Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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 When the syringe pi~ton i~ withdrawn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
,.,
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-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
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1 32946~
--7--
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
,
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, .
1 32946q
--8--
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
'
1 329469
--9
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
1 329469
--10--
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.
1 329469
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
-12-
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
-13
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
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-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
1 329469
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.
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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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