet to hybridization conditions and ligation, chain extension, or chain extension and ligation to generate a polynucleotide having a predefined sequence.   
     
     
         26 . The method of  claim 25 , further comprising:
 a) combining at least two secondary droplets, each droplet comprising a different polynucleotide thereby generating a third stage droplet; and   b) exposing the third stage droplet to hybridization conditions and ligation, chain extension, or chain extension and ligation to generate a longer polynucleotide having a predefined sequence.   
     
     
         27 . The method of  claim 25  wherein the oligonucleotides are at least about 20, at least about 50, at least about 100, at least about 200, at least about 300 nucleotides in length. 
     
     
         28 . The method of  claim 25  wherein the polynucleotides are at least about 1 kb, at least about 5 kb, at least about 10 kb, at least about 50 kb in length. 
     
     
         29 . The method of  claim 25  wherein the oligonucleotides comprise at least one primer binding site. 
     
     
         30 . The method of  claim 29  further comprising a pair of primers and wherein the pair of primers are universal primers. 
     
     
         31 . The method of  claim 29  further comprising a pair of primers and wherein the pair of primers are unique primers. 
     
     
         32 . The method of  claim 26  further comprising amplifying the oligonucleotides before combining the two primary droplets. 
     
     
         33 . The method of  claim 25  wherein each oligonucleotide is provided on different features of a solid support. 
     
     
         34 . The method of  claim 25  wherein the oligonucleotides are spotted onto a solid support. 
     
     
         35 . The method of  claim 25  wherein the oligonucleotides are immobilized onto a solid support. 
     
     
         36 . The method of  claim 25  wherein the oligonucleotides are synthesized onto a solid support. 
     
     
         37 . The method of  claim 25  wherein the droplets are separated to each others by surface tension. 
     
     
         38 . The method of  claim 34  wherein at least a portion of the features are spotted with a solution promoting amplification. 
     
     
         39 . The method of  claim 38  wherein the solution comprises a polymerase, a primer pair and dNTPs. 
     
     
         40 . The method of  claim 25  wherein at least one primary droplet is subjected to thermocycling promoting amplification. 
     
     
         41 . The method of  claim 25  or  26  wherein hybridization conditions comprise controlling the temperature of at least one droplet. 
     
     
         42 . The method of  claim 40  or  41  wherein the temperature control or the thermocycling is performed using a scanning laser. 
     
     
         43 . The method of  claim 42  wherein the thermocycling is modulated at individual features. 
     
     
         44 . The method of  claim 34  wherein spotting is performed using an acoustic liquid handler. 
     
     
         45 . The method of  claim 25  or  26  wherein the step of combining is performed by spotting a solution in between two first or two secondary droplets. 
     
     
         46 . The method of  claim 25  or  26  wherein a plurality of two primary or secondary droplets are combined in parallel. 
     
     
         47 . The method of  claim 25  or  26  further comprising determining the sequence of the assembled polynucleotides. 
     
     
         48 . The method of  claim 25  or  26  further comprising for each assembled polynucleotide, consecutively interrogating the identity of each of a plurality of contiguous nucleobases of the polynucleotide thereby determining a sequence for each polynucleotide. 
     
     
         49 . The method of  claim 48  further comprising selectively isolating the polynucleotides determined to have a desired sequence from one or more polynucleotide determined not to have the desired sequence. 
     
     
         50 . The method of  claim 49  wherein the step of isolating comprises selectively inactivating one or more polynucleotides determined not to have the desired sequence. 
     
     
         51 . The method of  claim 49  wherein the step of isolating comprises selectively irradiating one or more polynucleotides determined not to have the desired sequence. 
     
     
         52 . The method of  claim 49  wherein the polynucleotides determined to have the correct sequence are selectively captured using a laser tweezer. 
     
     
         53 . A polynucleotide assembly system comprising an acoustic liquid handling device operably connected to a microfluidic device, wherein the microfluidic device comprises an assembly station, a sequencing station and a polynucleotide isolation station. 
     
     
         54 . The polynucleotide assembly system of  claim 53  further comprising a scanning laser device. 
     
     
         55 . The polynucleotide assembly system of  claim 53  wherein the assembly station is a solid support. 
     
     
         56 . The polynucleotide assembly system of  claim 53  wherein the sequencing station is a sequencing by synthesis station. 
     
     
         57 . The polynucleotide assembly system of  claim 53  wherein the polynucleotide isolation station is a FACS. 
     
     
         58 . The polynucleotide assembly system of  claim 53  wherein the polynucleotide isolation station comprises a laser. 
     
     
         59 . The polynucleotide assembly system of  claim 58  wherein the laser is a laser tweezer. 
     
     
         60 . The method of  claim 1  or  25  further comprising the step of removing an error-containing oligonucleotide from a first plurality of oligonucleotides, comprising the steps of:
 a) contacting the first plurality of oligonucleotides with a second plurality of oligonucleotides immobilized on a solid support under hybridization conditions to form duplexes, wherein the first plurality of oligonucleotides comprises sequences that are complementary to at least portions of the second plurality of oligonucleotides, wherein a first duplex comprising said error-containing oligonucleotide comprises a mismatch in a complementary region, and wherein a second duplex does not comprise a mismatch in the complementary region; 
 b) denaturing said first duplex comprising said error-containing oligonucleotide under stringent melt conditions without denaturing said second duplex; 
 c) removing said error-containing oligonucleotide from the solid support; and 
 d) denaturing said second duplex, thereby forming a purified plurality of oligonucleotides. 
 
     
     
         61 . The method of  claim 60 , wherein the solid support is a microarray. 
     
     
         62 . The method of  claim 60 , wherein each of the first plurality of oligonucleotides comprises a detectable tag at one terminus. 
     
     
         63 . The method of  claim 60 , wherein each of the second plurality of oligonucleotides comprises a detectable tag at one terminus. 
     
     
         64 . The method of  claim 62  or  63 , wherein the detectable tag comprises a fluorescence tag, and wherein the stringent melt conditions are determined by a real-time melt curve. 
     
     
         65 . The method of  claim 60 , further comprising prior to step a):
 synthesizing the first plurality of oligonucleotides in a chain extension reaction, wherein the second plurality of oligonucleotides serve as templates in the chain extension reaction; and   denaturing products of the chain extension reaction.   
     
     
         66 . The method of  claim 65 , wherein said denaturing step comprises melting said products by raising temperature, using a helicase, or both. 
     
     
         67 . The method of  claim 60 , further comprising reusing the solid support in synthesizing oligonucleotides, hybridizing oligonucleotides, or both. 
     
     
         68 . The method of  claim 60 , further comprising using a digital mirror device to selectively heat different spots on the solid support. 
     
     
         69 . The method of  claim 60 , further comprising repeating a)-c) at least one time prior to forming the purified plurality of oligonucleotides. 
     
     
         70 . A method for removing error-containing oligonucleotides synthesized on a solid support, the method comprising:
 a) synthesizing a first plurality of oligonucleotides in a chain extension reaction, wherein a second plurality of oligonucleotides immobilized on said solid support serves as templates in the chain extension reaction;   b) denaturing products of the chain extension reaction;   c) contacting the first plurality of oligonucleotides with the second plurality of oligonucleotides under hybridization conditions to form duplexes; and   d) separating error-containing oligonucleotides from oligonucleotides with error-free sequences using a component which actively selects for a sequence error.   
     
     
         71 . The method of  claim 70 , wherein said sequence error selecting component comprises mismatch recognition protein MutS or a functional homolog of MutS. 
     
     
         72 . A method for removing error-containing oligonucleotides synthesized on a solid support, the method comprising:
 a) synthesizing a first plurality of oligonucleotides in a chain extension reaction on a first spot on said solid support, wherein a second plurality of oligonucleotides immobilized on said first spot on said solid support serves as templates in the chain extension reaction;   b) denaturing products of the chain extension reaction;   c) contacting the first plurality of oligonucleotides with a third plurality of oligonucleotides under hybridization conditions to form duplexes, wherein said third plurality of oligonucleotides are synthesized on a second spot on said solid support substantially in parallel to step a), and wherein said first and third plurality of oligonucleotides comprise sequences that are complementary; and   d) separating error-containing oligonucleotides from oligonucleotides with error-free sequences using a component which actively selects for a sequence error.   
     
     
         73 . The method of  claim 72 , wherein said sequence error selecting component comprises mismatch recognition protein MutS or a functional homolog of MutS. 
     
     
         74 . A method of assembling a polynucleotide product on a solid support comprising:
 a) moving a first droplet comprising a first plurality of oligonucleotides from a first spot on said solid support to a second spot on said solid support, wherein said second spot comprises a second plurality of oligonucleotides, wherein a terminal region of said second plurality of oligonucleotides comprise complementary sequences with a terminal region of said first plurality of oligonucleotides; and   b) contacting said first and second pluralities of oligonucleotides under conditions that allow one or more of: annealing, chain extension, and denaturing.   
     
     
         75 . The method of  claim 74 , further comprising moving a second droplet from a third spot to a fourth spot substantially in parallel to step a), wherein the second droplet comprises a third plurality of oligonucleotides, wherein said fourth spot comprises a fourth plurality of oligonucleotides, and wherein a terminal region of said third or fourth plurality of oligonucleotides comprises complementary sequences with a terminal region of said first or second plurality of oligonucleotides. 
     
     
         76 . The method of  claim 75 , further comprising hierarchically assembling said polynucleotide product that comprises sequences of said first, second, third, and fourth pluralities of oligonucleotides. 
     
     
         77 . The method of  claim 76 , wherein said first droplet is moved by a surface acoustic wave device. 
     
     
         78 . A method for qualitatively confirming a sequence of a polynucleotide product on a solid support comprising:
 a) synthesizing said polynucleotide product on said solid support, wherein said solid support comprises one of more spots comprising immobilized oligonucleotides, and wherein said polynucleotide product comprises a detectable tag;   b) recycling said solid support;   c) contacting said polynucleotide product with said recycled solid support under hybridization conditions to form duplexes between said polynucleotide product and said immobilized oligonucleotides; and   d) detecting a presence of said detectable tag at said one or more spots, thereby confirming the sequence of said polynucleotide product.   
     
     
         79 . A method for synthesizing a plurality of oligonucleotide having a predefined sequence, the method comprising:
 a) providing a plurality of support-bound template oligonucleotides in a solution comprising a primer, a polymerase and nucleotides, wherein each of the plurality of template oligonucleotides comprises a predefined sequence and includes a primer binding site, and wherein the primer comprises at least one nuclease recognition site;   b) exposing the plurality of template oligonucleotides to conditions suitable for primer hybridization and polymerase extension, thereby extending the primers to produce a complementary oligonucleotide for each of the plurality of template oligonucleotides;   c) releasing the complementary oligonuceotides;   d) exposing the complementary oligonucleotides to a nuclease under conditions suitable for the nuclease to bind to the nuclease recognition site on the primer and cleave the primer from complementary oligonucleotide; and   e) exposing the complementary and template oligonucleotides to conditions suitable for hybridization; thereby to produce a plurality of partially double-stranded oligonucleotides.   
     
     
         80 . The method of  claim 79 , further comprising washing the plurality of partially double-stranded oligonucleotides and releasing the complementary oligonucleotides. 
     
     
         81 . The method of  claim 79  wherein the plurality of template oligonucleotides are synthesized in situ on a support surface. 
     
     
         82 . The method of  claim 79  wherein the primer comprises at least two nuclease recognition sites. 
     
     
         83 . The method of  claim 82  further comprises the step of exposing the cleaved primer to a second nuclease under conditions suitable for the second nuclease to bind to the primer and subject the primer to further cleavage.