CA2044616A1 - Dna sequencing - Google PatentsDna sequencing
- Publication number
- CA2044616A1 CA2044616A1 CA 2044616 CA2044616A CA2044616A1 CA 2044616 A1 CA2044616 A1 CA 2044616A1 CA 2044616 CA2044616 CA 2044616 CA 2044616 A CA2044616 A CA 2044616A CA 2044616 A1 CA2044616 A1 CA 2044616A1
- Prior art keywords
- dna molecule
- complementary dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
nucleotide sequence without the use of gel electrophoresis.
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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 ' ~_ . ~ . ~ . . . " . . .
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' ' `2o~l46J'6 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). ~:
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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.
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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)~ ~;
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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~~
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.
J.F.M. De Rooiz, et al., Journal of ~^ Chromotoqraphy, Vol. 177, p. 380-384 (1987).
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 :.
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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 ~!
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~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
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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, - .
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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'.
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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
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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 ,.:
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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.
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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 '," ~
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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 ~
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.
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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~ ~
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 ~
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 ' ',:
..... . .. . . . .
": 204~61~ ~ ~
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 : - .
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 ~, 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 -, . ' ." ' ': ` : ' , ' . , ~
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 ! (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 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~ ~ ~
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
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:
F--N l RSH~ F--N l ~ \I~ ` SR
- O O
: 15 NONFI:,UORESOENT FLUORESOENT
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.
,, . :; ~ - - :
' '. ' . `
, - - . - : , , ' - ~ . ,,., ' :
~" . ,,, ,, "~ "~"~"~
-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~
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 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 ~
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 : .
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 ~
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 ' - - - ..... : . . . . . .
-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 ' .
synthesizing, in the presence of the subject DNA
molecule, the complementary DNA molecule, the synthesizing being carried out in a stepwise serial manner in which the identity of each deoxynucleotide triphosphate incorporated into the complementary DNA molecule is determined subsequent to its incorporation.
position of the complementary DNA molecule.
position of the complementary DNA molecule.
position of the complementary DNA molecule.
position of the complementary DNA molecule.
(a) synthesizing, in the presence of the subject DNA molecule, the complementary DNA molecule, the synthesizing being carried out in a stepwise serial manner in which the identity of each deoxynucleotide triphosphate incorporated into the complementary DNA molecule is determined subsequent to its incorporation;
(b) translating the identity of each deoxynucleotide triphosphate incorporated into the complementary molecule to the identity of its corresponding complement present in the subject molecule;
and (c) tabulating the identities of the corresponding complements thereby giving rise to the deoxyribonucleotide sequence of the subject DNA.
(a) synthesizing, in the presence of the subject DNA molecule an initial region of the complementary DNA
molecule, the synthesizing being carried out in a stepwise serial manner in which the identity of each deoxyribonucleotide triphosphate incorporated into the complementary DNA molecule is determined subsequent to its incorporation;
(b) tabulating the identities of the deoxyribonucleotides incorporated into the initial region of the complementary DNA molecule;
(c) removing the initial region of the complementary DNA molecule for the subject single stranded DNA molecule;
(d) separately synthesizing a DNA primer molecule corresponding in sequence to at least a part of the initial region of the complementary DNA molecule;
(e) annealing the DNA primer molecule to the subject single stranded DNA molecule;
(f) synthesizing, from the DNA primer molecule the next region of the complementary DNA molecule;
(g) tabulating the identities of the deoxyribonucleotides incorporated into the next region of the complementary DNA molecule; and (h) repeating steps c, d, e, f and g as needed to determine the entire structure of the subject single stranded DNA molecule.
Priority Applications (2)
|Application Number||Priority Date||Filing Date||Title|
|Publication Number||Publication Date|
|CA2044616A1 true CA2044616A1 (en)||1991-04-27|
Family Applications (1)
|Application Number||Title||Priority Date||Filing Date|
|CA 2044616 Abandoned CA2044616A1 (en)||1989-10-26||1990-10-26||Dna sequencing|
Country Status (3)
|EP (1)||EP0450060A1 (en)|
|CA (1)||CA2044616A1 (en)|
|WO (1)||WO1991006678A1 (en)|
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