Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly

Abstract

Methods and devices are provided for manipulating droplets on a support using surface tension properties, moving the droplets along a predetermined path and merging two droplets together enabling a number of chemical reactions. Disclosed are methods for controlling the droplets volumes. Disclosed are methods and devices for synthesizing at least one oligonucleotide having a predefined sequence. Disclosed are methods and devices for synthesizing and/or assembling at least one polynucleotide product having a predefined sequence from a plurality of different oligonucleotides having a predefined sequence. 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 oligonu-cleotides, 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 preparing a plurality of oligonucleotides on a support, the method comprising:
 a. providing a support comprising a plurality of surface-bound single-stranded oligonucleotides contained within one or more droplets of a predefined volume of solution;   b. exposing the plurality of surface-bound oligonucleotides to conditions suitable for a template-dependent synthesis reaction, thereby producing a plurality of complementary oligonucleotides; and   c. adjusting or substantially maintaining the volume of the one or more droplets of solution.   
     
     
         2 . The method of  claim 1  wherein the step of adjusting or substantially maintaining the volume of the one or more droplets of solution comprises maintaining the droplets under conditions that substantially limit solvent evaporation. 
     
     
         3 . The method of  claim 1  wherein the plurality of surface-bound oligonucleotides are coupled to the surface at a feature that is selectively coated with a coating material. 
     
     
         4 . The method of  claim 3  wherein the coating material has water trapping properties. 
     
     
         5 . The method of  claim 4  wherein the coating material is selected from the group of colloidal silica, peptide gel, agarose, solgel and polydimethylsiloxane. 
     
     
         6 . The method of  claim 2  wherein solvent evaporation is substantially limited by blocking the interface of the droplets with the atmosphere. 
     
     
         7 . The method of  claim 6  wherein the droplets are overlaid with a non-miscible liquid thereby preventing water evaporation of the solution. 
     
     
         8 . The method of  claim 7  wherein the non-miscible liquid forms a lipid bilayer. 
     
     
         9 . The method of  claim 7  wherein the non-miscible liquid forms a thin film at the surface of the droplet. 
     
     
         10 . The method of  claim 7  wherein the non-miscible liquid is a solvent. 
     
     
         11 . The method of  claim 7  wherein the non-miscible liquid is mineral oil. 
     
     
         12 . The method of  claim 7  wherein the non-miscible liquid is spotted onto the droplet. 
     
     
         13 . The method of  claim 12  wherein the step of spotting is performed with an inkjet or mechanical device. 
     
     
         14 . The method of  claim 1  further comprising monitoring the volume of the one or more droplets for evaporation. 
     
     
         15 . The method of  claim 1  wherein the step of adjusting or substantially maintaining the volume of the one or more droplets of solution comprises adjusting droplet volume by providing additional solution in response to evaporation. 
     
     
         16 . The method of  claim 15  wherein the step of adjusting droplet volume is performed with an inkjet device. 
     
     
         17 . The method of  claim 2  wherein solvent evaporation is limited by increasing the humidity around the one or more droplets. 
     
     
         18 . The method of  claim 17  wherein the humidity is increased locally by depositing satellite droplets in the vicinity of the one or more droplets. 
     
     
         19 . The method of  claim 1  wherein the plurality of surface-bound oligonucleotides comprise a primer binding site, and wherein the solution comprises a polymerase, at least one primer and dNTPs and wherein the primer is complementary to the primer binding site. 
     
     
         20 . The method of  claim 19  wherein the primer is a unique primer. 
     
     
         21 . The method of  claim 19  wherein the primer is a universal primer. 
     
     
         22 . The method of  claim 19  wherein the at least one primer is a pair of primer. 
     
     
         23 . The method of  claim 22  wherein the pair of primers are unique primers. 
     
     
         24 . The method of  claim 22  wherein the pair of primers are universal primers. 
     
     
         25 . The method of  claim 19  wherein the plurality of surface-bound oligonucleotides is subjected to thermocycling thereby promoting primer extension within a droplet. 
     
     
         26 . The method of  claim 25  further heating the surface to a denaturing temperature thereby providing a plurality of single-stranded complementary oligonucleotides within the one or more droplets. 
     
     
         27 . The method of  claim 1  wherein the support comprises a plurality of discrete features, and wherein each feature comprises a plurality of surface-bound single-stranded oligonucleotides contained within a droplet of a predefined volume of solution. 
     
     
         28 . The method of  claim 27  wherein each discrete feature comprises a plurality of surface-bound oligonucleotides having a different predefined sequence. 
     
     
         29 . The method for preparing a plurality of oligonucleotides, the method comprising the steps of:
 a. providing a support comprising a plurality of discrete features, each feature comprising a plurality of surface-bound single-stranded oligonucleotides having a predefined sequence, where each surface-bound oligonucleotide is hybridized to a synthesized oligonucleotide from a template-dependent reaction thereby forming a hybridized oligonucleotide duplex;   b. heating the support to a first melting temperature under stringent melt conditions thereby denaturing the hybridized oligonucleotide duplexes comprising error-containing oligonucleotides and releasing error-containing oligonucleotides;   c. removing the error-containing oligonucleotides from the solid support;   d. denaturing error-free duplexes; and   e. releasing error-free oligonucleotides in solution.   
     
     
         30 . The method of  claim 29  wherein the stringent melt conditions are determined by a real-time melt curve. 
     
     
         31 . The method of  claim 29  wherein the support is dehydrated and further comprising:
 hydrating at least one first feature of the support forming a droplet comprising hybridized oligonucleotides duplexes; and 
 optionally hydrating at least a second feature of the support and repeating steps b-e on at least one second different feature and at least one different melting condition. 
 
     
     
         32 . The method of  claim 29  wherein one or more discrete features are selectively heated. 
     
     
         33 . The method of  claim 32  wherein the one or more discrete features are selectively heated using a digital mirror device. 
     
     
         34 . A method for generating on a support a plurality of single-stranded oligonucleotides for conducting a plurality of specified reactions within a droplet, the method comprising:
 a. providing a plurality of surface-bound single-stranded oligonucleotides wherein the oligonucleotides are suitable for hydration and wherein each oligonucleotides is bound to a discrete feature of the surface, each oligonucleotide having a predefined sequence different from the predefined sequence of the oligonucleotide bound to a different feature;   b. selectively hydrating at least one predefined feature thereby providing hydrated oligonucleotides within at least one droplet; and   c. exposing the hydrated oligonucleotides to further processing.   
     
     
         35 . A method of generating a selective set of amplified oligonucleotides of predefined sequences, the method comprising:
 a. providing a plurality of surface-bound single-stranded oligonucleotides wherein the plurality of oligonucleotides are suitable for hydration and wherein each plurality of oligonucleotides is bound to a discrete feature of the surface, wherein the predefined sequence of each plurality of oligonucleotides attached to the feature is different from the predefined sequence of the plurality of oligonucleotides attached to a different feature;   b. selectively hydrating at least one selected feature thereby providing at least one plurality of hydrated oligonucleotides within a droplet; and   c. amplifying the at least one plurality of hydrated oligonucleotides without amplifying the oligonucleotides at unselected features thereby generating at least one selective set of amplified oligonucleotides within the droplet.   
     
     
         36 . The method of  claim 34  or  claim 35  wherein the step of selectively hydrating comprises selectively spotting a solution promoting primer extension onto at least one feature creating at least one first stage droplet. 
     
     
         37 . The method of  claim 36  wherein the solution comprises a polymerase, at least one primer and dNTPs wherein the primer is complementary to a primer binding site. 
     
     
         38 . The method of  claim 37  wherein the primer is a unique primer. 
     
     
         39 . The method of  claim 37  wherein the primer is a universal primer. 
     
     
         40 . The method of  claim 37  wherein the at least one primer is a pair of primer. 
     
     
         41 . The method of  claim 37  wherein the pair of primers are unique primers. 
     
     
         42 . The method of  claim 37  wherein the pair of primers are universal primers. 
     
     
         43 . The method of  claim 36  wherein at least one feature is subjected to thermocycling thereby promoting primer extension within a droplet. 
     
     
         44 . The method of  claim 36  wherein the surface is subjected to thermocycling. 
     
     
         45 . The method of  claim 43  or  claim 44  wherein the thermocycling is modulated at the discrete hydrated features. 
     
     
         46 . The method of  claim 34  further heating the surface to a denaturing temperature thereby providing a plurality of single-stranded complementary oligonucleotides within the at least one droplet. 
     
     
         47 . The method of  claim 34  or  claim 35  wherein the droplets are subjected to conditions limiting water evaporation. 
     
     
         48 . The method of  claim 34  or  claim 35  wherein the discrete features are selectively coated with a coating material. 
     
     
         49 . The method of  claim 48  wherein the coating material has water trapping properties. 
     
     
         50 . The method of  claim 49  wherein the coating material is selected from the group of colloidal silica, peptide gel, agarose, solgel and polydimethylsiloxane. 
     
     
         51 . The method of  claim 47  wherein water evaporation is limited by blocking the interface of the droplet with the atmosphere. 
     
     
         52 . The method of  claim 47  wherein the droplets are overlaid with a non-miscible liquid thereby preventing water evaporation of the solution. 
     
     
         53 . The method of  claim 52  wherein the non-miscible liquid forms a lipid bilayer. 
     
     
         54 . The method of  claim 52  wherein the non-miscible liquid forms a thin film at the surface of the droplet. 
     
     
         55 . The method of  claim 52  wherein the non-miscible liquid is a solvent. 
     
     
         56 . The method of  claim 52  wherein the non miscible liquid is mineral oil. 
     
     
         57 . The method of  claim 52  wherein the non-miscible liquid is spotted onto the droplet. 
     
     
         58 . The method of  claim 57  wherein the step of spotting is performed with an inkjet or mechanical device. 
     
     
         59 . The method of  claim 47  further comprising adjusting droplet volume by addition solution to said droplet. 
     
     
         60 . The method of  claim 59  wherein the addition is semi-continuous. 
     
     
         61 . The method of  claim 59  wherein the step of adjusting droplet volume is performed with an inkjet device. 
     
     
         62 . The method of  claim 47  wherein water evaporation is limited by controlling the humidity around the droplets. 
     
     
         63 . The method of  claim 63  wherein the humidity is locally increased by depositing satellite droplets in the vicinity of the droplets. 
     
     
         64 . The method of  claim 34  or  claim 35  further comprising the step of removing error-containing oligonucleotides from a first plurality of amplified oligonucleotides, the method comprising the steps of:
 a. hydrating at least one first feature of the support following the amplification step forming a droplet comprising oligonucleotides duplexes; 
 b. heating the surface to a first melting temperature under stringent melt conditions thereby denaturing duplexes comprising error-containing oligonucleotides and releasing error-containing oligonucleotides; 
 c. removing the error-containing oligonucleotides from the surface; 
 d. optionally repeating steps a through c on at least one second different feature and at least one different melting temperature; 
 e. denaturing error-free duplexes; and 
 f. releasing error-free oligonucleotides in solution. 
 
     
     
         65 . The method of  claim 64  wherein the stringent melt conditions are determined by a real-time melt curve. 
     
     
         66 . The method of  claim 64  wherein the surface is dried prior to step a. 
     
     
         67 . The method of  claim 64  wherein one or more discrete features are selectively heated. 
     
     
         68 . The method of  claim 67  wherein the one or more discrete features are selectively heated using a digital mirror device. 
     
     
         69 . A method for assembling at least one polynucleotide having a predefined sequence on a surface, the method comprising:
 a. providing a plurality of surface-bound single-stranded oligonucleotides having a predefined sequence wherein the plurality of oligonucleotides are suitable for hydration and wherein each plurality of oligonucleotides is bound to a discrete feature of the support, wherein the predefined sequence of each plurality of oligonucleotides attached to the feature is different from the predefined sequence of the plurality of oligonucleotides attached to a different feature;   b. selectively hydrating at least one selected feature thereby providing hydrated oligonucleotides;   c. synthesizing at least one plurality of oligonucleotides in a chain extension reaction on a first feature of the support by template-dependent synthesis;   d. subjecting the products of chain extension to at least on round of denaturation and annealing;   e. heating the support to a first melting temperature under stringent melt conditions thereby denaturing duplexes comprising error-containing oligonucleotides and releasing error-containing oligonucleotides in solution;   f. removing the error-containing oligonucleotides from the surface;   g. optionally repeating steps b-f on at least one second different feature and at least one different melting temperature;   h. denaturing error-free duplexes;   i. releasing error-free oligonucleotides in solution within a first stage droplet;   j. combining a first droplet comprising a first plurality of substantially error-free oligonucleotides to a second droplet comprising a second plurality of substantially error-free oligonucleotides, wherein a terminal region of the second plurality of oligonucleotides comprises complementary sequences with a terminal region of the first plurality of oligonucleotides; and   k. contacting the first and second plurality of oligonucleotides under conditions that allow one or more of annealing, chain extension and denaturing reaction.   
     
     
         70 . The method of  claim 69  wherein the first and second droplets are combined by merging the droplets. 
     
     
         71 . The method of  claim 69  wherein the step of combining comprises moving the droplet from a first feature to a second feature of the surface. 
     
     
         72 . The method of  claim 71  wherein the droplets are moved using surface tension properties. 
     
     
         73 . The method of  claim 69  wherein the surface is coated with a low melting-point substance for storage. 
     
     
         74 . The method of  claim 73  wherein the low melting point substance is wax. 
     
     
         75 . The method of  claim 69  wherein the reactions are initiated by heating the surface above the low-melting point. 
     
     
         76 . The method of  claim 69  wherein the reactions are initiated by hydrating the discrete features. 
     
     
         77 . A method of moving a droplet on a substrate, the method comprising:
 a. providing a support surface comprising a plurality of modifiers, wherein the plurality of modifiers comprises a plurality of first modifiers and plurality of second modifiers, wherein the plurality of first modifiers has a contact angle smaller than the plurality of second modifiers and wherein the first and second modifiers partition the substrate according to a pattern;   b. contacting a first modifier with a droplet, wherein the first modifier has a contact angle greater than the next first modifier; and   c. moving the droplet on the surface along a desired path in the direction of the first modifiers having smaller contact angles.   
     
     
         78 . The method of  claim 77  wherein each first modifier has a contact angle that is smaller than the previous one. 
     
     
         79 . The method of  claim 77  wherein the plurality of first modifiers forms a hydrophilic gradient. 
     
     
         80 . The method of  claim 78  wherein each first modifier has a contact angle that is at least 5° smaller than the previous one. 
     
     
         81 . The method of  claim 77  wherein the difference in contact angle value between the first plurality of modifiers and the second plurality of modifiers is greater than 30°. 
     
     
         82 . The method of  claim 77  wherein the first modifier comprises oligonucleotides. 
     
     
         83 . The method of  claim 77  wherein the step of contacting is performed with an inkjet device. 
     
     
         84 . The method of  claim 77  wherein the pattern is predetermined and wherein first modifiers alternate with second modifiers. 
     
     
         85 . The method of  claim 84  wherein the first modifiers are surrounded with second modifiers. 
     
     
         86 . The method of  claim 77  wherein the second modifier is the support surface. 
     
     
         87 . The method of  claim 77  wherein the pattern forms a predetermined path along which the droplet moves. 
     
     
         88 . The method of  claim 87  wherein the predetermined path is a hydrophilic gradient. 
     
     
         89 . A method of moving a droplet on support surface, the method comprising:
 a. providing a support surface comprising a plurality of features comprising a first modifier associated therewith, wherein the features are separated from each others by a second modifier and wherein the contact angle of the first modifier is different from the contact angle of the second modifier;   b. contacting a first feature with a first droplet;   c. contacting a second feature with a second droplet, wherein the second droplet volume is greater than the first droplet volume and wherein the second feature is adjacent to the first feature;   d. contacting the first droplet with a third droplet; and   e. merging the droplets into a fourth droplet, wherein the fourth droplet substantially covers the second feature surface thereby moving a first droplet from a first feature to a second feature using surface tension properties.   
     
     
         90 . A method of transferring droplets on support, the method comprising:
 a. providing a support comprising a plurality of addressable features, the features having different contact angles;   b. contacting the support with a first droplet at a first feature;   c. contacting the support with a second droplet at a second feature, wherein the second feature is adjacent to the first feature and the second feature contact angle is smaller than the first feature contact angle;   d. contacting the first droplet with a third droplet;   e. merging the first, second and third droplets into a fourth droplet, wherein the fourth droplet substantially covers the second feature thereby moving a first droplet along a desired path using surface tension directed properties.   
     
     
         91 . The method of  claim 89  wherein the first modifier comprises oligonucleotides. 
     
     
         92 . The method of  claim 89  wherein the contact angle of the second modifier is greater than the contact angle of the first modifier. 
     
     
         93 . The method of  claim 89  wherein the first modifier is more hydrophilic than the second modifier. 
     
     
         94 . The method of  claim 89  or  claim 90  wherein the step of contacting is performed with an inkjet device. 
     
     
         95 . The method of  claim 89  or  claim 90  wherein the third droplet volume is smaller than the second droplet volume. 
     
     
         96 . The method of  claim 89  wherein the difference in contact angles between the two modifiers is greater than 30°. 
     
     
         97 . The method of  claim 89  or  claim 90  wherein the fourth droplet volume comprises the first, second and third droplet volumes. 
     
     
         98 . The method of  claim 89  or  claim 90  further reducing the volume of the fourth droplet to the size of the first droplet volume and repeating steps c through e, thereby transferring the droplet along a desired path to a third feature by surface tension directed manipulation. 
     
     
         99 . A method for merging at least two droplets on a support, the method comprising:
 a. providing a support comprising a plurality of features;   b. providing a first droplet on a first feature and second droplet on a second feature wherein the first and second features are adjacent;   c. reducing the volume of the first droplet;   d. moving the first droplet toward the second droplet;   e. merging the first and the second droplet into a merged droplet; and   f. optionally repeating c through e with a third droplet.   
     
     
         100 . The method of  claim 99  wherein the first feature comprises a modifier having a contact angle greater than the modifier on the second feature. 
     
     
         101 . The method of  claim 100  wherein each feature is separated from the other with a second modifier. 
     
     
         102 . A method of preparing a plurality of oligonucleotides, the method comprising:
 a. providing a first support comprising a plurality of discrete features, each feature comprising a plurality of surface-bound single-stranded oligonucleotides having a predefined sequence;   b. providing a second support comprising an array of electrodes;   c. providing at least one droplet on a first selected feature;   d. synthesizing at least one plurality of oligonucleotides in a chain extension reaction on the first feature of the support by template-dependent synthesis;   e. subjecting the products of chain extension to at least one round of denaturation and annealing to form duplex oligonucleotides; and   d. exposing the duplexes to conditions promoting error reduction.   
     
     
         103 . The method of  claim 102  wherein the droplet is moved to a second selected feature by activating and deactivating a selected set of electrodes. 
     
     
         104 . The method of  claim 102  wherein the first and second support are the same. 
     
     
         105 . The method of  claim 102  wherein the two supports are arranged together relative to each other by a distance sufficient to define a space between the two supports and wherein the droplet is located within the space. 
     
     
         106 . The method of  claim 102  wherein the error reduction is an error filtration process. 
     
     
         107 . The method of  claim 102  wherein the error reduction is an error correction process. 
     
     
         108 . The method of  claim 102  wherein the error reduction is an error neutralization process. 
     
     
         109 . The method of  claim 102  wherein error reduction utilizes a mismatch endonuclease. 
     
     
         110 . The method of  claim 102  wherein the mismatch endonuclease is a CEL1 or a Surveyor™ endonuclease. 
     
     
         111 . The method of  claim 110  wherein the mismatch endonuclease cleaves heteroduplexes. 
     
     
         112 . The method of  claim 110  further comprising the steps of:
 a. exposing the error-containing duplexes with a mismatch endonuclease under conditions that permit cleavage of oligonucleotide duplexes having at least one mismatch; and 
 b. removing cleaved duplexes. 
 
     
     
         113 . The method of  claim 112  further comprising:
 a. denaturing surface-bound cleaved duplexes; 
 b. removing single-stranded cleaved oligonucleotides; 
 c. denaturing surface-bound substantially error free oligonucleotide duplexes; and 
 d. releasing a first plurality of substantially error-free complementary oligonucleotides in a first droplet volume. 
 
     
     
         114 . The method of  claim 112  further releasing a second plurality of substantially error-free oligonucleotides in a second droplet volume. 
     
     
         115 . The method of  claim 112  further merging the first and second droplets. 
     
     
         116 . The method of  claim 112  further activating and deactivating a set of electrodes to move the first and second droplet towards a third feature to form a merged droplet. 
     
     
         117 . The method of  claim 112  further activating and deactivating a set of electrodes to move the first droplet towards the second droplet to form a merged droplet. 
     
     
         118 . The method of  claim 115  wherein the step of forming a merged droplet mixes the first and second droplets composition together. 
     
     
         119 . The method of  claim 114  further combining a first droplet comprising a first plurality of substantially error-free oligonucleotides to a second droplet comprising a second plurality of substantially error-free oligonucleotides, wherein a terminal region of the second plurality of oligonucleotides comprises complementary sequences with a terminal region of the first set of plurality of oligonucleotides; and contacting the first and second plurality of oligonucleotides under conditions that allow one or more of annealing, chain extension and denaturing reaction. 
     
     
         120 . The method of  claim 102  wherein one or more discrete features are selectively heated. 
     
     
         121 . The method of  claim 102  wherein the one or more discrete features are selectively heated using a digital mirror device. 
     
     
         122 . A method for preparing of a plurality of oligonucleotides having a predefined sequence on a support, the method comprising:
 a. providing a plurality of surface-bound single-stranded oligonucleotides having a predefined sequence wherein the plurality of oligonucleotides are suitable for hydration and wherein each plurality of oligonucleotides is bound to a discrete feature of the support, wherein the predefined sequence of each plurality of oligonucleotides attached to the feature is different from the predefined sequence of the plurality of oligonucleotides attached to a different feature;   b. selectively inactivating at least one first feature by overlaying the first feature with an immiscible solution;   c. selectively hydrating at least one second feature thereby providing hydrated oligonucleotides;   d. synthesizing at least one plurality of oligonucleotides in a chain extension reaction on the second feature of the support by template-dependent synthesis;   e. subjecting oligonucleotide duplexes to error-reduction; and   f. releasing substantially error-free complementary oligonucleotides in a droplet volume.   
     
     
         123 . The method of  claim 122  further activating an inactivated first feature by removing the immiscible solution. 
     
     
         124 . The method of  claim 122  wherein the immiscible solution is oil. 
     
     
         125 . The method of  claim 123  further
 a. selectively hydrating the first feature thereby providing hydrated oligonucleotides; 
 b. synthesizing a plurality of oligonucleotides in a chain extension reaction on the first feature of the support by template-dependent synthesis; 
 c. subjecting oligonucleotide duplexes to error-reduction; and 
 d. releasing substantially error-free complementary oligonucleotides in a droplet volume. 
 
     
     
         126 . The method of  claim 125  further moving the droplets to a third feature by electrowetting. 
     
     
         127 . The method of  claim 1  wherein the plurality of single-stranded oligonucleotides are synthesized at each feature using high-voltage complementary semiconductor device. 
     
     
         128 . The method of  claim 102  wherein the plurality of single-stranded oligonucleotides are synthesized at each feature using emulsion droplets. 
     
     
         129 . A method of synthesizing at least one oligonucleotide of a predefined sequence onto a support, the method comprising
 a. providing a first support comprising a plurality of discrete features;   b. providing a second support comprising a high density array of electrodes;   c. providing a droplet on a selected feature, the droplet comprising a reagent for performing a step of oligonucleotide synthesis; and   d. moving the droplets using high voltage electronics to a second selected feature for performing a step of the oligonucleotide synthesis, thereby producing the oligonucleotide.   
     
     
         130 . A method of synthesizing at least one oligonucleotide of a predefined sequence onto a support, the method comprising
 a. providing a support comprising a plurality of discrete features;   b. providing a first emulsion droplet on a selected feature, the droplet comprising a reagent for performing a step of oligonucleotide synthesis; and   c. providing a second emulsion droplet onto the selected feature, the second droplet comprising a reagent for performing a step of oligonucleotide synthesis, thereby generating the oligonucleotide.   
     
     
         131 . The method of  claim 130  further comprising a step of forming a merged droplet wherein the step of merging mixes a first and second droplets composition together. 
     
     
         132 . The method of  claim 130  wherein each droplet comprises a reagent for the oligonucleotide synthesis, each reagent being encapsulated into an aqueous droplet within an immiscible compound. 
     
     
         133 . The method of  claim 132  wherein the immiscible compound is an oil. 
     
     
         134 . The method of  claim 129  or  claim 130  wherein the reagents are selected from the group consisting of A coupling reagent, T coupling reagent, C coupling reagent, G coupling reagent, U coupling reagent, deblocking reagent, oxidation reagent, capping reagent. 
     
     
         135 . A method for monitoring a plurality of isolated reaction volumes on a support, the method comprising:
 a. providing a first support comprising a plurality isolated reaction volumes having a predefined surface-to-volume ratio;   b. providing a second support comprising at least one monitoring isolated volume, wherein the monitoring volume has an identical surface-to-volume ratio to at least one of the reaction volume; and   c. monitoring the volume of the at least one monitoring isolated volume, wherein the modification of the isolated monitoring volume is indicative of the modification of at least one isolated reaction volume.   
     
     
         136 . The method of  claim 135  wherein the isolated volumes are droplets. 
     
     
         137 . The method of  claim 135  wherein the isolated reaction volume comprises a solvent and wherein the monitoring volume comprises the same solvent. 
     
     
         138 . The method of  claim 135  wherein the reaction volume comprises oligonucleotides. 
     
     
         139 . The method of  claim 135  wherein the modification is an increase in volume. 
     
     
         140 . The method of  claim 135  wherein the modification is a decrease in volume. 
     
     
         141 . The method of  claim 135  wherein the first and second support are the same. 
     
     
         142 . The method of  claim 135  wherein the isolated reaction volumes and the isolated monitoring volumes are placed on the same support. 
     
     
         143 . The method of  claim 135  wherein the isolated reaction volumes and the isolated monitoring volumes are subjected to preselected conditions. 
     
     
         144 . The method of  claim 143  wherein the preselected conditions include temperature, pressure, and gas mixture environment. 
     
     
         145 . The method of  claim 144  wherein the surfaces of the isolated reaction volumes and the isolated monitoring volumes are in contact with the preselected gas mixture. 
     
     
         146 . The method of  claim 145  wherein the gas mixture has a predefined molar ratio of solvent vapor and carrier gas. 
     
     
         147 . The method of  claim 144  wherein the conditions are modified to induce isolated volume growth. 
     
     
         148 . The method of  claim 144  wherein the conditions are modified to induce isolated volume evaporation. 
     
     
         149 . The method of  claim 135  wherein the second support is a mirror. 
     
     
         150 . The method of  claim 135  wherein the volume of the at least one monitoring isolated volume is monitored using an optical system. 
     
     
         151 . The method of  claim 150  wherein the volume of the at least one monitoring isolated volume is monitored by measuring the intensity of an optical beam reflected on the second support. 
     
     
         152 . A method for monitoring a plurality of isolated reaction volumes on a support, the method comprising:
 a. providing a first support comprising a plurality isolated reaction volumes;   b. providing a second support; and   c. monitoring the condensation on the second support using an optical system.   
     
     
         153 . The method of  claim 152  wherein the second support has a different surface tension property than the first support. 
     
     
         154 . The method of  claim 153  wherein the second support is a mirror and wherein the condensation is monitored by measuring the intensity of an optical beam reflected on the mirror.