CA1329469C - Polynucleotide synthesizer and method - Google Patents

Abstract

IMPROVED POLYNUCLEOTIDE SYNTHESIZER AND METHOD
ABSTRACT
There is disclosed a polynucleotide synthesizer and a method of using it. The synthesizer comprises supply means for supplying a) liquid reactants for synthesizing peptides or polynucleotides, b) liquid reagents that assist in the synthesis, and c) a wash liquid; a reactor column; connecting means for fluidly connecting the supply means with the column; means for positively displacing with gas pressure, liquid reactants and reagents, and wash liquid from the supply means into the connecting means; and controlling means connected to the column for allowing liquid to flow under the force of the gas pressure and at a controlled rate, into a temporary storage chamber and for thereafter emptying the chamber. The synthesizer is improved in one or both of the following features: In one aspect the controlling means is fluidly positioned only downstream of the reactor column, and the synthesizer further includes means preventing flow from the controlling means back to the column, whereby the column is precluded from contamination by reactants or reagents in the controlling means, and the controlling means needs no cleaning to prevent it from contaminating the column.
In another aspect the synthesizer further includes a parallel manifold having i) sufficient inlet means to receive either each of the liquid reactants or each of the liquid reagents, ii) a common inlet to receive the wash liquid, and iii) an outlet connected to the connecting means to supply reactant or reagent to the column or to the controlling means, each of the inlet means including a pair of tandem two-way valves, both of the valves of the pair having a common outlet that feeds into the manifold outlet, one portion of each pair of the tandem valves being connected to the common inlet for the wash liquid and the other portion of the valves being connected to the inlet means for one of the liquid reactants or one of the liquid reagents, and means for selectively opening one of the pairs of valves at a time to a source of wash liquid, in a predetermined sequence, whereby an adequate source of wash liquid can be provided for each of the valve pairs of the parallel manifold.
The method features the steps of a) drawing under the gas pressure a reactant from a source of supply first into an outlet common with all reactants, then into the column, and then into the controlling means, b) drawing under gas pressure a wash liquid through the common outlet, then into the column and then into the controlling means, c) and ejecting all liquid out of the controlling means into either a collection container or a waste disposal without passage through the column.

Description

1 3~9469 IMPROV~D POLYNUCL~Q~ NT~E~IZER AND METHOD
FIELD OF TEE INVENTION
This inYention relates to an improved apparatus and a method used to ~ynthe~ize polynucleotides or pep~ides, preferably in an ` automated manner.
ACKGRQ~D OF T~ INVENTION
Peptide ~ynthesizer~ and polynucleotide synthesizers have a ~ource of building blocks, which are, respectively, amino acids and nucleotide bases, ~hat are sequentially supplied to a reactor column where they are chemically reacted to form a peptide or an oligonucleotide. Such a reaction requires carefully controlled eondition~ in which the presence of an incorrect amino aeid (or nucleotide ba~e, re~pectively), lead~ to the syn~hesis of the wrong peptide or an incorrect oligonucleotide. A~ a result, each ~tepwiRe addition i8 followed by a washing of appropriate flow lines. Although the waghing delays the process, the delay iB minimized by minimizing the volume of the flow lines involved.
A further aspect of the problem i8 the method in which the amino acids for the peptide, or nucleotide bases for the oligonucleotide, are ~upplied from their containers. Positive gas displacement i8 preferred, inaamuch as any 8y8tem that attempt~ to "pull" them out U8 ing a vacuum, runs the risk of generating bubbles in the liquid, since the liquid amino acids or nucleotides are not dega~sed priox to use. However, ~uch po~itive gas di~placement ~ystem3 in turn render downstream control of the di~placed liquid difficult.
DNA ~ynthesizer& have been constructed to - overcome such a control problem and retain the use of gas displacement of the base~. To allow the bases to flow into the 8y8tem, including a reactor column, _3_ l 329 4 6q a controlled rate and volume, a syringe controlling means, also called a controller, has been positioned between the re?ctant (hereinafter, a "base"~ outlet line, fed from an 8-port rotary valve, and the reactor column. When the syringe pi~ton i~ withdrawn by a motor to create a storage chamber, the positive gas pressure on the supply bottles causes one or more bases to feed into the syringe. Thereafter, the syringe piston i8 reversed and the bases are injected into the reactor column. An example of 3uch a conventional synthesizer is the Autoinject module of the Cruachem DNA synthesizer.
A problem with ~uch an instrument is that the syringe becomes "contaminated" with the ba~e~
th~t are to be delivered to the reactor column, due to its up~tream location. Such a condition requires that the syringe be thoroughly cleaned (with acetonitrile) prior to the drawing of the ne~t base in the sequence, since the presence of the wrong base will ruin the DNA ~equence. Because of the relatively large volume of the syringe, such cleaning i8 time-consuming and difficult. Yet, if cleaning i8 minimized, contamination will occur.
Yet another problem with such instruments i9 that the syringe co~troller i8 not in a po~ition to control the feed of the reagents, such as iodine and the llke, needed in the synthesis, since the reagents are fed into the reactor via a line separate from the line supplying the base~.
Still another problem with such a synthesizer has been the use of an 8-port rotary valve to collect the bases ~equentially. As the valve wears, it leaks, and the base sequence i8 no longer free o~ eontamination from other bases. To deal with this problem, parallel manifold~ have been suggested as in U.S. Patent No. 4,598,049. ~owever, f . ,, . . , . . - ... .. ~ , . . - , _4_ ` l 329469 the manifolds taught therein do not provide for separate washing of each base inlet in sequence, ~o that there i8 the chance that one or more base inlet~
will be inadequately washed, and contamination will occur.
Thu~, prior to thi~ invention there has been a need for a DNA synthesizer that i~ free of these problems.
~UMMAR~ OF THE INV~NTION
This invention provides a solution to the contamination and/or time delay problems noted above.
More ~pecifically, in accord with one aspect of the invention, there i8 provided a synthesizer apparatu~ ~or synthe~iæing peptides or polynucleotide~, the apparatus including supply mean~
for supplying a) liquid reactants for synthesizing : the peptides or polynucleotides, b) liquid reagents that assist in the synthesis, and c) a wash liquid; a reactor column; mean3 for fluidly connecting the 20 Bupply meang with the column; and means for positively displacing with gas pressure, liquid reactants and reagents, and wash liquid from the , supply mean~ into the connecting means; and ; controlling means connected to the column for allowing liquid to flow under the force of the gas pressure and at a controlled rate, into a tem2orary s~orage chamber and for thereafter emptying the chamber. The apparatus i8 improved in that the controlling means i8 a positive displacement 30 controlling means and is fluidly positioned only ~`~
downstream of the reactor column, and further the synthesizer includes means for preventing flow from the chamber back to the column, whiereby the column is precluded from contamination by reactants or reagents : 35 in the controlling means, and the controlling means 1 32~46~
need~ no cleaning to prevent it from contaminating the column.
In accord with another a~pect of the invention, there i8 provided a ~ynthesi7er apparatus for syntheQizing peptide3 or polynucleotide~, the apparatus including supply means for aupplying a) liquid reactants for synthesizing the peptides or polynucleotide3, b) liquid reagents that assist in the synthesis, and c) a wash liquid; a reactor column; mean~ for fluidly connecting the supply mean3 with the column; means for positively di~placing with ga~ pre~sure, liquid reactant~ and reagent~, and wash liquid from the supply means into the connecting mean~; and controlling means connected to the column for allowing liquid to flow under the force of the gas pressure and at a controlled rate, into a temporary storage chamber and for thereafter emptying the chamber. Thi~ apparatu~ is improved in that the apparatus further includes a parallel manifold having i) sufficient inlet means to receive either each of . the liquid reactants or each of the liquid reagents, i, îi) a common inlet to receive the wash liquid, and .~ iii) an outlet connected to the connecting means to supply reactant or reagent to the column or the controlling means, each of the inlet means including ~ a pair-.of tandem two-way valves, both of the valves of the pair having a common outlet that feeds into -~ the maniXold outlet, one of each pair of the tandem valve~ being connected to the common inlet for the 30 wash liquid and the other sf the valve~ being connected to the inlet means for one of the liquid . reactants or one of the liquid reagents, and means `~ for ~electively opening only one of the pairs of valve~ at a time to a source of wash liquid, in a predetermined sequence, whereby an adequate source of ,., ,r: . . . ~ . .. . , : . j. :........ .. . .

-6- 1 32q 4 69 wa~h liquid can be provided for each of the valve pairs of the parallel manifold.
In accord with yet another aspect of the invention, there i8 provided a method ~or drawing reactant~ and reagents under gas pres~ure into a reactor column to synthesize a peptide or an polynucleotide, using a llquid controlling means fluidly connected to the column. The method comprise~ the steps of a) drawing under the gas pressure a reactant or reagent from a source of supply first into an outlet common with all reactants or reagent~, then into the column, and then into the controlling means, lS b) drawing under the gas pressure a wa~h liquid through the common outlet, then into the column and then into the controlling means, c) and ejecting all liquid out of the controlling mean~ into either a collection container or a waste disposal without passage through the column.
Thu~, it i~ an advantageous feature of the invention that a synthesizer i8 provided with a positive displacement fluid controlling mean~, which gives rigid lî~uid advancement within the synthesizer, that need not be thsroughly washed between base introduction, and still will not contaminate the material that is formed.
It is a related advantageous feature of the invention that such a synthesizer i~ provided that allows one liquid controlling means to control flow advancement of all the liquids used i~ the synthesi~.
- ret another advantageous feature of the invention i~ that such a synthesizer i8 provided that minimizes the chance of contamination occurring in a parallel manifold for supplying reactants sr reagents .. . . , . .. - ~ . : . - . .. . : . . - . . ..... - .

,, .- . . . . . : . .

1 32946~

to a reaction column, by providing for the individual washing of each reactant or reagent inlet line.
Yet another advantageous feature of the invention i~ the use of valves that eliminate pressure ~pikes in the downstream fluid line~.
Other advantageous features will become apparent upon reference to the following Description of the Preferred Embodiments, when read in light of the attached drawings.
BRI~F ~ESCRIPTION OF 1~ DRAWINGS
Figure 1 i8 a schematic illustration o~ a DNA sy~thesizer constructed in accordance with the - prior art;
Fi~ure 2 ~A and B) is a schematic illustration of a DNA synthesizer constructed in accordance with the invention;
Figure 3 is section view o~ the parallel manifold used to supply the base reactants for the synthe3izer;
Figure 4 i8 a view partially in ~ection, ~, taken generally along the line IV-IV of Figure 3;
Figure 5A i8 a plan view of a ~eries manifold used in the supply of reagents to the synthesizer;
Figure 5B i8 a partial section view taken along the line VB-VB of Figure 5A;
Figure 6 (A and B) i8 a ~chematic illustration of an alternative embodiment for the manifold connected to the reagents, wherein the manifold i8 a parallel manifold rather than series manifold; and Figure 7 i8 a fragmentary schematic illustration ~imilar to Figure 2, illustrating an alternative embodiment.
~SCRIPTION 0~ T~E PREF~RRED EMBODIMENTS
The invention iB described hereinafter with respect to the pre~erred embodiments of apparatus , ,,.,. -~ . :: : -, .

1 32946q used for DNA synthesizing. In addition, the invention i8 applicable to apparatu~ for synthe~izing any polynucleotide sequence, be it DNA or RNA, and to the synthesis of peptides using amino acids. In the case of peptides, instead of using ~he four nucleotide bases a~ hereinafter enumerated, the apparatus has as its reaetants, 80me or all of the twenty amino acids used to synthesize peptides, and a different set of reagents and reactio~ sequences.
A conventional DNA synthesizer apparatu~
illustrated in Figure 1. In such a device, reactant supply means 10 8upplie8 the four bases A, C, G and T
in a particular sequence to a reaction column 30 via a controlling means 20 and two-way valve 40.
Preferably, supply means lQ i8 an 8-port rotary valve and controlling means 20 i8 a ~yringe operated by motor 25 and suitable electronic controls, not shown. The capping reagents, along with iodine for oxidation and dichloracetic acid for detritylation, reguired by the conventional cyanoethylphoaphoramidite reaction chemistry, are added separately from supply means 50, and not under the control of controlling means 20. Such an apparatus is provided by the Cruachem DNA Synthesizer noted above, and e~counter~ the disadvantages noted . above.
In accord with the invention, the DNA
synthesizer 60 preferably comprise~, Figure 2A, supply manifold 70 supplying reactants A, C, G, T, X
. 30 and Y from bottles 72, 74, 76, 78, 79 and 80, a~ well a8 reactant ACT, a tetrazole activator, from bottle 82; ~upply ~anifold 90 supplying reagen~s from bottles 92, 94, ~6, 98 and 100, reaction column 110, gas ~upply manifold 62 for supplying ga~ pressure to :~
displace liquids from their bottles through the supply manifolds under constant pressure, and ' positive displacement controlling means 150 for drawing liquid under the force of the gas pressure in response to the actuation of controlling means 150.
Positive displacement controlling means 150 compri~es a piston cylinder 158 and a piston 160, and causes positive displacement of liquid due to its down~tream location relative to column 110 and to the positive gas pressure applied on the system. That i8, as the piston 160 is withdrawn, it allows liquid to flow due to the aforementioned po8itive pressure.
.~ This creates a temporary storage chamber within chamber 158. At the same time, pressure~ within all ; the feed lines, such as lines 85 and 114 (referred to hereinafter), remain above atmospheric pre~sure, as the movement of piston 160 proceed~ at a rate that i8 le~s than the maximum flow achievable for the pressure of the feed lines in thi~ eonfiguration. As a result, the flow through the entire system i8 es~entially rigid, providing precise flow through any gingle fluid path.
Any particular synthesis chemistry is usable with this invention, the reagent~ being selected, of course, to fit the desired scheme. Preferred i~ the conventional beta cyanoethylphosphoramidite reaction, which uses, for example, iodine for base o~idation and dichloroacetic acid for detritylation. Further detail of the phosphoramidite reaction scheme can be found in &iles and Morrison, "An Economical System for Automated DNA Synthesi~ , Vol. 5, p. 16-25 ~March/~pril 1987~.
I~ such a reactio~, reactants A, C, G, T and X and Y ~tand for the following ba~es: A is adenine, C i8 cytosine, T i8 thymine, G i8 guanine, and X and ~ are variables which ~or example can be ~ for uracil usually used only in RNA, a mixture of ba~es, or a modified version of any of the bases. Becau~e the chemistry of such a reaction i8 well-known, hereinafter details will be directed to the apparatua only.
Gas manifold 62 supplies a non-reactive ga~
5 such a~ helium, argon or nitrogen gas, from an ~:
e~ternal source 64, through a drier 65 and a preRsure regulator valve 66 that maintains the pressure between 0 and 207 kilopascals. Within manifold 62 the incoming line i~ split into two lines 67 and 68.
Lines 67 and 68 are u~ed to pressurize the content~
of bottles 72, 74, 76, 78-80 and 92, 94, 96, 98 and 100, respectively, 90 that when liquid valves are opened down~tream, the liquid will exit from the bottles under the influence of the pressure and the appropriate action of controlling means 150. Return lines 69 and 69~ vent the ga~ pressure bac~ through manifold 62 and then to waste. The ga~ feed line 67 associated with vent line 69 and feed line 68 associated with vent line 69' pressurize their respective manifolds independently, allowing one of two manifold~ 70 and 90 to be vented for a bottle change without disturbing the other.
Turning now to manifolds 70 and 90, each of these is connected to supply bottles via valves, as ' 25 follows:
For the ba~e manifold 70, preferably each ; ba~e port thereof ha~ a pair of independently ; actuated tandem, two-way valve~ 84. For the reagent manifold 90, each port has a three-way valve 102, - :
except that valve 103 fsr the C~3CN can be a two-way valve. These valves utilize non~reactive material such as Teflon~. For e~ample, valves 84 can be "SV-24"~ valves manufactured by Valcor ~ :
Scientific, and valves 102 can be "Valcor Series 20"~ valves manufactured al80 by Valcor Scientific.

In accord with one a~pect of the invention ?
the arrangeme~t of valves 84 on manifold 70 i8 as follows:
Fir~t, manifold 70 is a parallel manifold, Figures 2-4. Incoming line 85, Figure 2A, enters port 86, to deliver wash liguid acetonitrile aa it i~
supplied from manifold 90, Figure 2B. From port 86 such liquid passes to each pair of valves 84 via individual channels 87. Each channel 87 feeds to valve portion 88 of each tandem pair 84, Figure 4.
Line 89 ~upplies a base to a valve portion 91 in each tandem pair 84. Valve portions 88 and 91 include a diaphragm 280 that seats on a valve seat 282, to supply liquid to a common channel 284 that feeds channel 9S, if the re~pective valve portion i8 open.
AR i8 conventional, the diaphragms are solenoid operated, each being independently acting 80 as to isolate the separate flow path~ with minimal dead volume. ~owever, outlet channel 95 carries whatever the output of the two valve portions 88 and 91 provide to it (ba3e or wash liquid).
The two-way valves are preferred over three-way valves for the base manifold 70 in that the dead volume i8 minimized. Often the base volumes may be legg than 100 ~1 delivered in less than a second. Three-way valves tend to di~Elace 100 ~1 when they close, ~hic~ can upset or alter the delivered volume.
The operation of manifold 70 will be apparent from the above description. First, a valve of a valve pair open~ to allow infeed from a bottle, only one at a time, since if two valves were 80 ope~ed simultaneously, precise flow from each bottle could not be assured. When a particular base is needed in column 110, say for e~ample baRe A, the correspo~ding valve portion for that base i8 opened, " 1 32946q valve portion 91 in this case of the pair of valves 84 associated with bottle 72, Figure 2. When piston 160 is withdrawn, as described hereinafter, base A i8 pushed out of bottle 72 by the con~tant gaa pre~ure from manifold ~2. Base A travel~ through valve portion 91 into channel 95, common outlet 93, and then line 114. Then an equal volume of activator i8 allo~ed to flow by closing valve portion 91 of the valve pair 84 for bottle 72, and opening valve portion 91 of the valve pair 84 for bottle 82. Other bases are added similarly. Wh,en the appropriate amou,nt of each ba~e A and activator i~ passed into the system, including line 95, valve portion 91 iB
clo3ed and valve portion 88 of the valve pair for bottle 72 i8 opened, along with the valves of manifold 90 that allow wash liquid, for example C~3CN, to flow into inlet 86. The first gtep in the wash i8 to pa88 the wash liquid through the common, channel 284 of valve pair 84 and line 95. The valve portions of pair 84 for bottle 82 are then washed similarly. In this fashion wash liguid i~, used to push the last bit of each base and activator all the way into column 110. Even some of the wash liquid pas~es into the column for this purpose. Then the wagh side of each valve pair 84 i~ clo~ed and the wash liquid is directed through each valve 102, 102' and 103 of manifold 90.
Thu~, the tandem valve pairs in manifold 90 allow washing, in sequence around the ~anifold, to insure that ~11 of the base lines (for base3 C, G, T, X and ~ as well as A~ are washed in sequence. Thi~
insures that any minor leakage of valve portion 91 of each valve pair 84, is washed away. That is, the invention preferably sequences the washing because each valve orifice may be ~lightly different, such .

.. ~i,~.. , . ~ ' , .. '. ,. ", ...... ' .' ': ' .; ' ... ' ,': , '. '', . ' ., . '' ' ' ' . . . : ' ' ~ 32q46q that an all-at-once wash will ~ot guarantee a wa~h o~
leaked base out of each valve portion 91.
Thi~ sequence of washing i8 repeated after each o~ the ba~es i~ passed through column 110.
As shown in Figures 2, 5A ~nd 5B, manifold 90 i~ a series manifold, 80 that ~ach valve 102 or 103 delivers reagent in series a~ permitted by the positions of each of valves 102. Each valve 102 i8 preferably a three-way valve and receives liquid from a supply line 104, e~cept for valve 102' that feed~
the output of manifold 90 (or manifold 70) via line 106 to column 110 or to line 107 to controlling means 150. Valve 102' receives as its input from a channel 108, Figure 5A, the liquid 8upplied through any one of the valves 102.
There is a port 112 in manifold 90, Figure 2, that i8 independent of any valve 102, and this i8 the inlet port for the reactants ~upplied via line 114 and outlet 93 from manifold 70.
; 20 Valve~ 102, 102~ and 103 operate a3 follow~:
In no case i8 it de3irable for more than one valve 102, 102' or 103 to be "open" at any one time, that i8, to allow input from the bottle ~ed by that valve. Pas~-through of reagent from another valve 102 is still poasible, however. The valve portion "8", Figure 2, for wash bottle 92 opens o~ command to ; ~eed C~3CN into manifold 90, when pi~ton 160 is withdrawn. If val~e portion "13" i8 ener~ized, that wa8h goe8 to line 85 and manifold 70. When valve . 30 portion 13 i8 dee~ergized, the wash i8 shunted on to pa83 through the porting of all the other valves in manifold 90 that are down~tream. To obtain other reagents sequentially, the valve portion a~sociated with that reagent bottle i8 energized. Valve 102' is of particular i~portance. It will supply the liquid from Yalye~ 102 only either to line 106 or to line -14- l 329469 107. Furthermore, flow from manifold 70 is ~hut off when supplying a reagent from a bottle of manifold 90 by deenergizing valve portion ~13~.
Preferably, however, a~ shown and de~cribed in Figure 6, the manifold for the reagent supply mean~ can b~ a parallel manifold similar to that used for the base3. The reason i~ that a parallel manifold reduce~ the ~ystem volume~, and contamination of one reagent by another i8 less likely t~han in a series manifold. That i8, in order to wash manifold 90, the dead volumes of each valve therein must be flushed completely since the next reagent shares the flow volume. The above-noted advantage3 of two-way valve~ over three-way valve~
apply here al80. Regarding contamination, any small orifice or leakage at a valve in such a series manifold will contaminate the next (different) reagent that must perforce flow pa~t it.
In Figure 6, parts similar to those . ~.
previougly described bear the same reference numeral, to which the di~tinguishing suffi~ "a" has been appended.
I Thus, base manifold 70a receives wash liquid I from line 85a and delivers base or wash liquid to valve 102~a, and then to column llOa or controlling j mean~ 150a, a~ described above. Each of the reagents i8 supplied to its manifold 90a from a 2espective bottle 92a, 94a, 96a, 98a and lOOa, also as described.
~owever, manifold 90a, Figure 6A, is a parallel manifold, and each of the valve~ 102a i8 a tandem pair of substantially the same construction as i8 provided for in valve~ 84 of Figures 2 and 4.
Furthermore, no valve i8 interposed on line 104a between bottle 92a and manifold 90a. Instead, that line feeds wash liquid direct to eommo~ inlet 200 for manifold 90a. From there channels 202 carry the wash . . .

1 32~6q ~ s -liquid to each of tandem pair valves 102a. TheRe are con~tructed identically to ~he pair ~hown in Figure 4, that is, each pair 102a comprises a portion 91 and a portion 88 (not shown in Figure 6). Each tandem pair valve 102a has an individual outlet channel 204 feeding to a common outlet 206 for manifold 90a, which supplies liquid to line 208 that goes to valve 102'a.
The operation of manifold 90a i8 readily apparent from the preceding. For example, to detritylate a base in column llOa, Figure 6B, valve 102'a i~ energized to connect line 106a to and di~connect line 107a from line 208, and valve portion 91, Figure 4, of the valve pair 102a for DCA i8 opened. When the piston i8 withdrawn in controlling means lSOa, Figure 6B, ga~ preQ~ure pushes DCA into its valve pair 102a and then into common outlet 206, and line 208, Figure 6A, and eventually into the column. After detritylation, valve portion 91 of valve pair 102a for DCA i8 closed and portion 88 (Figure 4) is opened to allow wash liquid from common inlet 200 to wash that valve pair. This wa~h continues on to column llOa and through it.
Thereafter valve 102'a i8 reversed 80 that the wash liquid now goes directly to line 107a and controlling means lSOa, by-passing column llOa. Valve 102'a îs then closed, and a valve 220 (used to combine the function~ of valves 154 and 156) i8 operated to direct the contents of controlling means 150a to wagte, ag the plunger is advanced. As par~ of this wash step, wash can be cycled through each of the other valves 102a on the manifold, jUBt as i~ done on manifold 70a, to be sure to clean up any leaking reagent. ~ -Ne$t, a ~econd ba~e and activator i8 added, and then the base linkage i~ column llOa is o~idized, using the iodi~e of bottle 98a. To thi~ end, valve portion 91 i8 opened in the valve pair 102 for that ~ . .. , ., ~ . .. , ., .. . - . .. ..... .. . , .. , , .. - .. --16- ~ 329469 bottle, to supply iodi~e to outlet 206, line 208, valve 102~a and column llOa ~a~ the plunger of controlling means 150a is withdrawn.) Thus, the cycle of reagent/wash, reagent/wash i~ repeated as was used in the previously described embodiment.
In this embodiment, valve 152 of controlling means 150a has been replaced with a check valve 152a, although as noted below, it too can be eliminated.
Column 110 i8 a conventional reactor column pogitioned to receive as input, the output of line 106 from manifold 90 or 70. The finished ; polynucleotide remains on column 110 for future extraction, Examples of columns that can be used herein include control pore glass types.
In accord with another aspect of the invention, liquid controlling means lSO iB positioned fluidly do~nstream onlyg from column 110. Means 150 preferably uses a conventional drive or motor means 161, for example, one using a lead screw ant a stepper motor. Any conventional low pressl~re piston and pistol~ chamber can be uæed. Preferably, however, means 161 include a linear displaeement transducer (not ahown) to provide abaolute indication of piston position, ~o that motor pulse count i8 not the ~ole measure of piston movement or position. In a preferred mode of operation, means are also provided that insure that controlling means 150 only receives -~
liquid from, and does not send liquid to, c~lumn 110. One mechanism for doing this i~ the use of two-way valves 152, 154 and 156. Valves 152-156 are conventional two-way valves, and function as follows:
When column 110 iB to dçliver ~aterial via line 120, valve 152 is opened. Otherwise it i8 . .
preferably clo~ed. Because valve 102' preferably does not connect line 107 with line 106, controlling ~eans 150 i8 thus prevented from delivering liquid to column 110. Line 107, however, connects directly to controlling means 150, 80 that when pi~ton 160 is withdrawn, the liguid supplied via manifold 90 enters the chamber 158 when valve 102' i~ deenergized, asauming a valve to a bottle in either manifold ~a~
been opened. Thus controlling means 150 acts to : control the supply of all liquid, not just liquid from manifold 70. Because liquid delivered via line 107 is waste material, piston 160 preferably ejects such liquid collected, via valve 156 to waste only.
After piston 160 has emptied any waste in such a fashion, chamber 158 can receive material from column 110. Such material includes protecting groups cleaved from the base by an acid environment. To this end, valve 152 is opened. At this point, valve 102~ is energized to allow flow from manifold 90 to line 106, but what iB supplied to manifold 90 is only the wash liquid CH3CN from a valve 102. As piston 160 is withdrawn, material i8 drawn through column 110. Thereafter, valve 152 i8 closed and valve 154 opened (valve 156 remains closed) and piston 160 is advanced to eject that material from chamber 158 to "collection", where the efficiency of the reaction can be measured as a function of the color density of the cleaved material, as iæ well-known. For e~ample "collection" can be a fraction collector that separate~ sample , or such samples can be collected and sent to a spectrophotometer to monitor reaction e~ficiency. Sampling and monitoring can also be done through a spectrophotometer on line at the external : collection port.
: Al~ernatively, valve 152 can be omitted entirely, with the understanding that when piston 160 i~ advanced to eject liquid from controlling means 150, all valves upstream from column 110 are clo~ed, e.g., valve 102' or all the valves in manifolds 70 -18 ~ 329469 and 90. In that case, there i8 nowhere for liquid in column 110 to flow out, as would be nece~sary fo~
controlling means to pu~h its liguid back into column 110. Thus, the clo~ing of tho~e upstream valves in a hard liquid flow-path constitutes the means that, in the reacting mode, prevents controlling mean~ 150 from sending liquid from its chamber 153 back into the column.
Preferably a pressure transducer 162 i~
aggociated with controlling means 150. The purpose ; of thi~ transducer i8 to detect changeR in pressure as will occur if a valve operation fails. That i~, if a valve fails to energize to allow liquid to feed i~to a manifold, or is partially blocked, the pre3gure when piston 160 i8 withdrawn will drcp below a set threshhold valve indicating a 8y8tem error. If a valve 154 or 156 fails to open when piston 160 advances, pre~sure will increase beyond a pre~et value. The ~ynthe~izer further preferably includes appropriate circuitry 163 for comparing the signal generated by transducer 162, with the preset safety levels. When the signal from transducer 162 varies beyond the present level by a predetermined amount, the circuitry shuts down the synthesizer and issues ; 25 an error code, allowing for real time diagnostics.
The following i5 a repre~entative e~ample illustrating the step of operation using synthe~izer 60. These ~teps are synchronized by computing means (not ~hown), which can be any conventional programmed computer system, using for example one or more microproce~ors.
Column 110 i8 provided with a ~upport having a tritylated base already on it. Thu~, the first step in the proce~s i8 to detritylate that ba~e.
This is accomplished by opening only valve 102~ ~o the column and a reagent valve portion "11" of a -19- l 329469 valve 102, 80 that when pi8ton 160 i8 withdrawn, the extra volume that i8 created allow~ the gas pres~ure from manifold 62 to push DCA into valve 102~ and line 106, and then into the column. Following the requi~ite amount of DCA being drawn into the system, valve portion "11" is closed and valve portion l'8" of valve 102 is opened, to allow enough wash liquid into manifold 90 and column 110 to pu~h all the DCA
through that i8 needed. This also serves to wash out valve 102~ and line 106 of any DCA, prior to using the next reagent. The detritylate waste i8 al80 drawn into chamber 158 from column 110. Valve 102' i8 then closed, valve 154 or 156 i~ opened, and piston 160 is advanced. This causes chamber 158 to empty into the waste line or to the collection line where the trityl groups can be collected and analyzed.
Next, a new base is added, along with ACT in a predetermined ratio. This is done as follows:
Line 114 already has wash liquid in it, B0 Z0 that the energizing of valve 102~ with all the valves 102 of manifold 90 being closed, and the opening of a valve portion 91 of tandem valves 84 will cause one of the base~ and then ACT to be drawn sequentially into outlet 93 of manifold 70, and then on into column 110. (Valve 102~ i8 energized to pass liquid to line 106 of the column rather than to syringe 158.) When the appropriate amount of that base and ACT has been drawn into line 114 and column 110 (by the operation of piston 160), the re~pective valve portion 91 in manifold 70 or 70a i8 closed and the wa~h portion 8~ for that particular tandem pair i8 opened to allow CE3CN to pass into that valve pair, line 95 and outlet 93. After enough wash liquid has been drawn to clear line 114 and line 106 of that base (and then of ACT) into column 110, each of the other wash valves around manifold 70 are opened ' 20- 1 32~469 seguentially (with valve 102~ operated to divert flow directly into chamber 158) a~ described above. This cau~es the other base line~ 95 ~o be washed of any possible leaking bases, which wash i8 Bent via S chamber 15B and valve 156 to wa~te.
Thereafter, the linkage between the two base~ in column 110 i~ oxidized, u~ing the iod;ne reagent. The process i8 essentially identical to that deæcribed above for DCA, except that valve portion "12'l of the valves 102, a~sociated with the iodine, controls its flow. Washing occurs after this, using the proces~ described for DCA wherein CH3CN is used to pu~h all the iodine through the ,, column.
Next, capping of the base in column 110 is achieved by allowing flow of both cap A and cap B
reagents in a predetermined ratio into manifold 90.
This i6 done by energizing valve portions 9 and 10, respectively, for those reagents and valve 102~ to a~low flow from manifold 90 to column 110, when piston 160 of chamber 158 is withdrawn. Each cap reagent ~ completely drawn into the column by following it with wagh liguid C~3CN, that also washes the valves 102 corresponding with the cap reagents.
Alternatively, after each of the reagents has been drawn into manifold 90 and column 110, the wash valve 8 i3 energized to wash all of the valves 102 even though only one such valve has been u~ed for the particular reagent.
It will be appreciated that after each of t,he above reagent~ and wash has flowed into column 110 and chamber 158, the chamber is emptied into the waste line by opening valve 156.

, The proce~R i8 then repeated for each of the other base~ from bottles 72, 74, 76, 78, 79 and 80, as needed for the particular polynucleotide sequence.
Alternatively, Figure 7, dual or ~ultiple columns 300 mounted in parallel, can be u~ed. If two columns are present, the second one can be mounted to receive reactant from the parallel manifold via a valve 302 in the reagent manifold, which then feeds via a normally closed valve 304 to a second controlling means not shown, as described for the first column. Such multiple columns can be used with accurate flow rate and volumes, even if both columns are producing two different sequences simultaneou~ly.
The invention has been described in detail with particular reference to pre~erred embodiments thereof, but it will be under~tood that variations ; and modifications can be effected within the ~pirit and scope of the invention.

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Claims (14)

1. In a synthesizer apparatus for synthesizing peptides or polynucleotides, the apparatus including supply means for supplying a) liquid reactants for synthesizing the peptides or polynucleotides, b) liquid reagents that assist in said synthesis, and c) a wash liquid; a first reactor column; means for connecting said supply means with said column; means for positively displacing with gas pressure, liquid reactants and reagents, and wash liquid from said supply means into said connecting means; and controlling means connected to said column for allowing liquid to flow under the force of said gas pressure and at a controlled rate, into a temporary storage chamber and for thereafter emptying the chamber;
the improvement wherein said controlling means is a positive displacement controlling means and is fluidly positioned only downstream of said reactor column, and further including means for preventing flow from said chamber back to said column, whereby said column is precluded from contamination by reactants or reagents in said controlling means, and said controlling means needs no cleaning to prevent it from contaminating said column.

2. A synthesizer apparatus as defined in claim 1, wherein said controlling means include a piston and cylinder, and means for driving said piston.

3. A synthesizer apparatus as defined in claim 1, wherein said preventing means comprise a valve and means for activating said valve when said chamber is being emptied.

4. A synthesizer apparatus as defined in claim 1, and further including means in said controlling means for detecting pressure changes, and further including comparator means for comparing the pressure detected by said detecting means with a preset level, whereby the improper performance of a value can be detected.

5. In a synthesizer apparatus for synthesizing peptides or polynucleotides, the apparatus including supply means for supplying a) liquid reactants for synthesizing the peptides or polynucleotides, b) liquid reagents that assist in said synthesis, and c) a wash liquid; a reactor column; connecting means for fluidly connecting said supply means with said column; means for positively displacing with gas pressure, liquid reactants and reagents from said supply means, and wash liquid into said connecting means; and controlling means connected to said column for allowing liquid to flow under the force of said gas pressure and at a controlled rate, into a temporary storage chamber and for thereafter emptying the chamber;
the improvement wherein said apparatus further includes a parallel manifold having i) sufficient inlet means to receive either each of said liquid reactants or said liquid reagents, ii) a common inlet to receive the wash liquid, and iii) an outlet connected to said connecting means to supply reactant or reagent to said column or to said controlling means, each of said inlet means including a pair of tandem two-way valves, both of the valves of said pair having a common outlet that feeds into said manifold outlet, one portion of each pair of said tandem valves being connected to said common inlet for said wash liquid and the other portion of said valves being connected to said inlet means for one of said liquid reactants or one of said liquid reagents, and means for selectively opening one of the pairs of valves at a time to a source of wash liquid, in a predetermined sequence, whereby an adequate source of wash liquid can be provided for each of said valve pairs of said parallel manifold.

6. Synthesizer apparatus as defined in Claim 5, wherein said pairs of tandem valves comprise solenoid valves.

7. In a synthesizer apparatus for synthesizing peptides or polynucleotides, the apparatus including supply means for supplying a) liquid reactants for synthesizing the peptides or polynucleotides, b) liquid reagents that assist in said synthesis, and c) a wash liquid; a reactor column; means for fluidly connecting said supply means with said column; means for positively displacing with gas pressure, liquid reactants and reagents, and wash liquid from said supply means into said connecting means; and controlling means connected to said column for allowing liquid to flow under the force of said gas pressure and at a controlled rate, into a temporary storage chamber and for thereafter emptying the chamber;
the improvement wherein said controlling means is a positive displacement controlling means and is fluidly positioned only downstream of said reactor column, and further including means for preventing flow from said chamber back to said column, so that said column is precluded from contamination by reactants or reagents in said controlling means, and said controlling means needs no cleaning to prevent it from contaminating said column, and wherein said apparatus further includes a parallel manifold having i) sufficient inlet means to receive each of said liquid reactants, ii) a common inlet to receive the wash liquid, and iii) an outlet connected to said connecting means to supply reactant or reagent to said column or said controlling means, each of said inlet means including a pair of tandem two-way valves, both of the valves of said pair having a common outlet that feeds into said manifold outlet, one portion of each pair of said tandem valves being connected to said common inlet for said wash liquid and the other portion of said valves being connected to said inlet means for one of said liquid reactants, and means for selectively opening only one of the pairs of tandem valves at a time to a source of wash liquid, in a predetermined sequence, whereby an adequate source of wash liquid can be provided for each of said valve pairs of said parallel manifold.

8. Apparatus as defined in claim 6, wherein said controlling means include a piston and cylinder, and means for driving said piston.

9. Apparatus as defined in claim 6, and further including means in said controlling means for detecting pressure changes, and further including comparator means for comparing the pressure detected by said detecting means with a preset safety level, whereby the improper performance of a value can be detected.

10. Apparatus as defined in claim 6, wherein said pairs of tandem valves comprise solenoid valves.

11. Apparatus as defined in claim 1, 4 or 6, and further including an additional reactor column connected in parallel with said first reactor column, to a controlling means.

12. Apparatus as defined in claim 1 or 6, wherein said controlling means include means for providing absolute indication of piston position.

13. A method for drawing reactants and reagents under gas pressure into a reactor column to synthesize a peptide or an polynucleotide, using a liquid controlling means fluidly connected to said column, the method comprising the steps of a) drawing under said gas pressure a reactant or reagent from a source of supply first into an outlet common with all reactants or reagents, then into said column, and then into said controlling means, b) drawing under said gas pressure a wash liquid through said common outlet, then into said column and then into said controlling means, c) and ejecting all liquid out of said controlling means into either a collection container or a waste disposal without passage through said column.

14. A method as defined in claim 13, wherein said reactants each feed into a separate supply line that is fluidly connected to said common outlet, and wherein said method further includes the step, after step b), of washing each of said separate supply lines sequentially, before drawing a different reactant into said common outlet and said column, whereby contamination from leaking reactant suppliers is minimized.