Note: Descriptions are shown in the official language in which they were submitted.
~ 1/06678 PC-r/VS90/0617X
2 ~ 6 ~ ~ '
'' ~
. .- ~
' :
DNA SEQUENCING
Backqround Of The Invention
Field of the Invention
This invention relates to DNA sequencing. More
particularly it relates to methods and apparatus for
determining the sequence of deoxyribonucleotides within
DNA molecules.
Description of Backqround Art
DNA sequencing is an important tool. A current
goal Oc the biological community in general is the
~`; 20 determination of the complete structure of the DNA of a
number of organisms including man. This information will
-~s aid in the understanding diagnosis prevention and
treatment of disease.
Current DNA sequencing methods employ either
chemical or enzy~matic procedures to produce labeled
fragments of DNA molecules. In the chemical method
reactions are performed that specifically modify certain
: of the nucleotide bases present in the end-labeled DN~.
These reactions are carried ~ut nnly partially tc-
~ 30 comple ion so that only a porti^n of the bases present in
; the molecules are reactecl. These modified bases are then
~ treated with piperidine to clsave the DNA cllaills at the
`~ modified bases producina four sets of nested fragments.
These fragments are the]l separate~ from one another
35 according to size by electrophoresis in polyacrylamide
gels. The fragments can then be visualized ln the gels by
'
~_ . ~ . ~ . . . " . . .
. ~ . .. . .
W O 91/06678 PC~r/~'S90/06178 ~
' ' `2o~l46J'6
--2--
means of radioactive labels. The position of the
fragments in the gel indicates the identity of the last
nucleotide in each fragment so that on the gel a ~ladder"
of fragments, with each step identified, is assembled to
provide the overall sequence.
In the enzymatic method, the DNA to be sequenced
is enzymatically copied by the Klenow fragment of DNA
polymerase I or by a similar polymerase enzyme such as Taq
polymerase or Sequenase-. The enzymatic copying is carried
out in quadruplicate. In each of the four reactions a low
concentration of a chain terminating dideoxynucleotide is
present, a different dideoxynucleotide being present in
each of the four reactions (ddATP, ddCTP, ddGTP and
ddTTP). Whenever a dideoxynucleotide is incorporated, the
polymerase reaction is terminated, again producing sets of
nested fragments. Again, the nested fragments have to be
:~ separated from one another by electrophoresis to determine
' the sequence.
Recently, new advances in sequencing technology
have introduced automated methods. Applied Biosystems has
developed an instrument based on the use of fluorescent
labels and a laser-and computer-based detection system
(Smith et al., 1986; 5mith, 1987). An automated system
developed by E.E: du Pont de Nemours ~ Company, Inc.
' 25 (Prober et al., 1987) is similar to the Applied Biosystems
instrument but uses fluorescently labeled ddNTPs to
terminate the reaction instead of fluorescent primers.
. Hitachi (Japan) and EMBL (West Germany) have developed
similar systems (Ansorge et al., 1986). Other approaches
involve multiplexing technology (Church and
` Kieffer-Higgins, 1988), detection of radioactively labeled
DNA fragments by sensitive Beta-detectors (EG~G),
automated gel readers (BioRad), and automated liquid
: handlers (Beckman Instruments; Seiko; Goodeno~, University
35 of California, Berkeley). ~:
:
-
. . . . . , , -
. - . , . : . . ~ ~ .
- -: . . ~ . - :: : :
~ 1/06678 PCT/~'S90/0617~
2 ~
--3--
.
The need to rely on electrophoresis and a
separation according to size as part of the analytical
scheme is a severe limitation. The gel electrophoresis is
- a time-consuming step and requires very highly trained
skilled personnel to carry it out correctly. The present
.~ invention provides methods and apparatus for sequencing
- DNA which do not require electrophoresis or similar
separation according to size as part of their methodology.
References of Interest
The following articles and patents relate to the
general field of DNA sequencing and are provided as a
general summary of the ~ackground art. From tinle to time
reference will be made to these items for their teaching
~~ 15 of synthetic methods, coupling and detection
- methodologies, and the like. In these cases, they will
- generally be referred to by author and year.
W.B. Ansorge, et al., (1987) Nucleic A id
Research, 15:4593-4602.
-:~
W.B. Ansorge, et al., (1986) Journal of
Biochemical and Biophysical Methods, 13:325-323.
J. T. Arndt-Jovin, et al., (1975) European
~ Journal of BiochemistrY, 54:411-413.
.i H. Bunemann, et al. (1982) Nucleic Acids
Research, 10:7163-7180.,
. L.D. Cama, et al., (1978) Journal of the
American Chemical Society, 100:8006.
G. M. Church, et al., (1988) Science
240:185-188.
S.A. Chuvpilo, et al., (1984) A Simple and
- Rapid Method for Sequencing DNA, FE~S 179:34-36.
L.F. Clerici, et al., (1979) Nucleic Acids
Research, 6:247-258.
L.A. Cohen, et al., (1966) Journal of Orqanic
Chemistry, 31:2333.
~ - :
~'' ,.
. - . . ~ - ., ., ~ .
'. ' ' ,. ~: ~'~ '' . :
' .' . . ' . ' '~:
W O 91/06678 PC~r/~'S90/0617X ~
: ` ~ 6
B.A. Connolly, (1987) Nucleic Acids Research,
15:3131-3139.
C.G. Cruse, et al., (1978) Journal of Oraanic
Chemistry, 43:3548-3553.
P.T. Englund, et al., (1969) Journal of
Biolo~ical Chemistry, 244:3038-3044.
B.C. Froehler, et al., (1986) Nucleic Acids
- Research, 14:5399-5407.
R. Gigg, et al., (1968) Journal of the Chemical
10 Society, C14:1903-1911.
P.T. Gilham, (1968) Biochemistry, 7:2809-2813.
M.L. Goldberg, et al., (1979) Methods in
Enzymoloqy, 68:206-220.
T. Goldkorn, et al., (1986) Nucleic Acids
15 Research 14:9171-9191.
T.W. Greene, (1981) Protective Groups in Organic
Synthesis, John Wiley and Sons, Inc., New York, New ~ork.
'~ E. Hansbury, et al., (1970) Biochemical &
~ Biophysical Acta, 199:322-329.
.
C. Hansen, et al., (1987) Analytical
Biochemistry, 162:130-136.
W.D. Henner, et al., (1983) Journal of
Bioloqical Chemistry, 258:151198-15205.
J.A. Huberman, et al., (1970) Journal of
Bioloqical Chemistry, 245:5326-5334.
Y. Kanaoka, (1977), Anqewante Chemie
International Edition English, 16:137-147.
A. ~ornberg, (1974), DNA Synthesis, w. H.
Freeman and Company, San Francisco.
A.A. Kraevskii, et al., (1987) M^lecular
Biolec~-, 21:25-29.
A.A. Kraevsky, et al., (1987) Biophosphates and
; Their Analogues--Synthesis, Structure, Metabolism and
Activity, K.S. Bruzik and W.J. Stec (Eds.), Elsevier,
35 Amsterdam, pp. 379-390 (and references therein)~ ~;
,
'~- : . ' . . , ' -' ' ', ~ .," . '. . . - :
-: . . . .
- : .. : , ,
W091/06678 PCT/US90/0617X
_5_ 2~
J.N. Kremsky, et al., (1987) Nucleic Acids
Research, 15:2891-2909.
T.V. Kutateladze, et al., (1987) Molecular
Bioloqy, 20:222-231.
J.A. Langdale, et al., (1985) Gene 36:201-210.
R.T. Letsinger, et al. (1964) Journal of Oraani~~
Chemistry, 29:2615-2618.
J.K. Mackey, et al., (1971) Nature, 233:551-553.
T. Maniatis, et al., (1982) Molecular Cloninq, A
Laboratorv Handbook, Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York.
A. M. Maxam, et al., (1980) Methods in
Enzymoloqy, 65:499-560.
^ E. Ohtsuka, et al., (1978) Journal of the
American Chemical Society, 100:8210-8213.
` A.V. Papchikhin, et al., (1985) Bioorqanic
Chemistry, 11:716-727.
S. Pochet, et al., (1987), Tetrahedron,
43:3481-3490.
R. Polsky-Cynkin, et al., (1985) Clinical
; Chemistry, 31:1438-1443.
J.M. Prober, et al., (1987) Science,
;~ 238:336-341.
C.B. Reese, et al. (1968) Tetrahedron Letters,
. 25 40:4273-4276.
-' T.A. Rezovskaya, et al., (1977) Molecular
Bioloqy, 11:455-466.
F. Sanger, et al., (1977) Proceedinqs of the
National AcademY of Science US~, 7~:5~63-546,.
., .
S.R. Sarfati, ~t al. (1987) Tetrahedron Letters,
43 3491-3497.
B. Seed, (198~) Nucleic Acidi Research,
10:179?-1810.
A.J.H. Smith, (1980) Meth~ds in Enzym~loqy,
~ 35 65:560-580.
,, .
' :~
-:: - ~ .. . . . . . .
WO 91/0667~ 0 d~ ~ 6 1 6 PCT/~IS90/061 7X
.
. --6--
L.M. Smith, et al., (1986) Nature, 321-'674-679.
L.M. Smith, (1987) Science, 235:G89.
E.M. Southern, (1975) Journal of Molecular
Bioloqy, 98:503-517.
S. Tabor, et al., (1987) Proceedlnqs_of the
National Academy of Sciences USA, 84:4767-4771.
R.I. Zhdanov, et al., (1975) S~nthesis,
1975:2~2-245.
: .
Additional references of interest are:
' N. Dattagupta, U.S. Patent No. 4,670,380 issued
June 2, 1987.
W.J. Martin, European Patent Application No.
018769", published July 16, 1986.
Japan Kokai Tokyo Kobo JP 58/87,452 (May 25,
1983); Chem. Abs, Vol. 99, No. 172376n.
' R. Lewis, "Computerizing Gene Analyses~ Hiqh
Technoloqy, December 1986, p. 46 ff.
C. Connell, et al. "Automated DNA Sequence
Analysis", BioTechniques, Vol. 5, No. 4, p. 342 ff.
(1987).
J.F.M. De Rooiz, et al., Journal of
~^ Chromotoqraphy, Vol. 177, p. 380-384 (1987).
9 :
Statement of the Invention
.. :
'~ The present invention provides methods and
~:~ apparatus for determininq the sequence of
deoxyribonucleotides in a DNA molecule. A ke~-
charac_eristic of this invention is that it determines the
DNA sequence without recourse to electrophoresis or other
, size-based separation teshniques.
- In one aspect, the present invention provides a
method for determining the deoxyribonucleotide sequence of
a single stranded DNA subject molecule. This method
:.
~'~
.
, s , . .. , . .. : . . . . : . . . .... : .
- . . - ' , ,, ' . - .
, .
W O 91/0667X PC~r/~S90/06178
_7_ 2~
involves synthesizing, in the presence of a multitude of
identical copies of the subject DNA, the DNA molecule
which is complementary to it. This synthesis is carried
out using deoxyribonucleotide triphosphates (dNTP) in a
stepwise serial manner so as to simultaneously build up
numerous copies of the complementary molecule, dNTP by
dNTP. As each dNTP is added to the growing complementary
molecules, it is identified by way of an appropriate label
(i.e., reporter group). By noting the identity of the
bases present in this complementary molecule and using
standard rules of DNA complementation, one can translate
from the complementary molecule to the corresponding
original subject molecule and thus obtain the
deoxyribonucleotide sequence of the subject molecule.
In an additional aspect, this invention provides
apparatus for carrying GUt the above-described method.
. As will be seen in the Detailed Description of
the Invention which follows, this method and apparatus for
, carrying it out can take many different configurations. A
key to all of them, however, is the fact that the DNA
~ sequence is determined not by generating a series of
.~i nested fragments which must be separated according to size
but rather by direct identification of the dNTPs as they
are incorporated into the growing complementary DNA chain.
: 25 This invention can be carried out in a single
reaction zone with multiple differentiable reporters or in
multiple reaction zones with a single reporter in each
zone. It can be carried out by detecting the incremental
signal change after addition of reporters or by noting
-.......... 30 each added reporter separately. The various reporters can
be measured in the reaction zones while attached to the
~` growing molecule or they can be separated from the
molecule and then measured.
The invention can be practiced to create the
growina complementary DNA chain without interruption or it
/ can be practiced in stages wherein a portion of the
~!
,'......... . '
~ . :
.
. . . ~
W O 91/06678 PC~r/~'S90/0617X
2~46~6 ~
~8-
complementary chain is created and its sequence
determined; this portion of the chain is then removed; a
sequence corresponding to a region of the removed chain is
separately synthesized and used to prime the template
chain for subsequent chain growth. The latter method can
be repeated as needed to grow out in portions the complete
- complementary chain.
Detailed Description of the invention
Brief 3escription of the Drawinqs
The invention will be further described with
reference being made to the accompanying drawings in
- 15 which:
- Figures lA and lB are schematic diagrams of the
process of this invention on a molecular level.
Figure 2 is a schematic representation of one
form of apparatus for practising the invention. In this
embodiment the DNA growth takes place in a single reaction
~, zone. This embodiment uses separate, distinguishable
.~ reporters associated with each of the four nucleotides
; incorporated into the growing molecule. The four
different reporters are measured after each addition to
detect which base has just been added to that position of
the complementary chain.
Figure 3 is a schematic representation of
another form of apparatus for practising the invention.
This embodiment employs f^ur rQaction zoneC in which the
molecular arowth is carried out in guadruplicate. In each
of the four zones, a different one of the four nucleotides
is associated with a reporter (with the remaining three
,' being unlabeled) so that the identity of the nucleotide
incorp^rated at each stagQ can be determined.
Figure 4 is a schematic representation of an
adoption of the apparatus for practising the invention
J
~' ,','.. .
~ , ' ' ' ' ., ' ~
~ - :' . . ' , "~ ' . '. " " ' ~ '`
'' '.
' ~ , , . , . ' ' ~ ' , ' ~ ' : : ,
~ 9l/06678 PCT/~S90/06178
_g~
particularly adapted for carrying out the invention to
grow a series of portions of the complementary molecule as
opposed to a single continuous complementary molecule.
Figures 5 through 8 are pictorial
representations of chemical reaction sequences which can
be used to synthesize representative labeled nucleotide
building blocks for use in the practice of this invention.
Orqanization of this Section
This Detailed Description of the Invention is --
;~ organized as follows:
First, several terms are defined in a
Nomenclature section.
. Second, a series of Representative Apparatus
Confiqurations and Process Embodiments for carrying out
:. .
the invention are described.
Third, Materials and Reaqents and Methods of Vse
- employed in the process of the invention are set forth,
including;
. 20 Enzymes and Couplinq Conditions,
Blockinq Groups and Methods for Incorporation,
. Deblockinq Methods,
f Reporter Groups, their IncorPoration and
Detection, and
Immobilization of Subiect DNA.
Thereafter, a series of nonlimiting EXAMPLES is
provided.
Nomenclature
A number of relate-J and gellerally conventional
~ abbreviations and define~ termC appear in thiC
.. speci.fication and claims. The four nuclectides are at
` times referred to in shorthand by way cf thelr nucleoside
- bases, adenosine, cytidine, guanosine and thymidine, or
"A'-, "C", "G and "T . DeoY~ynucleotide triphosphates
"dNTPs' of these materials are abbreviated as dATP, dCTP,
- .
' , ' ' ,' ~ " ' ~ . ' ' , ' '~' ' ,
'. .
. ' ' ~ ~ ~ ' ` . ' , '
WO91/0667X PCT/~S90/0617
2o~461~ f-,-`~
--10--
dGTP and dTTP. When these materials are blocked in their
3'-OH position they are shown as 3'blockeddATP,
3'blockeddCTP, 3'blockeddGTP and 3'blockeddTTP.
Similarly, when they are each tagged or labeled with a
5 common reporter group, such as a single fluorescent group,
they are represented as dA'TP, dC'TP, dG'TP and dT'TP~
When they are each tagged or labeled with different
reporter groups, such as different fluorescent groups,
they are represented as dA'TP, dC''TP, dG'''TP and
10 dT''''TP. As will be explained in more detail below, the
fact that the indication of labeling appears associated
with the "nucleoside base part" of these abbreviations
does not imply that this is the sole place where labeling
can occur. Labeling could occur as well in other parts of
15 the molecule.
Representative Apparatus Confiqurations and Process
Embodiments
In the specification and claims, reference is
20 made to a "subject" DNA or ~template~ DNA to define the
DNA for which the sequence is desired. In practice, this
material is contained within.a vector of known sequence. ~-
A primer, which is complementary to the known sequence of
.~ the vector is used to start the growth of the unknown
25 complementary chain. Two embodiments of this process are
illustrated on a molecular level in Figures lA and lB.
In Figure lA, a solid support 1 is illustrated
with a reactive group A attached to its surface via tether
, 2. This attachment can be covalent, ionic or the like. A
A 30 second reactive group ~, capable of bondin~ to ~roup A,
again via a covalent, ionis or the like bond, is attached
to the 5~ end of a DNA primer 4. This primer has a known
DNA se~uence. When coupled to the substrate via the A-X
bond it forms immobilized primer 5. Primer 5 is then
35 hybridized to template DNA strand 6 which is made up of an
unknown region 7 inserted between regions 8 and 8'.
., , , :, ~- ~ ~ . . :- . :
: ~ . ,,: : :
WO 91/06678 PCT/~'S90/06178
2 ~
Regions 8 and 8' are located at the 5' and 3~ ends of the
unknown region and have known sequences. The 8' region's
known sequence is complementary to the sequence of primer
4 so that those regions hybridize to form immobilized
template DNA 9. Therefore the individual dNTPs are
serially added to form the DNA sequence complementary to
the unknown region of the template. 11 and 12 represent
the first two such dNTPs incorporated into the ~rowing
molecule. These in turn provide the identity of their
complements 11' and 12' respectively. This growth
continues until the entire complementary DNA molecule has
been constructed. Completion can be noted by identifying
the sequence corresponding to the 8 region of template 6.
Turning to Figure lB, a variation of this
chemistry is shown in that the template 6* carries the
-
reactive group X which bonds to the substrate via the A-X
~ bond to form an immobilized template 5*. This is then
; hybridi-ed with primer 3* to give the immobilized, primed
. template 9* upon which the desired adding of dNTPs takes
., 20 place to add units 11 and 12 and thus identify the
.~ sequence and identity of units 11' and 12'. While in the
chemistry illustrated in Figure lB reference is made to
i~ coupling template DNA 6* via an X group on its 3' end to
.~j the A group on the substrate, it will be appreciated that
the template DNA 6* could just as well be coupled through
its 5' end. ~he chemistry for such an attachment is known
in the art.
Referring now to Figure 2, a device 13 for
carryin~ out the invention is shown schematically. In
this schematic representation, and the representation
provided in Figure , many components such ac mixers,
valves and the like are omitted to facilitate a clear
focus on the invention. Device 13 includes a reaction
zone la which carries inside it a surface 15. A plurality
of copies of a subject primed single stranded DNA are
immobilized on this surface 15. This is the strand of DNA
., .
:
. . . - - - -. -- ~ - ,
:; . . , ~ . - . - . ~ - . :, , .
WO 91/0667~i PCI/~1590/06178
20 ~6~6 -12-
for which the sequence is desired. The immobilized DNA is
depicted fancifully on surface 15 as if it were present as
a series of separately visible attached strands. As will
: be appreciated, this is not in fact the case and is only
done to guide the reader as to the location of the DNA
strands. The reaction zone 14 may be configured to permit
direct reading of reporter signals emanating from within.
` Examples of this configuration include equipping the
: reaction zone to permit measuring fluorescence or
luminescence through one or more transparent walls or
detecting radionuclide decay. Reaction zone lq is fitted
with inlet 16 for the addition of pol,vmerase or another
suitable enzyme capable of moderating the templat-
` e-directing coupling of nucleotides to one another. The
reaction zone is also accessed by inlet lines, laa-18d for
four differently labeled blocked dNTPs, that is
3'blockeddA'TP, 3'blockeddC''TP, 3'blockeddG'''TP, and
3'blockeddT''''TP. These materials can be added in four
separate lines, as shown, or can be premixed, if desired,
and added via a single line. Buffer and other suitable
~ reaction medium components are added via line 20.
`~. In practice, the polymerase and the four labeled
dNTPs are added to the reaction zone 14 under conditions
. adequate to permit the enzyme to bring about addition of :
~` 25 the on~, and only the one, of the four laheled blocked
dNTPs which is complementary to the first available ..
template nucleotide following the primer. The blocking
- group present on the 3'-hydroxyl position of the added
dNTP prevents inadvertent multiple additions. After thi~ -
first addition reaction is complete, the liquid in
~ reaction zone 14 is drained throuah line 22 either to
ii waste or if desired to storage for reuse. The reaction
zone and the surface 15 are rinsed as appropriate to ..
remove unreacted, uncoupled labeled blocked dNTPs. At
35 this point the first member of the complementary chain is ~ -
now in place associated with the subject chain attached to
,.:
:::. , : : . .,~- . - . :. . . ,.. : . .- ., ., , :-.: , : , : -.-
~ 1/06678 PCT/~l590/0617~
~. .
-13- 2 ~
surface 15. The identity of this first nucleotide can be
determined by detecting and identifying the label attached
to it.
This detection and identification can be carried
- 5 out in the case of a fluorescent label by irradiating the
surface with a fluorescence-exciting beam from light
source 24 and detecting the resulting fluorescence with
detector 26. The detected florescence is then correlated
to the fluorescence properties of the four different
labels present on the four different deoxynucleotide
triphosphates to identify exactly which one of the four
materials was incorporated at the first position of the -
. complementary chain. This identity is then noted.
. In the next step, a reaction is carried out to
` lS remove the blocking group and label from the 3~ position
on the first deoxynucleotide triphosphate. This reaction
is carried out in reaction zone 14. A deblocking solution
is added via line 28 to remove the 3~ hydroxyl labeled
~ blocking group. This then generates an active 3' hydroxyl
i 20 position on the first nucleotide present in the
complementary chain and makes it available for coupling to
the 5' position of the second nucleotide. After
completion of the deblocking, removal of the deblocking
solution via line 22 and rins~ng as needed, the four
blocked, labeled deoxynucleotide triphosphates, buffer and
. polymerase are again added and the appropriate second
member is then coupled into the growing complementary
chain. Following rinsin~, the second member of the chain
can be identified based on its label.
This proces~ i~ then repeated as needed until
the complementary chain has been completed. At the
completion of the construction of the complementary chain,
~- the se~uence of incorporated deoxynucleotides i5 known,
and therefore so is the sequence of the complement which
;! 35 is the subject chain.
~ , .
'. .
WO91/06678 PCT/US90/06178
2~ ~ 4~
-14-
It will be appreciated that this process is
easily automated. It is a series of fluid additions and
removals from a reaction zone. This can be easily
accomplished by a series of timer-controlled valves and
the like. This technology has been well developed in the
area of oligonucleotide synthesizers, peptide
synthesizers, and the like. In such an automated system,
the timing can be controlled by a microprocessor or, in
most cases, by a simple programmable timer. The rate and
exten~ of reaction can be monitored by measurement of the
reporter concentration at various stages.
The labels present in the blocked dNTPs can be
incorporated in one of several manners. For one, they can
- be incorporated directly and irremovably in the
. 15 deoxynucleotide triphosphate unit itself. Thus, as the
complementary chain grows there is a summing of signals
and one identifies each added nucleotide by noting the
chanqe in signal observed after each nucleotide is added.
Alternatively, and in many cases preferably, the
label is incorporated within the blocking group or is
other~-ise incorporated in a way which allows it to be
removed between each addition. This permits the detection ~
to be substantially simpler in that one is noting the ` -
presence of one of the four reporter groups after each
addition rather than a chanqe in the sum of a group of
;. reporter groups.
In the embodiment shown in Figure 2, the
presence of reporter signal is noted directly in the
reaction zone 14 by the analytical system notod as source
`~ 30 24 and detector 26. It will bo appreciated, howover, that
in embodiments where the reporter group is removed during
each c-ycle, it is possible to read or detect the reporter
at a remote site after it has been carried out of the
reaction zone 14. For example, drain line 22 could be
. 35 valved to a sample collector (not shown) which would
isolate and store the individual delabeling product
'," ~
.. . .
.. . .
., . . . - , , - . : .
- . . . . . -
~9t/06678 PC~ S9O/06178
-15- 2~
solutions for subsequent reading. Alternatively, if the
nature of the label permitted, the various removed labels
could be read as they flowed out of the reaction zone by
equipping line 22 with an in-line measurement cell such as
source 24' and detector 26' or the like.
A second embodiment of this invention employs
four separate parallel reaction zones. This method has
the advantage of requiring only one type of labeling and
- being able to use it with all four dNTPs. Figure 3 shows
a schematic representation of a device 30 which has the
four reaction zone configuration. In this configuration
there are four reaction zones 32a through 32d, each of
which resembles the reaction zone I4 in Figure 2. In
these cases each of the four reaction zones contains a
surface 34a-d to which is immobilized numerous copies of a
primed subject single stranded DNA. Each reaction zone is
supplied with polymerase via lines 36a-d. Each zone is
supplied with suitable reaction medium v a lines 38a-38d.
The four dNTPs are supplied in blocked form to each zone,
20 as well. In zone 32a one of the blocked dNTPs is labeled, -
for example "A'"; in zone 32b a second dNTP is labeled,
for example "C'"; in zone 32c a third dNTP is labeled, for
.' example "G'"; and in 32d the fourth labeled dNTP "T'" is
present. These labeled materials are supplied via lines
40a through 40d respectively. Unlabeled blocked dNTPs are
;~ supplied via lines 42a-d so that each of the four reaction
zones contains three unlabeled blocked dNTPs and one
labeled blocked dNTP. Again, as noted with refarence to
Figure 2, the various labeled and unlabcled dNTP's can be
premixgd. These premi~ad materials can be added to the
variou~ reaction zones ~-ia singla addition lines.
Using the same general methodology described
. with reference to Figure 2, tha sinale stranded DNA
hybridized to a primar and attached to each of surfaces
34a-34d is contacted with polymerase (supplied via lines
36a-36d)l buffer (supplied via lines 38a-38d) and the four
, , - ,. , . : - : - : . .
W O 91/06678 20 44~ PC~r/US90/06178 ~
.
-16-
bases in each of the four reaction zones. The blocked
dNTP which complements the first base on the subject chain
couples. In one of the four reaction zones, this base is
labeled. By noting in which of the four zones this label
is incorporated into the growing chain, one can determine
the identity of the dNTP which is incorporated at the
first position. This determination of the identity of the
first unit of the chain can be carried out using signal
:. .
sources and detectors such as 44a-44d and 46a-46d, :~
respectively. Deblocking is carried out by adding
deblocking solution to the reaction zone through lines
48a-48d. Lines 50a-50d are drain lines for removing
; material from the reaction zones following each step.
In this second configuration, all of the..
variations noted with reference to the device described in
Figure 2 can also be used including cumulating reporter
signals and generating reporter signals away from the
reaction zone by removing the reporter groups as part of
each of the sequential couplings. Clearly, this
- 20 embodiment can be readily automated, as well.
One obvious potential shortcoming of the present
invention is that it employs a long sequence of serial
reactions. Even if the efficiency and yield of each of
these reactions are relatively high, the overall yield
becomes the product of a large number of numbers, each of
which is somewhat less than 1.00, and thus can become
unacceptably low. For example if the yield of a given
' addition step is 98% and the deblocking is 98Q! as well,
the overall yield aftor 15 additions is 48~, after 30
additions it is 23~ and aftel- 60 additions it is 5.3
:~ This limitatio~ can be alleviated ~}
period.ically halting tho D~A moloculo growth and using the
sequence data obtained prior t^ halting the growth to --
externally recreato a portion of the molecule which can ~--;
; 35 then be used as a primer for renewed DNA fabrication.
:j ' - '
' ~
. ' ' , : .
:- . . . . :.
W~91/06678 P(~r/~lS90/0617~
-17- 2~
This process is illustrated in Figure 4. Figure
4 shows a schematic of an automated sequencer 52 employing
the present invention. Sequencer 52 has a single reaction
zone 14 combining the subject primed DNA, immobilized
therein such as on surface 15. The four 3-blocked DNTP's,
suitably detachably labeled, are fed to the reaction zone
through line 18. Polymerase and buffers are added via
lines 16 and 20, respectively. Additionally, the dNTP's,
polymerase and buffer can be recycled from step to step
via lines 54 and 56 and holding vessel 58. All of the
valves admitting and removing fluids from reaction zone 14
. can be controlled by central computer 60 which functions
as a valve control cloc~. This computer 60 can also
control the addition of deblocker from line 2~, deblocl~ing
. 15 eluent with cleaved labels (as obtained when the label is
present in the blocking group) is removed via line 22 and
detected via detector system 24/26 reading label values in
detector vessel 62.
~- This embodiment illustrates the use of a
"! 20 fluorescent label system and shows the addition of
- fluorescent sensitizer (flooder) via line 64 to the
fluorescent detection zone 62.
. Following detection of the label in vessel 62,
rS the deblocking solution and detected label are discarded
, 25 via line 66.
'''~5 The signal presented by the label identified by
~ detector 26 is passed to analog/digital converter 68 and
.3, therein to a memory in central computer 60 where it is
~` stored. After a number of iterations, the memory in
computer 60 contains the sequence of an initial portion of
. the complementary DNA molecule which has been constructed
in association with the subject or target DNA molecule
contained within reactor 14. After some number of units
have keen assembled - typicallY 25 to 300, or more;
preferably 50 to 300, or more; and more preferably 100 tc
300, or more - the growing complementary DNA molecule is
,, .
, . .. , , . , , . , . . . . . - . : ~ .
- . . .. : : .. - - ., . : ., . - : .- ,. , , , . -
WO91/06678 PCT/~'S9n/0617~ ~
- 20~46~ ~
-18-
.
stripped from the immobilized subject DNA molecule and
discarded. This stripping ~denaturing) can be done by
art-known methods such as by warming the reaction zone to
75C or higher (preferably 90-95C) for a few (1-15)
minutes. Other equivalent methods can be used. The
sequence information stored in computer 60 is used to
drive DNA synthesizer 70 to externally create a new DNA ~-
primer corresponding to at least a portion of the
discarded DNA molecule. (The sequence can also be read
on printer 72, if desired.) This newly constructed DNA
primer molecule is fed through line 74 to reaction zone 14
under hybridization conditions so as to join to the
complementary region of the subject DN~ molecule as a new
primer.
The length of the primer must be adequate to
unambiguously and strongly hybridize with a single region
of the subject DNA. As is known in the hybridization art, -
this can depend upon factors such as the sequence,
environmental conditions, and the length of the subject
DNA. For efficiency of operation, the primer should
,~,tl ideally be as short as possible. Primer lengths typically
!`',4 range from about 10 bases to about 30 bases, although
shorter primers would certainly be attractive if they met
the above criteria, and longer primers could be used
~; 25 albeit with an increase in cost and time. Good results
generally are achieved with primers from 12 to 20 bases
long. This gives the molecular growth reaction a "new
start" with a large number of properly primed identical
molecules. This allows a strong si?nal to be generated
when the next dNTP is coupled.
This restartin? of the growth can be carried out
as often as needed to assure a strona consistent label
: signal.
., :
~,
. .:
, - - .~ ~ ,
', ~
~ l/06678 PC~r/US90/06178
2 ~
--19--
Materials and Reaqents and Methods of Use
` En~ymes and Couplinq Conditions
The coupling process employed in this invention
to incorporate each of the blocked deoxynucleotide
triphosphates into the growing complementary chain is an
enzyme moderated process. Each member of the
complementary DNA chain is added using a suitable
template-dependent enzyme. One enzyme which can be used
is SequenaseTM enzyme (an enzyme derived from
bacteriophage T7 DNA polymerase that is modified to
improve its sequencing properties - see Tabor and
Richarson, Proc. Nat. Acad. Sci. USA, 84:4767-4771
- (1987j--sold by United States Biochemical Corporation,
Cleveland, Ohio). Other polymerases which can be used
instead of Sec~uenaseTM include but are not limited to
` Xlenow fragment of DNA polymerase I, AMV reverse
transcriptase, and Taq polymerase.
.~ Typically the coupling conditions which are
20 employed are those known in the art for these enzymes. In -
; the case of SequenaseTM these include temperatures in the
range of from about room temperature to about 45C; a
buffer of pH 7 to 8 and preferably pH 7.3 to 7.7; an
enzyme concentration of from about 0.01 units per
microliter to about 1 unit per microliter and a reaction
~`~ time of from about 1 to about 20 minutes and preferable 1
~ to 5 minutes. A typical buffer for use with SequenaseTM
;"r is made up of
0.040 M Tris HC1 (pH 7.
0.050 M sodium chlorido
0.010 M maanesium chloride
0.010 M dithiothreitol
In the case of ~;lenow fragment of DNA polymerase
I, these typical conditions include temperatures in the
range of from about 10CC to about 45C and preferably from
about 15C to about 40C; a buffer of pH 6.8 to 7.4 and
' ',:
..... . .. . . . .
WO9l/06678 PCT/US90/0617~
": 204~61~ ~ ~
-20-
preferably pH 7.0 to 7.4; an enzyme concentration of from -~
about 0.01 units per microliter to about 1 unit per
microliter and preferably from about 0.02 to about 0.15
- units per microliter and a reaction time of from about l
to about 40 minutes. A typical buffer for use with Klenow
fragment of DNA polymerase I is made up of
0.05 M Tris chloride, pH 7.5
0.05 M magnesium chloride
0.05 M sodium chloride
0.010 M dithiothreitol
These conditions are representative. When other
enzymes are employed, one should use the conditions
~ optimal for them since it is generally desirable to run
; the addition reaction as quickly as possible. To this
:- 15 end, it is often desirable to use temperatures of 42C for
reverse transcriptase; 24C for Klenow polymerase; 37C .
with SequenaseTM and 72C with Taq polymerase. In
i addition, to force the reaction, especially with `
`~ derivatized dNTP's it may often be helpful to use
:~ 20 substantial excesses (over stoichiometry) of the dNTP's,
or to modify other condi~ions such as the salt
: - .
concentration.
Blockinq Groups .and Methods for Incorporation
The coupling reaction generally employs
3'hydroxyl-blocked dNTPs to prevent inadvertent extra
;~ additions.
The criteria for the successful use ~f
3'-blocking groups include:
(l) the ability of a polymerase enzyllle to
accurately and efficiently incorrorate the dNTP~ carrying
i the 3'-blocking groups into the cDNA chain,
(2) the availabilit~ of mild conditions for
rapid and quantitative deblocking, and
~,
.1 .
W O 91/06678 PC~r/~'S90/06178
-21- 2~
(3) the ability of a polymerase enzyme to
reinitiate the cDNA synthesis subsequent to the deblocking
stage.
In addition, if the 3'-blocking group carries a
reporter group, it is desirable that the reporter permit
sensitive detection either when part of the cDNA chain
before deblocking or subsequent to deblocking in the
reaction eluant.
For the present invention, 3'-blocked dNTPs are
used that can be incorporated in a template-dependent
fashion and easily deblocked to yield a viable 3'-OH
terminus. The most common 3'-hydroxyl blocking groups are
esters and ethers. Other blocking modifications to the
3'-OH position of dNTPs include the introduction of groups
15 such as -F, -NH~, -OCH3, ~N3~ -OPO3 ~ -NHCOCH3~ 2-
nitrobenzene carbonate, 2,4-dinitrobenzene sulfenyl and
tetrahydrofuranyl ether. Incorporation and chain
termination have been demonstrated with dNTPs containing
many of these blocking groups (Kraevskii et al., 1987).
Presently preferred embodiments focus on the
ester blocking groups such as lower (1-4 carbon) alkanoic
acid and substituted lower alkanoic acid esters, for
.~ example formyl, acetyl, isopropanoyl, alpha fluoro- and
alpha chloroacetyl esters and the like; ether blocking
groups such as alkyl ethers; phosphate blocking groups;
carbonate blocking groups such as 2-nitrobenzyl;
2,4-dinitrobenzene-sulfenyl and tetrahydrothiofuranyl
ether blocking groups. Blocking groups can be modified to
incorporate reporter moieties, if desired, including
radiolabels (tritium, C1 or P~~, for example), enzymes,
fluor^phores and chromophores.
These blocking materials in their fundamental
forms have all been described in the literature as has
their use as blockers in chemical DNA synthesis settings.
Two representative blockers, esters and phosphate, can be
incorporated into dNTP's as follows:
.
- ~ ..................... .. , , ~
- ,
WO91/06678 PCT/~S90/06178
~,
6 1 6 -22-
The general procedure for synthesis of 3'-O-acyl
dNTPs is outlined in Reaction Scheme 1 set forth in ~igure
5 for ~'-O-acetyl TTP. 5'-Dimethoxytrityl (DMT) thymidine
2 is prepared from thymidine I by reaction with DMT
chloride in pyridine, followed by acetylation of the 3~-OH
function using acetic anhydride in pyridine to yield 3
(Zhdanov and Zhenodarova, 1975). Treatment of the 5'-DMT
! group with 2% benzene-sulfonic acid yields 4, which i5
converted into the phosphomonoester 5 by reaction with :
POC13 in trimethyl phosphate (Papchikhin et al., 1985) and
by purification using chromatography. The 5'-
monophosphate is converted into the 5'-triphosphate 6 by
activation with N,N'-carbonyldiimidazole, followed by
pyrophosphorylation with tri(n-butylammonium)
pyrophosphate (Papchikhin et al., 1985) and purification
by chromatography.
Preparation of 3'-O-acetyl derivatives of dATP,
dCTP, and dGTP follows the same general scheme, with
additional steps to protect and deprotect the primary
amino functions (see below). Because 5'-triphosphate
derivatives of nucleosides are often unstable, the final
preparative steps outlined above may be optionally carried
out just before introducing the dNTPs into the reaction
cell. If radioiabeled acetic anhydride is used, this
serves to introduce a label into the ester blocking group.
When carrying out this ester~blocking of the 3'-
OH group it should be borne in mind that the primary amino
- residues in cytosine, adenine, and guanine are also
suscep~ible to attac}; by electropllllic reagents such as
acetic anhydride and may be advanta~eously protected. I
chemi-al oligonucleotide synthesis (phosphotriester or
.~ phosrh^ramidite approaches), various N-acyl aLoups are
-` commonly used for protection ^f the primary amine
(Papchikhin et al., 1985). Because the N-acyls are stable
in acidic and neutral solutions, removal is typically
effected by ammonolysis. These conditions are li~.ely to
-
,
. ' ." '
': ` : ' , ' . , ~
WO91/06678 PCT/US90/06178
-23- 2~
cleave 3'-O-acyl blocking groups and other blocking groups
hydrolyzable under basic conditions, so alternative
N-protection should be used if it is desired to
selectively remove the amino group protection. Several
selectively-removable amine protection groups include
carbamates cleavable by acid hydrolysis [t-butyl,
2-(biphenyl)isopropyl] and certain amides susceptible to
acid cleavage (formamide, trichloroacetamide) (Greene,
1981).
- 10 The synthesis of 3'-monophosphate dNTPs is
outlined in Reaction Scheme 2 set forth in Figure 6 for
TTP and is a modification of reported procedures for
chemical oligonucleotide synthesis using the H-phosphonate
method (Froehler et al., 1986). 5'-DMT-3'-thymidine
, 15 H-phosphonate 7 is prepared by reaction of 5'-DMT
thymidine 2 with phosphorous trichloride, 1,2,4-triazole,
and N-methylmorpholine. Removal of the 5'-protecting
group and formation of the 5'-triphosphate moiety (7 to
} 11) is achieved as shown in Scheme 1. The 3'-OH
20 phosphonate TTP 11 is converted to the 3'-O-monophosphate
12 by oxidation with iodine in basic solution.
For other nucleotide derivatives, protection of
the primary amino groups is performed prior to
phosphonation. In this preparation, standard amino
: 25 protecting groups cleavable by ammonolysis may be used.
. .
; Deblockinq Methods
After successfully lncorporating a 3'-blocked
.~ nucleotide into the DNA chain, the sequencing scheme
; 30 requires the blockinq group to be removed to ~-ield a
; viable 3~-OH site for continued chain synthecis. The
deblocking method should:
(a) proceed rapidly,
(b) yield a viable 3'-OH function in high
yield, and,
:
,
- ..:
.
~ - , ~ ., .; ;. -. ~ . . . . . ... . . . .
: .: : : . . . . : ... - . ; .. . ; . - - . ~ .-
- . , . .. . , : .. .: . :: - .
. .. . : . . -.: . ~ - .
WO 91 /06678 PCl /~IS90/061 78
2Q~6~6
-24-
! (C) not interfere with future enzyme function
or denature the DNA strand.
(d) the exact deblocking chemistry selected
will, of course, depend to a large extent upon the
5 blocking group employed. For example, removal of ester
blocking groups from the 3'hydroxyl function is usually ~;
achieved by base hydrolysis. The ease of removal varies
widely; generally, the greater the electro-negativity of
substituents on the carbonyl carbon, the greater the ease
- 10 of removal. For example, the highly electronegative group
trifluoroacetate is cleaved rapidly from 3' hydroxyls in
methanol at pH 7 (Cramer et al., 1963) and thus would not
be stable during coupling at that pH. Phenoxyacetate -
groups are cleaved in less than one minute but require
15 substantially higher pH such as is achieved with NH3/
.-. methanol (Reese and Steward, 1968). To prevent
:~ significant premature deblocking and DNA degradation, the
.~ ester deblocking rate is advantageously selected so as to
.~ exhibit a deblocking rate of less than 10 3s 1 during the
20 incorporation, and at least 10 ls during the deblocking
stage. Ideally, this rate change is achieved by changing
the buffer pH from 7 to about 10, but care must be taken
3 not to denature the DNA.
A wide variety of hydroxyl blocking groups are
25 cleaved selectively using chemical procedures other than
base hydrolysis. 2,4-Dinitrobenzenesulfenyl groups are
t cleaved rapidly by treatment with nucleophiles such as
.' thiophenol and thiosulfate (Letsinger et al., 1964).
Allyl ethers are cleaved by treatment with Hg(II) in
30 acetone/water (Gigg and warrell, l9h8)
Tetrahydrothiofuranyl ethers are removed under neutra1
conditions using Ag(I) or Hg(II) (cohen and ~teele, 1966;
Cruse et al., 1978). These ~rotecting groups, which are
stable to the conditions used in the synthesis of dNTP
35 analogues and in the sequence incorporation steps, have
some advantages over groups cleavable by base hydrolysis -
., -
-
.
': '- . ~ ~, '- ' ' .
- , . , :
~, ' ' --' '- '': ' '; - :' -
W091/06678 PCT/~'S90/0617~
20~6~ 6
-25-
deblocking occurs only when the specific deblocking
reagent is present and premature deblocking during
incorporation is minimized.
Photochemical deblocking can be used with
photochemically-cleavable blocking groups. Several
blocking groups are available for such an approach. The
use of o-nitrobenzylethers as protecting groups for
2'-hydroxyl functions of ribonucleosides is known and
demonstrated (Ohtsuka et al., 1978); removal occurs b~
irradiation at 260 nm. Alkyl o-nitrobenzyl carbonate
protecting groups are also cleaved by irradiation at pH ,
(Cama and Christensen, 1978).
Enzymatic deblocking of 3'-OH blocking groups is
also possible. It has been demonstrated that T4
polynucleotide kinase can convert 3'-phosphate termini to
3'-hydroxyl termini that can then serve as primers for DNA
polymerase I (Henner et al., 1983). This 3'-phosphatase
- activity is use~ to remove the 3'-blocking group of those
dNTP analogues that contain a phosphate as the blocking -
group; the radioactive label enables the incorporation of
the nucleotide analogue and the removal of the phosphate
group to be followed easily. If the use of radioisotopes
~ represents too great a drawback, it is possible to use
:. unlabeled phosphate monoesters with a cleavable
fluorescent label (see below).
This method is improved by increasing the
efficiency and speed of each step. Upon selection of the
optimal methodology for incorporation and deblocking,
other nonchemical assistance may be used to accelerate
chemical deblocking. This may include, for eY.a~le
applying controlled ultrasonic irradiation of the reaction
chambe- to increase the rate of the deblockincl ste~ if
mass transport limitations are sictnificant and raising the
reaction temperature up to about 50~C for a short period.
~; 35
-
:. . .. . . .... . .. .... . ..
,~ - . . ... : :.- . . ... . . .
-: : - :: : -:
. : .: ', - -. : - : ' ~ : : : : :
~ W O 91/06678 PC~r/~lS90/0617~ ~ ~
` ;20~61~
-26-
Reporter Groups, their Incorporation and Detection
As part of this invention, the incorporation of
each dNTP into the complementary chain is noted by
detecting a label or reporter group present in or
5 associated with the incorporated dNTP. The labels or
markers are ~innocuous . An ~innocuous marker or label or
- reporter" refers to a radioactive, fluorescent, or the ~`
like marker or reporter which has physical and chemical
properties which do not interfere with either the
10 enzymatic addition of the marked nucleotide to the cDNA,
or the subsequent deblocking to yield a viable 3'-OH
terminus.
One simple labeling approach is to incorporate a
- radioactive species within the blocking group or in some
15 other location of the dNTP units. This can be done easily -
by C14 labeling or p32 labeling.
Another labeling approach employs fluorescent
labels. These can be attached to the dNTP's via the 3'OH-
blocking groups or attached in other positions. There are
20 two general routes available using fluorescent tags:
(1) the use of a labeling group that is itself
hs fluorescent and detected either before or after
. deblocking, and
~2) the use of a nonfluorescent labeling group
25 that is detected by its fluorescent interaction with a
nonfluorescent probe or other moiety.
The first route is fairly straightforward and
can employ a range of known fluorophores such as
rhodamines, fluoresceins and the like, typicall~ including
30 those fluorophores known as useful in labelina dNTP's and
the like. One caution however, ic to try to sele-t
fluorcphores which are not so larae and bulky that the
labeled dNTP can not be incorporated readily into the
growina DNA chain by a polymerase or similarly functioning
35 enzyme. The second route can employ a fluorophore where
only a fragment is attac-hed to the dNTP. This can reduce
:
~.... . . - ~ ~ - :
. ~, . ~ ' - ' ~ - - - ' .
.
WO9l/06678 PCT/US90/06178
-27- 2 ~ t~
size and minimize steric interference. In the second
route, rapid reaction of a normally nonfluorescent probe
or molecule with specific functional group(s) found only
on the label fragment leads to the formation of a
fluorescent addition product. Thls leads to a signal only
when the particular labeling group is present.
One system that is applicable to this scheme is
the thiol/maleimide interaction:
O O
11
~ ~\
F--N l RSH~ F--N l
~ \I~ ` SR
- O O
: 15 NONFI:,UORESOENT FLUORESOENT
'-;
~Me2N
Certain N-substituted maleimides which are
normally nonfluorescent react readily with various thiols
to form fluorescent products (Kanaoka, 1977). Blocking
groups or other label fragment groups containing free
thiol functions, such as -COCH2SH, can be used for this
approach. ~lternatively, the blocking group or other
label fragment can contain a metal-binding liaand, e.g. a
: 30 carboxylic acid group which will react with added rare
earth metal ions such as europium or terbium ions to yield
~ a fluorescent species.
-~ While the above-described approaches to labeling
focus on incorporating the label into the 3'-hydroxyl
blocking group, there are a number of alternatives -
particularly the formation of a 3'-blocked dNTP analogue-
:,
.
- , , . . . .. , , - -
:, . - . .. .
W O 91/06678 PC~r/~'S90/06178
~ '
2 ~ 6 -28-
containing a label such as a fluorescent group coupled to
a remote position such as the base. This dNTP can be
incorporated and the fluorescence measured and removed
according to the methods described below.
One method involves the use of a fluorescent tag
attached to the base moiety. The tag may be chemically
- cleaved (either separately from or simultaneously wit.~ the
; deblocking step) and measured either in the reaction zone
before deblocking or in the reaction eluant after
i 10 cleavage. This method is included because a number of
base moiety derivatized dNTP analogues have been reported
to exhibit enzymatic competence. Sarfati et al, (1987)
demonstrates the incorporation of biotinylated dATP in
nick translations, and other biotinylated derivatives such
~- 15 as 5-biotin (l9)-dUTP (Calbiochem) are incorporated by
polymerases and reverse transcriptase. Prober et al.
(1987) show enzyrnatic incorporatic3n of fluorescent ddNTPs
by reverse transcriptase and SequenaseTM
In another type of remote labeling the
fluorescent moiety or other innocuous label can be
attached to the dNTP through a spacer or tether. The
tether can be cleavable if de.sired to release the
, fluorophore or other label on demand. There are several
cleavable tethers that permit removing the fluorescent
group before the next successive nucleotide is added--for
example, silyl ethers are suitable tethers which are
cleavable by base or fluoride, allyl ethers are cleavable
. by Hg(II), or 2,4-dinitrophenylsulfenyls are cleavable by
` thiols or thiosulfate. Cleavages using acidic conditions
are undesirable becauso ~NA is mc!re labile in acid than in
base. Long tethers may bo used Sn that tho large
fluorescent groups are spaced sufficiently far away from
the base and triphosphate moieties and do not interfere
with the binding of the dNTP to the polymerase or with
proper base pairing during complementary chain growth.
.
,, . :; ~ - - :
' '. ' . `
,
- - . - : , , ' - ~ . ,,., ' :
~" . ,,, ,, "~ "~"~"~
WO91/06678 PCT/~IS90/0617X
-29- 2~ 6
Typical tethers are from about 2 to about 20, and
preferably from about 3 to about 10 atoms in length.
The C-8 position of the purine structure
presents an ideal position for attachment of a label.
5 Sarfati et al. (1987) describes a derivatization of
deoxyadenosine at C-8 of the purine to prepare,
- ultimately, an 8-substituted biotin aldylamino dATP. TheSarfati et al. (1987) approach can be used to prepare the
- . appropriate fluorescent, rather than biotinylated,
10 analogues. A number of approaches are possible to produce -~
fluorescent derivatives of thymidine and deoxycytidine.
One quite versatile scheme is based on an approach used by
Prober et al. (1987) to prepare ddNTPs with fluorescent
tags. Structures A, B, C and D below illustrate the type
~ 15 of fluorescent dNTPs that result from these synthetic
- approaches. The synthetic routes have a great flexibility
in that the linker can be varied with respect to length or -
functionality. The terminal fluorescent moiety ~an also
be varied according to need.
; ~
~, -.
., .
~ 25
. ~ .
~ 30
.. . .
~ 35
J
f
-- ' ' . .
,, ~, . _ . . .' ' ' ''
.
' . ', ~: ' . ' ' ' - ':
' ': ' . : . . : ' . ' ' ' : ' . ' ' - ' . . '`
.. , , . : , . , . . ... : , . . . . .
WO 91 /0667X PCI /~1S90/061 7~
20~4~6
.
o=~
10 ~ ~
~ 15~ 0~
~ Z Z7~
~ L O
." ~
;~j 20
~50=~
;. 30
35=?~o~
,x C~ '
~ .
:.'` .
, . . : . ~ : .,
WO91/06678 PCT/~IS90/06t78
-31- 2~
The labels so incorporated in the growing cDNA
chain are detected by conventional analytical methods. In
many cases, particularly with fluorescent labels,
increased detection sensitivity is a major advantage of
the present method. When the fluorescent signal is
detected in sequencing gels, the signal is based on a low
level of fluorophores and is superimposed on a background
of scatter from the gel and glass plates. This decreases
sensitivity and often constrains current methods to the ~-
use of laser illumination to maximize sensitivity (Smith
et al., 1986; Prober et al., 1987; Ansorge et al., 1986).
Detection of fluorophores ls readily achievable in
commercial non-excited spectrofluorometers, such as are
sold by Perkin-Elmer. In these devices, the requirement
for a laser light source is eliminated (although one can
of course be used if desired) allowing use of llght-
emitting diodes (LED) or a conventional xenon arc lamp,
the choice being dictated primarily by the fluorochromes
decided upon and the excitation frequency they require.
Typical LEDs include:
(1) Red LED, emitting at approximately 650 nm -
with a radiance of 40 mw/cm2/steradian;
(2) Green LED, emitting at approximately 540 nm;
' and
(3) Blue LED, emitting at approximately 450 nm.
:` Although fluorescent and radioactive detection
;, methods form the basis of the preferred approaches, other
Ç detection procedures are contemplated. Chemiluminescence
can be used as the detection method. Interaction of
30 specific (cleaved) blocJ~in~ ~roups with immobilized
lumin^l derivatives could also be detected
spectr^electrochemically. .. -
~ In another approach, using mass spectrometric
s detection, the solution containing cleaved blocking groups ~-
i 35 or nucleotides is directly injected into a field
ionization mass spectrometer. Identification of the ~-
- : , , . , . , : . : : . - - .
WO91/06678 PCT/~IS90/06178
2~4d6~6
-32-
particular nucleotide incorporated or cleaved is achieved
by monitoring the relative abundance of molecular ion
peaks corresponding to the specific nucleotides or
blocking groups; for example, four distinct acetyl
blocking groups differing by one mass unlt (replacement of
0 to 3 hydrogens by deuterium) could be detected by
monito ing a small "window.~
Immobilization of Subject DNA.
In the present invention, single stranded
subject DNA or its primer is immobilized. One approach to
this immobilization is to attach the DNA to a solid
substrate. Many of the techniques of modern molecular
biology involve immobilization of DNA onto a solid
support. DNA and RNA are commonly attached noncovalently
through ionic interactions along their length to various
types of membranes (Southern, 1975; Maniatis, Fritsch, an~
, Sambrook, 1982; Chuvpilo and Kravchenko, 1984).
` Similarly, polynucleotides are covalently attached along
`~ 20 their length to membranes (Goldberg, et al., 1979), resins
- (Seed, 1982; Arndt-Jovin, et al., 1975), or plastic
` (Polsky-Cynkin, et al., 1985). These methods may be
employed subject to the caution that this multipoint
attachment may, in some cases, introduce interference with
~5 the subsequent synthesis of the complementary DNA strand.
A single-point covalent attachment of DNA to a solid
polymer or glass support is possible. Such single-point
methods are preferred for immobilizing the subject DNA,
since this leaves the chain free for interactions with the
-~ 3~ polymerase and similar enzymes used herein.
To effect a single point coupling ^f ~NA t^
glass ~r quartz it is often preferred to treat the glass
or quartz to assure an inert bond and prevent loss of the
DNA during the reactions and rinses carried out in the
present method. Pochet et al. (1987) have shown that a
very efficient immobilizatlon of DNA occurred on a
,
,
.
. . . ~ :
- . .. . . .. : . .
~ l/06678 PCT/US90/0517~
-33- 2 Q ~
silanized glass surface. Therefore, the inner quartz or
glass surface can be advantageously functionalized using
: silanizing reagents such as triethoxysilylpropylamine or
dichlorodimethylsilane. This is followed by covalent
attachment of a long-chain alkylamine to these
functionalizing groups. The single stranded subject DNA
is attached to the long chain amine. The attached single
stranded DNA then serves as the template for the formation
of the complementary chain.
In another embodiment, immobilization is carried
out bv attaching the subject DNA to a plastic surface. A
thin polypropylene chamber wall designed to pass Cerenko~ -~
radiation from 32p, for example, can serve as a suitable
- substrate for DNA immobilization. With a plastic surface,
it is preferable-to use the method of Kremsky et al.
~1987), wherein the surface is coated with streptavidin,
to which an alkylbiotinylated oligonucleotide will bind.
The immobilized oligonucleotide is annealed to the
template DNA as a primer.
-~ 20 In addition to retaining the subject single
strand DNA by means of immobilizing it to a surface, the
~` subject DNA can also be entrapped by the use of membranes
which retain it. In this embodiment, the reaction zone
, has one or more openings covered with a membrane such as
an ultrafiltration membrane, for example, Amicon's PM-5 or
PM-10 membranes which have nominal molecular weight cut
offs of 5000 and 10,000 respectively. That is, they are
capable of passing materials having molecular weights of
less than 5,000 and 1~,0~0 respoctivel)~ while rotaining
3n materials above these sizes. Other ultrafiltration or
dialysis membranes such as thoso marketed b~ Do~ or Abcor
can also be used. In this embodiment, the sin~lo ctranded
DNA is suspended in liquid in the reaction zone. The
labeled and unlabeled dNTPs and other coupling reaaents
are flowed into the zone. Materials are removed from the
zone through such a filter which retains the DNA chalns. -
, . .
,. . ~ .
W O 91/06678 PC~r/~S90/0617X 2 0 ~
-34-
In this method, the polymerase or other enzyme which is
used to effect coupling is generally of a size to be
retained by the membrane. This scheme works for chemical
but not enzymatic deblocking, since in enzymatic
deblocking the polymerase and phosphatase must be cycled
separately through the cell.
In an alternative embodiment the DNA can be
immobilized on particles of resin or polymer microspheres
and these particles retained within the chamber. In this
embodiment, the filter material is unimportant as long as
the DNA is attached to resin particles which are of a size
that cannot penetrate the filter pores. There are several
methods that couple DNA to resins through the 5' terminus
(Pochet, 1987; Polysky-Cynkin, 1985). For example,
oligonucleotides or polynucleotides are linked through
their 5~ end to cellulose (Gilham, 1968; Clerici et al.
; 1979), ~ephacryl (Langdale and Malcolm, 1985), or latex
microspheres (Kremsky et al., 1987). In these methods,
the D~A is available for interactions with other nucleic
acids or proteins. Of particular interest for our
application is the method of Goldkorn and Prockop (1986)
- for covalent coupling of DNA to oligo(dT)-cellulose.
Alternatively, t.he DNA is coupled covalently to
streptavidin-agarose beads by an alkylbiotinylated
. 25 oligonucleotide (Kremsky et al., 1987).
In yet another embodiment, the single-stranded
DNA is coupled to DBM paper such as a filter in the
presence of a protecting strand. After cou~ling, the
protecting strand is released, leavin? the immobilized
templa~e and priming site free for successi~e enzymatic
reactions (Hansen et al., 19~7). This method and the
other single-point methods described above are useful for
immobilizing DNA while leaving it free for interactions
with enzy~mes used in DN~ sequencina-
. :: - .. : . . . . - .. . . . . -
:-- : : -
- .
WO 91 /06678 PCI /~IS90/061 7X
20~a~6
-35-
: .
Examples
~xample l
Synthesis of 3~-PO ~32Pl Thymidine Triphosphate:
To a stirred solution of phosphorus trichloride
(32p) (75 mmole) and N-methyl morpholine (750 n~ole,
- Aldrich) in 750 ml dry methylene chloride (CH2Cl2) is -
added 1,2,4-triazole (250 mmole) at room temperature. The
` reaction mixture is stirred one hour, cooled to 0C and 15
mmole of 5~-dimethoxytrityl thymidine I (Sigma) in 200 ml
of anhydrous acetonitrile is added dropwise over 30
minutes. (See Reaction Scheme 3 given in Figure 7). The
solution is stirred an additional 30 minutes, and poured
into 600 ml of lM triethylammonium bicarbonate (TEAB, pH,
15 8.5). The organic layer is separated and the aqueous ~ -
layer washed with 2 x 200 ml CH2Cl2 The combined CH2C12
extracts are dried over magnesium sulfate (MgSO4),
filtered and evaporated to dryness under vacuum at room
temperature. The crude 5'-dimethoxytrityl-3'thymidine -
H-phosphonate II is then treated with 2~ benzenesulfonic
acid in CH2Cl2:methanol (MeOH) (7:3) (200 ml) for one
hour. The solution is washed with 10% sodium bicarbonate
(NaHCO3) and water, dried over magnesium sulfate and
evaporated to dryness. The crude 3~-thymidine- -
~ 25 H-phosphonate III is recrystallized 'crom ethanol/ether.
-~ To a solution of l ml Orc phosphorus
oxytrichloride (POC13) in 30 ml of triethylphosphate at
-s 0C is added 10 mmole of the 3'-thymidine H-phosphonate.
The mixtu.re is stirred fcr l~ hours at 4C, neutralized
~ 30 with ~aHCO3 solution, and addecl to 15Q ml water. The
- aqueous sclution is washed with l-~enzenQ (2 v lQ0 ml) and
ether !2 x 100 ml), and diluted to n . 8 liters with water
and charged on a 2.5 v~ 5~ cm ~olumn of DEAE-cellulose.
The products ar~ eluted usincl a linear gradient of pH 8.5
~ 35 ammonium bicarbonate solution (0.05 to 0.25 M). The
;~ fractions collected are analyzed by HPLC to determine the :.
:
. .
WO91/06678 PCT/~'S90/06178
204~6~ 6 ~
. .
-36-
desired product-containing fractions, and these are
evaporated to dryness under vacuum. The residue is
repeatedly re-evaporated with water to remove salts.
The 5'-monophosphate IV (16 mmole) is then
dissolved in 30 ml of dimethylformamide (DMF) and treated
` with ~,N~-carbonyldiimidazole (30 mmole) at room
temperature for one hour. The reaction is quenched by
addition of 5 ml methanol, and 60 ml of a O.SM solu~ion of
bis(tri-n-butyl-ammonium) pyrophosphate in DMF is added
dropwise over 10 minutes. After stirring for 24 hours,
the solution is diluted with water to 1 liter and treated
with 100 ml of a solution of 0.1 M iodine (I2) in 5~
pyridine/water. After one hour, the solution is deposited
: on a DEAE-cellulose column from Sigma (Sx50cm) or
'. 15 Sephradex from Pharmacia. The column is washed with water
and eluted with triethylammonium bicarbonate solution
0.05 to 0.5M). The 5'-triphosphate-3'-phosphate
thymidine product V is obtained by evaporation of the
appropriate fractions collected.
-} 20
Example 2
~ Synthesis of 3'-labelled (fluorescent)
.~ thymidine t~phos~hate
~ A solution of 5-dimethoxytrityl thymidine I (2.5
25 mmole) in 10 ml dry pyridine is treated with succinic
.~ anhydride (8 mmole) at 4C for 24 hours. Cold water
(150ml) is addedj and after 30 minutes the solution is
~' filtered. The washed, dried, precipitate is taken up in
30 ml CH2C12, extracted with water (2 x 25ml), dried over
30 MgSO~ and evaporated to drynes~ ee Reaction Scheme 4
r shown in Figure 8.)
1' The 5'-dimethoxytrityl-thymidine 3'-succinate VI
(2mmolo) is dissolved in 15 ml dry CHqCl , cooled to 0C
and troated with a fivefold eXcesC of N,N'-dicyclohexyl-
35 carbodiimide and N-hydroxybenzotriazole. After one hour,
an equivalent amount of the fluorescent labeling group
,. .
: .
-
, .. , ... . . ~ . . . ~ . - - . - - - . .. . -
-: . .. ,: . : ... , . , , : - ., ., -
WO 91 /06678 PCl /~'S90/061 7~S
'.~
2 O L~ 6
containing a pendant amino function, dansylcadaverine, is
added and the solution stirred for 8 hours at 10C. The
solution is then washed with water (2 x 10 ml). The
CH2C12 layer is dried over MgSO4 and evaporated to
S dryness to yield the product VII. Removal of the
dimethoxytrityl protecting group and conversion to the
5'triphosphate VIII is accomplished in the same manner as
described for the 3'-phosphate thymidine triphosphate ~'.
;- This reaction is carried out in similar fashion
using the other three nucleosides to give the
corresponding labeled materials.
Example 3
Quartz Surface Immobilization of Subject DNA . .
lS Four 25 microliter volume quartz cuvette
reaction chambers are prepared. These chambers are
configured like chamber 32 in Figure 3 with the exception
that t'ey use their inner walls as the surface to which
the D~A is affixed.
The inner surfaces are cleaned and dried.
.:~ Triethoxysilylpropylamine (5 microliter in 20 microliter
CHC13) is added and held at 5C for 120 minutes under
anhydrous conditions. This couples the
triethoxysilylpropylamine to the surface and gives an
amine character to the surface.
The subject DNA is then attached to the amine
. surface. This is carried out by first attaching a long
chain alkyl amine (n-octylamine) to the base at the S' end
of the subject DNA molecule Cl to the base at the 5~ end
~; 30 of a suitable primer, such as an Ml'~ primer for example
the l~-mer dGTAAAACGACGGCCAGT, and then joining the
al~ylamine to the aminopropylsilane surface groups by
reaction with glutaraldehyde (1.5 e~uivalents, 25CC, 120
minutes). Other functional groups pendant to the base :.
moiety or attached to the S' position can also be used
[for example: aldehydes or carboxylic acids (Kremsky et
. :
.
h'
WO91/06678 PCT/~IS90/0617
` 2 0 ~46 16 -3a-
al)] for covalent immobilization on derivatized quartz or
glass surfaces.
Example 4
Incorporation of Labeled Nucleotide Analoas into DNA
The 25 microliter reaction zones are charged
with a reaction mixture which contains three Units of
SequenaseTM enzyme. The reaction mixture also contains an
appropriate buffer for this enzyme (20 mM Tris-~Cl pH 7.5,
10 mM MgC12 25 mM NaCl, 0.01 M dithiothreitol), the
single-stranded primed subject DNA is present at a
concentration of approximately 0.1 M attached to the
surface of the reaction chamber at its 5~ end, (see
Example 3), three unlabeled, 3'-blocked deoxynucleotide
" 15 triphosphate (dNTP) analogs at a concentration of 1.5
micromolar each, and one 3'-blocked, fluorescently labeled
; dNTP analog of Example 2 at a concentration of 30
micromolar are each present in each of the four reaction
~ zones. In each zone a different one of the four dNTPs is
;~ 20 labeled. The reaction proceeds at room temperature for
- one minute. Then the reaction zones are drained and
',.f~ rinsed with buffers.
,q
` In one embodiment the identity of the added dNTP
r~ is determined by-exciting the fluorophores present in the
one cuvette which incorporated its fluorescently-labeled
. dNTP. Alternatively, the fluorescent group is removed
before measurement.
.',, ' .
E.YamrlQ 5
Chemical ~eblocl.i.n~
The 2,4-dinitrobenzenesulfen~rl fluorescent
blockina groups are removed with a deblocking reagent
which consists of 0.1 M pyridine/pyridinium chloride
buffer (pH 7.8) containing thiourea 0.05 M. The
debloc.king reaction is allowed to proceed for one minute
at 40C. The reaction chamber is then drained and washed
'
- - - ..... : . . . . . .
1/06678 PCT/US90/06178
-39- 2 ~L~
twice with 100 mM Tris-HCl buffer, pH 6.5. The release of
the fluorescent blocking group is measured in the initial
eluate from the reaction chamber using a flow-through
cell. Depending on the cell in which the fluorescent
group is present, the identity of the nucleotide which has
been added to the DNA chain is determined. Similarly, if
the blocking group were a dansylcadaverine type ester such
as in reaction scheme 4, it could be removed by treatment
with 50~ methanol/50~ water pH lO.0 for one minute.
Example 6
Enzymatic Deblockinq
The blocking group can also be removed
enzymatically.
For enzymatic deblocking, the deblocker fed into
the reaction chamber contains lO0 mM Tris-HCl (pH 6.5) lO
mM MgCl2, 5 mM 2-mercaptoethanol, and one Vnit T4
polynucleotide kinase. The reaction proceeds for one :
minute at a temperature of 37C. The 3'-phosphatase
activity of T4 polynucleotide kinase converts 3'-phosphate
termini to 3'-hydroxyl termini which then serve as primers
for further synthesis. .
While in these examples, the invention has been ...
shown as practiced in a manual manner with each step being
carried out sequentially, it can readily be appreciated
. that this process can be easily automated. A simple clock
mechanism or microprocessor driven timer circuit can be
! used to actuate a plurality of electrically controlled
valves in sequence to add the various reaaents for addiny
buildina blocks, deblocking and the like with the result
that the sequence of the taraet DNA single strand can be
obtained with minimum invnlvement of lab personnel.
hile only a few embodiments of the invention
have been shown and described herein, it will become
- 35 apparent to those skilled in the art that various
modifications and changes can be made in the present
,
-
-. - ,
WO91/06678 PCT/~'S90/06178 ~
20 44~6 -40-
invention to methods to determine the sequence of
.. deoxyribonucleotides in a deoxyribonucleotide chain (DNA)
without the use of a sequencing gel without departing from
the spirit and scope of the present invention.
'
:
~' 20
:` ' ` ~'
.:' 25
~t
' .
:
-
