EP0648221A1

EP0648221A1 - Trityl monitor for automated polynucleotide synthesis

Info

Publication number: EP0648221A1
Application number: EP93916801A
Authority: EP
Inventors: William A. Andrus; Jay L. Kaufman; Minh Q. Le
Original assignee: Perkin Elmer Corp; Applied Biosystems Inc
Current assignee: Applied Biosystems Inc
Priority date: 1992-06-30
Filing date: 1993-06-25
Publication date: 1995-04-19
Also published as: EP0648221A4; JPH07508282A; WO1994000471A1

Abstract

A method and apparatus are provided for indirectly monitoring nucleoside monomer coupling yields by measuring the conductance of trityl cations released after a deprotection step in solid phase procedures for nucleic acid synthesis.

Description

TRITYL MONITOR FOR AUTOMATED POLYNUCLEOTIDE SYNTHESIS

The present invention relates generally to the synthesis of polynucleotides, and more particularly, to automated techniques for solid phase synthesis of polynucleotides.

Background The development of reliable and convenient methods for solid phase synthesis of polynucleotides has led to many advances in molecular biology and related fields, e.g. Itakura, Science, Vol. 209, pgs. 1401-1405 (1980); Caruthers, Science, Vol. 230, pgs 281 -285 (1985); Narang, ed., Synthesis and Applications of DNA and RNA (Academic Press, New York, 1987); and Eckstein, ed., Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991). As the use of synthetic polynucleotides has increased, the demand for even greater convenience in the preparation of pure, ready-to-use polynucleotides has also increased. This demand has stimulated the development of many improvements in the solid phase chemistry, e.g. Sinha et al, Nucleic Acids Research. Vol. 12, pgs. 4539-4557 (1984)(beta- cyanoethyl in phosphoramidite chemistries); Froehler et al, Tetrahedron Letters. Vol. 27, pgs. 469-472 (1986)(H-phosphonate chemistry); Germann et al, Anal. Biochem.. Vol. 165, pgs. 399-405 (1987); and Ikuta et al, Anal. Chem.. Vol. 56, pgs. 2253- 2256 (1984)(rapid purification of synthetic oligonucleotides by way of trityl moieties); Molko et al, U.S. patent 4,980,460 (improved base-labile acyl protection groups for exocyclic amines), Brill et al, J. Amer. Chem. Soc, Vol. 111 , pg. 2321 (1989)(nucleoside phosphorothioamidite monomers for synthesizing oligonucleotide phosphorodithioates), Stec et al, PCT Intern'l. patent appl. US91/01010, and Beaucage et al, U.S. patent 5,003,097 (more efficient sufurizing agents for synthesis of oligonucleotide phosphorothioates and -dithioates), and the like. An important aspect of automated procedures for oligonucleotide synthesis is the monitoring of coupling yields during synthesis. Such monitoring not only provides an initial estimate of the purity and yield of the oligonucleotide end product, but also permits one to terminate synthesis if one or more coupling yields are unacceptably low, thereby saving the cost of the reagents that otherwise would have been wasted were the synthesis to continue. On currently available synthesizers, such monitoring is typically carried out "off line" by collecting waste stream samples after the 5'- hydroxyl deprotection step, which releases a trityl cation into a mildly acidic deprotection reagent, usually trichloroacetic acid. An indirect measure of the trityl concentration can be determined spectrophotometrically by the absorbance of the trityl cation at 498 nm. Unfortunately, however, such off line monitoring is inconvenient and for long syntheses is impractical. Moreover, the trityl cation is released from the synthesis support in very high concentrations which are not amenable to accurate quantitation by absorbance measurements. Consequently, trityl samples must be diluted prior to making absorbance measurements, thereby further complicating the monitoring process and introducing error.

Summary of the Invention The invention is directed to a method and apparatus for monitoring coupling yields in oligonucleotide synthesis by measuring trityl cation conductivity in a waste mixture produced from the cleavage of the trityl moiety from a growing oligonucleotide chain. Important features of the invention include flushing the synthesis chamber prior to cleaving the trityl to remove residual reagents, such as acetonitrile, iodine, and the like, that contribute to measurement noise and alternating the polarity of the electrodes to prevent the accumulation of conductivity-altering ions.

Another important feature of the invention is the integration of conductance over time without imparting flow resistance or backpressure into the fluidics system of the synthesis apparatus.

Brief Description of the Drawings Figure 1 is a diagram of a preferred apparatus for implementing the method of the invention. Figure 2 is a diagram of a preferred apparatus for carrying out conductivity measurements on the deprotection waste mixture.

DETAILED DESCRIPTION OF THE INVENTION The invention includes a method and apparatus for synthesizing polynucleotides wherein coupling yields between trityl-protected nucleoside monomers and a growing polynucleotide chain can be monitored by measuring the conductivity of a waste fluid containing trityl cation. The waste fluid is referred herein as the "deprotection waste mixture."

The term "polynucleotide" as used herein includes linear polymers of natural or modified nucleosides, including deoxyribonucleosides, ribonucleosides, alpha-anomeric forms thereof, and the like, usually linked by phosphodiester bonds or analogs thereof ranging in size from a few monomeric units, e.g. 3-4, to several hundreds of monomeri units. In particular, "polynucleotides" as used herein include polymers synthesized on solid phase support by repeated cycles of monomer addition i) wherein the monomer contains at least one trityl moiety as a hydroxyl protecting group, and ii) wherein the coupling chemistry involves the condensation of a free hydroxyl on the growing polynucleotide and a reactive phosphorus-containing functionality, e.g. a phosphoramidi on the monomer. Whenever an oligonucleotide is represented by a sequence of letters, s as "ATGCCTG," it will be understood that the nucleotides are in 5'->3' order from left t right. Polynucleotide as used herein also includes abasic sugar-phosphate or sugar- phosphorothioate polymers as described by Iyer et al, Nucleic Acids Research, Vol. 18, 2855-2859 (1 990).

Phosphorus linkages between nucleosidic monomers include phosphodiester bonds analogs of phosphodiester bonds, such as phosphorothioate, phosphorodithioate, alkyl phosphonate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, and the like. Preferably, the monomers of the polynucleotides of the invention are linked by phosphodiester, phosphorothioate, or phosphorodithioate linkages

As used herein, "nucleoside" includes the natural nucleosides, including 2'-deoxy 2'-hydroxyl forms, e.g. as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" in reference to nucleosides includes synthe nucleosides having modified base moieties and/or modified sugar moieties, e.g. describe generally by Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogs include the natural and synthetic nucleosides with or without appropriate protecting gro for synthesis in accordance with the invention. An exemplary list of nucleoside analogs includes 2-aminopurine, deoxyinosine, N⁴-methoxydeoxycytidine, 5-fluorodeoxyuridine and the like.

Solid phase DNA and RNA synthesis techniques are described in the following references which provide extensive guidance in the selection of reagents, e.g. solvents, nucleoside monomers, cleavage reagents, activators, and the like; and other materials, e.g. solid phase supports, phosphorus protection groups, exocyclic amine protection groups, and the like: Caruthers et al, U.S. patents 4,973,679, 4,415,732 and 4,458,066; Itakura, U.S. patents 4,373,071 and 4,401 ,796; Koster et al, U.S. patent 4,725,677; Molko et al, U.S. patent 4,980,460; Beaucage et al, Tetrahedron Letters, Vol. 22, pgs. 1859-1862 (1981 ); Froehler et al, Tetrahedron Letters, Vol. 27, pgs. 469-472 (1986) and U.S. patent 4,959,463; Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Washington, D.C., 1984); Sinha, editor, DNA and RNA Synthesis and Applications (Academic Press, New York, 1987); Vinayak et al, Nucleic Acids Research, Vol. 20, pgs. 1265-1269 (1992); Slim et al, Nucleic Acids Research, Vol. 19, pgs. 1183-1188 (1991); Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991 ); and Beaucage and Iyer, Tetrahedron, Vol. 48, pgs. 2223-2311 (1992).

Generally, solid phase polynucleotide synthesis involves repeated cycles of the following steps until a polynucleotide of a predetermined sequence is obtained: (1 ) cleaving a trityl moiety from a trityl-protected hydroxyl on the correct-sequence chain, or on the initial monomer attached to a solid phase support, to form a free hydroxyl on the correct-sequence chain and a deprotection waste mixture, (2) reacting a trityl-protected nucleoside monomer or analog thereof with the free hydroxyl of the correct-sequence chain, and (3) capping unreacted free hydroxyls with a capping agent. When the coupling step (2) results in the formation of a phosphorus(lll) linkage between the coupled monomer and the correct-sequence chain, e.g. as would occur when nucleoside phosphoramidite monomers are employed, the synthetic cycle further includes the step of oxidizing the newly formed phosphorus(lll) linkage to form a pentacoordinate, or phosphorus(V), linkage. The step of oxidizing is understood to include sulfurization, or other processes that result in the formation of a phosphorus(V) linkage, whether the phosphorus(V) linkage is phosphate or an analog thereof. After synthesis is completed, the desired polynucleotide is cleaved from the solid phase support, e.g. by treatment with concentrated ammonium hydroxide for 4-5 hours at room temperature or for about 1 hour at 55°C .

In accordance with the invention, the step of cleaving a trityl moiety further includes the steps of (i) flushing the synthesis chamber and conductivity cell to remove conductive reagents and/or products, and (ii) measuring the conductivity of the deprotection waste stream after cleaving the trityl moiety from the correct- sequence chain. Conductive reagents and/or products that may be present in the synthesis chamber in residual amounts include acetonitrile, iodine, pyridinium acetate, lutidinium acetate, and other charged species that may contribute to the conductance.

An important feature of the invention is the flushing of the synthesis chamber prior to trityl cleavage to remove such conductive components. It was discovered that significant noise was introduced into the conductivity measurements by the presence of such non-trityl conductive components when present in the deprotection waste mixture. Preferably, flushing is carried out with a flushing agent comprising a non-ionizing solvent, such as dichloromethane, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylsulphoxide, methanol, or the like, e.g. Riddick et al, Organic Solvents, Fourth Edition (John Wiley, New York, 1986) provides guidance in selecting suitable solvents. Most preferably, flushing is carried out with dichloromethane. In the preferred embodiment, the synthesis chamber is flushed for 30-60 seconds with a flow rate of about 2.5 mL/min for 30 nmole to 1 micromole scale syntheses. Generally, the larger the scale of synthesis, the more flushing required. The conductance, or equivalently resistance (conductance=i/resistance), of the deprotection waste mixture can be measured by standard techniques for measuring the conductance of electrolyte solutions, e.g. as disclosed generally by Christian et al, Instrumental Analysis, 2nd edition (Allyn and Bacon, Boston, 1986). Usually, the deprotection waste mixture is directed to a conductivity cell (described more particularly below for one embodiment) which contains first and second electrodes across which an electrical potential is maintained. In the presence of the deprotection waste mixture, a closed electric circuit, or conductive pathway, is formed such that an electric current can flow between the first and second electrodes. With such a configuration, the amount of current flowing across the electrodes is monotonically related to conductance of the deprotection waste mixture which, in turn, is monotonically related to the trityl concentration in the deprotection waste mixture. A relative measure of the total amount of trityl released during deprotection is readily determined by integrating the conductance between the electrodes over the interval when the deprotection waste mixture flows through the conductivity cell. Such an integration of the conductance values is referred to herein as a "timed integration." Preferably, the integration starts when the trityl cations start to enter the conductivity cell and finishes immediately after the deprotection waste mixture has finished passing through the conductivity cell. Thus, the timed integration takes place in the interval of a coupling cycle defined by the step of cleaving the trityl moiety and the step of coupling the nucleoside monomer. It is 71

understood that the precise current measured depend on many variables such as the distance between the electrodes, temperature, the size of the electrodes and volume of fluid between them, the volume of deprotection reagent used, the scale of the synthesis, i.e. how much polynucleotide is being made, and the like. Moreover, when the value of the conductance is integrated over time, the integrated value depends strongly on the flow rate of the deprotection waste mixture through the measurement region, or conductivity cell as described in the preferred embodiment below. In embodiments where the conductance is integrated the flow rate should be constant and consistent from coupling cycle to coupling cycle. In accordance with the method of the invention, relative values of conductance, or resistance, (or integrated values thereof) are related to coupling yields. The refative values are usually determined with respect to conductivity measurements carried out in the first and/or second cycles of the synthesis.

Alternatively, a constant current can be maintained across the first and second electrodes and the conductance can be related to changes in potential between the first and second electrodes.

In the implementation of the method of the invention, conductivity measurements need not be made in every coupling cycle of the synthesis. Such measurements, and hence, flushings to remove conductive materials, can be implemented in a subset of coupling cycles, either uniformly distributed throughout the synthesis, or distributed by way of a pre-programmed pattern.

Another important feature of the invention is the periodic reversal of the polarity of the voltage between the first and second electrodes. This inhibits the accumulation of charged impurities at the electrode surfaces which would otherwise alter the measured conductance. Such charged impurities are refer to herein as

"conductance-altering ions." This frequency of the polarity reversal are not critical features. Preferably, the electrode polarity is reversed after every measurement. As used herein "trityl" refers to the triphenylmethyl radical and its electron- donating-substituted derivatives. "Electron-donating" denotes the tendency of a substituent to release valence electrons to the molecule of which it is apart, i.e. it is electropositve, March, Advanced Organic Chemistry, pgs. 16-18 (John Wiley, New York, 1985). Preferably, electron-donating substituents include amino, alkyl having from 1 to 6 carbon atoms, aryl having from 6 to 12 carbon atoms, alkoxy having from 1 to 6 carbon atoms, and the like. More preferably, the electron-donating substituents are methoxy. Exemplary trityls include 4,4-dimethoxytrityl (i.e. bis(p-anisyl)phenylmethyl), moπomethoxytrityl, alpha-naphthyldiphenylmethyl, tri(p- methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"- tris(benzoyloxyphenyl)methyl, and the like. Attachment and cleavage conditions for these and other trityls can be found in Greene and Wuts, Protective Groups in Organic Synthesis, 2nd Edition (John Wiley, New York, 1991 ). Generally, trityls are cleaved with a deprotection reagent comprising a mild acid solution, such as a saturated solution of ZnBr2 in methanol, a 2-3% solution of di- or trihaloacetic acid in dichloromethane, and the like. Preferably, 4,4-dimethoxytrityl (DMT) is employed and is cleaved with a solution of 2% (w/v) trichloroacetic acid in dichloromethane for about 3 minutes at room temperature. In selecting the deprotection conditions, care must be taken to avoid conditions that would lead to depurinations, or other undesirable side reactions.

As used herein, "correct-sequence chain" refers to a chain of nucleosides which is capable of reacting with an additional monomeric nucleoside intermediate via a free hydroxyl (i.e. it is uncapped), usually a free 5'-hydroxyl, and whose sequence corresponds to that of the desired polynucleotide. The term includes the first nucleoside attached to the solid phase support (i.e. a nucleoside chain of one unit) as well as the completed polynucleotide product of a predetermined sequence. As used herein, "failure sequence" refers to chains of nucleosides which have not reacted with a monomeric nucleoside intermediate during an addition step and which are subsequently capped. The term also includes polynucleotide chains whose growth was initiated at an extraneous site of the solid phase support.

Thiophosphate analogs of polynucleotides can be synthesized in accordance with the invention following the sulfurization steps taught by Froehler, Tetrahedron Letters. Vol. 27, 5575-5578 (1986), for H-phosphonate chemistry, or the sulfurizaton steps taught by Stec et al, J. Am. Chem. Soc. Vol. 106, pgs. 6077-6079 (1984), or Stec et al, PCT patent appl. US91/01010 for phosphoramidite chemistry.

As used herein, the term capping refers to reacting either the free 5' hydroxyl of a 3' to 5' growing nucleotide chain or the free 3' hydroxyl of a 5' to 3' growing nucleotide chain with a capping agent to render the chain incapable of participating in subsequent condensation steps. Capping can be achieved by acetylation of the free hydroxyls, e.g. by exposure of the free hydroxyls to a solution of acetic anhydride in 2,6-lutidine delivered concurrently to the synthesis chamber with a 16% (v/v) solution of N-methylimidazole in anhydrous tetrahydrofuran. Capping can also be achieved by reacting the free hydroxyls with a phosphitylating agent as taught by Andrus et al, U.S. patent 4,816,571.

Preferably, the method of the invention is automated. The apparatus for automating can take several forms. Generally, the apparatus comprises a series of reagent reservoirs, a synthesis chamber containing a solid phase support, a conductivity cell downstream of the synthesis chamber, and a computer controlled means for transferring in a predetermined manner reagents from the reagent reservoirs to and from the synthesis chamber and the conductivity cell. The computer controlled means for transferring reagents can be implemented by a general purpose laboratory robot, such as that disclosed by Wilson et al, BioTechniques. Vol. 6, pg. 779 (1988), or by a dedicated system of tubing, and electronically controlled valves. Preferably, the computer controlled means is implemented by a dedicated system of valves and tubing connecting the various reservoirs and chambers. In further preference, the reagents are driven through the tubing by maintaining a positive pressure in the reagent reservoirs by means of a pressurized inert gas. such as argon, as is used by many widely available automated synthesizers, e.g. Applied Biosystems, Inc. models 392 or 394 DNA synthesizers.

A diagrammatic representation of a preferred embodiment of such an apparatus is illustrated in Figure 1. The apparatus of Figure 1 is set forth as if the phosphoramidite chemistry were being employed and as if the polynucleotide is a deoxyribonucleotide and is being synthesized in the 3'->5' direction. It is understood that the method and apparatus of the invention are adaptable to other nucleotide synthesis chemistries, e.g. based on H-phosphonate monomers, or the like, with obvious modifications, e.g. different synthesis reagents are used, different reaction times are required, and the like. These modifications are readily implemented via programmable controller 48, or like means. 5'-tritylated nucleoside intermediates are stored in reservoirs 2 through 10, one reservoir each for the four natural nucleosides. Optionally, an additional reservoir 10 is provided for a 5'-tritylated nucleoside analog, e.g. deoxyinosine, a linking agent, e.g. U.S. patent 4,757,141 , or like intermediates. The reservoirs 2 through 10 containing the synthesis intermediates are connected to synthesis chamber 28 by way of valve block 24 whose operation is controlled by controller 48. Synthesis reagents are stored in reservoirs 12 through 19. For example, in phosphoramidite chemistry these can be 12 trichloroacetic acid in dichloromethane for deblocking, 13 iodine/pyridine/water/tetrahydrofuran solution for oxidizing internucleoside phosphorous, 14 tetrazole/acetonitrile solution for activating the nucleoside intermediates, 15 ammonium hydroxide for cleaving the completed chain from the synthesis support, 16 1 -methylimidazole/tetrahydrofuran solution and 1 7 tetrahydrofuran/lutidine/acetic anhydride solution for capping, and 18 acetronitrile for washing. Reservoir 19 contains a flushing agent. These reagent reservoirs are connected to synthesis chamber 28 by way of valve block 22 which is controlled by controller 48. Synthesis proceeds under programmed control with each nucleotide being added to the growing chain by successive cycles deblocking, addition, capping, and oxidizing. Reagents removed from synthesis chamber 28 pass through conductivity cell 30 then to waste reservoir 38. When actuated by microprocessor 48 power supply 32 establishes an electric circuit whose current is measured by current measuring device 34. Preferably, conductivity cell 30 is located as close to synthesis chamber 28 as possible to minimize the transfer time of reagents from the synthesis chamber to the conductivity cell and to minimize the volume of reagents required to bring about the transfer. When synthesis is complete, the synthesis support is treated with concentrated ammonium hydroxide to deprotect and cleave the polynucleotide chains.

A preferred embodiment of the conductivity cell is illustrated diagrammatically in Figure 2. Annular insulator 56 is coaxially sandwiched between annular electrodes 54 and 58 in housing 50 so that fluids can flow through their central orifices. Cap 52 presses electrodes 54 and 58 and insulator 56 sealably together and against the inside wall of housing 50 so that a leak proof pathway is formed for fluids to enter the conductivity cell via fitting 60 and flow through the central orifices of electrodes 54 and 58 and insulator 56. Electical contacts 66 and 68 carries current to or from electrodes 54 and 58, respectively, through lead wire 64 to power supply 32 and/or current meter 34. Preferably, noncorrosive inert materials are employed in the construction of the conductivity cell, e.g. plastics, stainless steels, and the like. Preferably, insulator 56 is made of teflon, and first and second electrode 54 and 58 are made of gold or of a gold plated conductive material. In the preferred embodiment, a constant voltage is maintained across first and second electrodes 54 and 58. The precise voltage selected is not a crucial feature. Factors related to the selection of a voltage includes size and distance between the electrodes, the scale of synthesis, shock hazard, and the like. Preferably, the voltage is within the range of 5 to 25 volts. EXAMPLE

Comparison of Absorbance and Conductivity Monitoring in 0.2 and 1.0 micromole Scale DNA Synthesis To compare conductance-base trityl monitoring with spectrophotometric trityl monitoring, the 18-mer, 5'-TCACAGTCTGATCTCGAT, was synthesized at 0.2 and 1.0 micromole scales on an Applied Biosystems, Inc. model 392 DNA synthesizer using standard protocols, with the following exceptions: The DNA synthesizer was modified by the insertion of a conductivity cell in the waste line from the synthesis chamber and the DNA synthesizer was programmed to accommodate the flushing step prior to detritylation. When spectrophotometric trityl monitoring was employed the synthesis chamber was not flushed prior to detritylation. Flushing was accomplished with dichloromethane whenever conductivity measurements were made. Dichloromethane was driven through the synthesis chamber at a flow rate of 2.5 mL/min for 60 sec. Spectrophotometric monitoring was based on absorbance at 498 nm of diluted samples of the deprotection waste mixture prepared according to manufacturer's protocols (Models 392 and 394 DNA/RNA Synthesizers User's Manual, Applied Biosystems, Inc., Part. No. 901237, Revision C, May 1991). Table I below sets forth the stepwise calculated coupling yields based on both monitoring approaches which were obtained during the synthesis of the indicated oligonucleotide. Table II below sets forth the final average stepwise yields based both on conductivity and absorbance for several syntheses of varying scale of of varying sized oligonucleotides.

TABLE I

Comparison of calculated coupling yields based on conductance and absorbance

Absorbance measurements were performed as described in the Trityl Cation Assay in the 392/394 user's manual, at 490 πm. All of the above numbers were derived using the following formula:

OY = (lowest yield value)/(highest yield value) ASWY = OY ^{1 n} where n = # of couplings.

The data above comes from the following sequence synthesized at the 0.2 μmole scale: 5'- TCA CAG TCT GAT CTC GAT - 3' TABLE II

Claims

We claim:

1. in a method for synthesizing a polynucleotide wherein (i) a correct-sequence chain is attached to a solid phase support in a synthesis chamber, (ii) a nucleoside monomer having a reactive functionality protected by a trityl moiety is coupled to the correct-sequence chain to form a trityl-protected correct-sequence chain, and (iii) the trityl moiety is cleaved from the correct-sequence chain by a deprotection reagent to form a deprotection waste mixture, an improvement comprising the steps of : flushing the synthesis chamber prior to cleaving the trityl moiety from the correct-sequence chain to remove conductive reagents and/or products; and measuring the conductivity of the deprotection waste stream after cleaving the trityl moiety from the correct-sequence chain.

2. The method of claim 1 wherein said step of measuring includes: providing a first electrode and a second electrode such that said deprotection waste mixture forms a conductive pathway between the first and second electrodes when said deprotection waste mixture is expelled from said synthesis chamber; and establishing a voltage between the first electrode and the second electrode, the voltage having a polarity with respect to the first and second electrodes.

3. The method of claim 2 wherein said step of measuring further includes alternating the polarity of the voltage between said first and second electrodes to prevent the accumulation of conductance-altering ions at either said first or second electrodes.

4. The method of claim 3 wherein said step of measuring includes a timed integration of said conductivity between said first and second electrodes such that the timed integration occurs within the interval of between said cleavage of the trityl moiety and said coupling of said nucleoside monomer.

5. An apparatus for synthesizing a polynucleotide of a predetermined sequence, the apparatus comprising: a solid phase support in a synthesis chamber, the solid phase support having a trityl-protected correct-sequence chain attached; one or more reactant reservoirs containing trityl-protected nucleoside monomers; one or more first reagent reservoirs containing synthesis reagents for attaching the trityl-protected nucleoside monomers to the trityl-protected correct- sequence chain after a trityl moiety of the trityl-protected correct-sequence chain has been cleaved, the synthesis reagents including a deprotection reagent for cleaving the trityl moiety from the trityl-protected correct-sequence chain; a conductivity cell capable of holding a deprotection waste mixture formed after cleavage of the trityl moiety from the trityl-protected correct-sequence chain, the conductivity cell comprising a first electrode and a second electrode disposed within the conductivity cell so that the deprotection waste mixture is capable of forming a conductive pathway between the first and second electrodes; means for establishing a voltage between the first electrode and the second electrode so that the conductance and/or the resistivity of the deprotection waste mixture can be measured; and fluid transfer means for transferring trityl-protected nucleoside monomers from the one or more reactant reservoirs to the synthesis chamber, for transferring synthesis reagents to the synthesis cham.ber, and for transferring the deprotection waste mixture from the synthesis chamber to the conductivity cell so that the polynucleotide of the predetermined sequence is synthesized.

6. The apparatus of claim 5 wherein said one or more first reagent reservoirs further include a flushing reagent for removing conductive reagents and/or products from said synthesis chamber.

7. The apparatus of claim 6 wherein said means for establishing a voltage between said first electrode and said second electrode establishes a voltage having a polarity with respect to said first and second electrodes and includes means for reversing the polarity of the voltage between said first and second electrodes.