WO 98/33939
`
` PCT/JP97/00239
`
`World Intellectual Property Organization
`International Office
`International Application Laid Open [WIPO seal]
`According to Patent Cooperation Treaty
`(51) Int’l Patent Classification 6
`(11) Int’l KOKAI No.
`C12Q 1/68, C12M 1/00, G01N 33/50,
`C12N 15/10
`(21) Int’l Application No.
`
` WO98/33939
`
`(43) Int’l KOKAI Date
` August 6,1998
`(81) Designated countries: CN, JP, KR, US, European
`Patent (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT,
`LU, MC, NL, PT, SE).
`
`Attached KOKAI documents
` International Search Report
`
`PCT
`
`A1
`
` PCT/JP97/00239
`
`(22) Int’l Application Date
` January 31, 1997
`(71) Applicant (all countries except the United States)
`Hitachi, Ltd. [JP/JP] 4-6 Kanda Surugadai, Chiyoda-ku,
`Tokyo 101 (JP)
`(72) Inventor; and
`(75) Inventor/Applicant (the United States only)
`ANAZAWA, Takeshi [JP/JP]
`UEMATSU, Chihiro [JP/JP]
`3-8-1 Koigakubo, Kokubunji-shi, Tokyo 185 (JP)
`OKANO, Kazunori [JP/JP]
`5-17-2-402 Honcho, Shiki-shi, Saitama-ken 353 (JP)
`KAMBARA, Hideki [JP/JP]
`1-4-3 Kitanodai, Hachioji-shi, Tokyo 192 (JP)
`(74) Agent
`OGAWA, Katsuo, patent attorney
`c/o Hitachi, Ltd., Tokyo (JP)
`1-5-1 Marunouchi, Chiyoda-ku, Tokyo 100
`
`(54) Title of Invention: Method for Determining Nucleic Acid Base Sequences and Apparatus for Determining Nucleic
`Acid Base Sequences
`
`Illumina, Inc.
`Exhibit No. 1011
`
`Page 1
`
`Illumina Ex. 1073
`IPR Petition - USP 10,435,742
`
`

`

`WO 98/33939 PCT/JP97/00239
`
`
`(57) Abstract
`
`
`
` A single molecule of single-stranded sample DNA (7) having a bead (5) at one end
`thereof and a magnetic bead (6) at the other end thereof is elongated and fixed in the
`field of view of a fluorescence microscope by a magnetic force (11) and a laser trap (3),
`a primer (8) is bound thereto, and an elongation reaction (10) is conducted with
`polymerase. Only a single chemically modified nucleotide (9) that is labeled with a
`fluorophore that is different for each base type is incorporated. Only the single
`fluorophore incorporated is measured, by evanescent irradiation from an ultraviolet
`laser (2), as a fluorescence microscopic image, and the base type is determined from the
`fluorophore type. By the evanescent irradiation (13) from the excitation laser (1), the
`fluorophore that labels the incorporated nucleotide is released, and the next nucleotide
`is incorporated. DNA base sequences are determined by repeating this process. Base
`sequence determination can be performed using a single DNA molecule, and base
`sequences can be efficiently determined for DNA having several hundreds of thousands
`of bases or more.
`------------------------------------------------------------------------------------------------------------------------------
`
`Codes used for identifying PCT member nations listed on page 1 of the pamphlet for the
`international application laid open on the basis of the PCT (reference information)
`AL Albania
`DK Denmark
`KZ Kazakhstan
`PT Portugal
`AM Armenia
`EE Estonia
`LC Saint Lucia
`RO Romania
`AT Austria
`ES Spain
`LI Liechtenstein
`RU Russian Federation
`AU Australia
`FI Finland
`LK Sri Lanka
`SD Sudan
`AZ Azerbaijan
`FR France
`LR Liberia
`SE Sweden
`BA Bosnia and
`GA Gabon
`LS Lesotho
`SG Singapore
`Herzegovina
`GB United Kingdom
`LT Lithuania
`SI Slovenia
`BB Barbados
`GE Georgia
`LU Luxembourg
`SK Slovakia
`BE Belgium
`GH Ghana
`LV Latvia
`SL Sierra Leone
`BF Burkina Faso
`GM Gambia
`MC Monaco
`SN Senegal
`BG Bulgaria
`GN Guinea
`MD Republic of
`SZ Swaziland
`BJ Benin
`GW Guinea-Bissau
`Moldova
`TD Chad
`BR Brazil
`GR Greece
`MG Madagascar
`TG Togo
`BY Belarus
`HU Hungary
`MK The former
`TJ Tajikistan
`CA Canada
`ID Indonesia
`Yugoslav Republic
`TM Turkmenistan
`CF Central African
`IE Ireland
`of Macedonia
`TR Turkey
`Republic
`IL Israel
`ML Mali
`TT Trinidad and
`CG Congo
`IS Iceland
`MN Mongolia
`Tobago
`CH Switzerland
`IT Italy
`MR Mauritania
`UA Ukraine
`CI Côte d'Ivoire
`JP Japan
`MW Malawi
`UG Uganda
`CM Cameroon
`KE Kenya
`MX Mexico
`US United States of
`CN China
`KG Kyrgyzstan
`NE Niger
`America
`CU Cuba
`KP Democratic
`NL Netherlands
`UZ Uzbekistan
`CY Cyprus
`People’s Republic
`NO Norway
`VN Viet Nam
`CZ Czech Republic
`of Korea
`NZ New Zealand
`YU Yugoslavia
`DE Germany
`KR Republic of Korea
`PL Poland
`ZW Zimbabwe
`
`
`
`
`Exhibit No. 1011
`Page 2
`
`Page 2
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`WO 98/33939 PCT/JP97/00239
`1
`
`Specification
`
`Method for Determining Nucleic Acid Base Sequences and Apparatus for Determining
`Nucleic Acid Base Sequences
`
`Technical Field
`
`This invention relates to an apparatus for analyzing DNA and RNA and the like, and
`more particularly to an apparatus that is effective for determining DNA and RNA base
`sequences or, alternatively, analyzing limited enzyme fragments or specific fragments.
`
`Technical Background
`
`Techniques for analyzing DNA, RNA, and the like are important in the fields of
`
`medicine and biology, inclusive of gene analysis and genetic diagnosis. The determination
`or DNA and RNA base sequences, and the analysis of limited enzyme fragments and
`specific fragments, are both based on separation by molecular weight by electrophoresis. A
`fragment or fragment group is subjected beforehand to radioactive labeling or fluorescent
`labeling, and, after conducting electrophoresis, or during electrophoresis, analysis is
`effected by measuring molecular weight-separation development patterns. Recently, in
`connection with genome analysis, the demand for DNA base sequence determining
`apparatuses, in particular, has grown, and the development of such devices is on-going.
`DNA base sequence determination using fluorescent labeling is now described. Prior to
`electrophoretic separation, a dideoxy reaction is carried out by the Sanger method. A
`nucleotide having a length of approximately 20 bases that is complementary to a known
`portion of the base sequence of the sample DNA to be analyzed is synthesized and labeled
`with a fluorophore [lit. a fluorophore is labeled]. This oligonucleotide is complementary-
`chain bound to approximately 10í12 mol of the sample DNA as a primer, and a
`complementary chain elongation reaction is conducted using polymerase. At this time, four
`deoxynucleotide triphosphates are added as substrates, namely deoxyadenosine
`triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate
`(dGTP), deoxythymidine (dTTP), and, in addition thereto, dideoxyadenosie triphosphate
`(ddATP). When ddATP is incorporated by complementary-chain elongation, because the
`complementary chain is not elongated beyond that, fragments of various lengths and
`terminated by adenine (A) are prepared. Reactions are conducted independently wherein,
`instead of ddATP in the reaction noted above, dideoxycytidine triphosphate (ddCTP),
`dideoxyguanosine triphosphate (ddGTP), and dideoxyguanosine triphosphate (ddTTP),
`respectively, are added. However, while the primers used in each reaction have the same
`base sequence, four types of fluorophores, which can be mutually distinguished by
`separating fluorescence, are used for labeling [lit. are labeled].
` When the four types of reactant noted above are mixed, fragments complementary to
`the sample DNA, having a length of up to approximately 1000 bases, and with lengths
`differing one base at a time, are prepared, with four types of fluorophores being used in
`labeling [lit. labeled], according to the terminal base type. The number of fragments of
`each base length, respectively, is approximately 10í15 mol. Next, the samples prepared are
`
`Exhibit No. 1011
`Page 3
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`WO 98/33939 PCT/JP97/00239
`2
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`separated with a resolving power of 1 base by electrophoresis. In electrophoresis, wide use
`is made of a slab gel wherein acrylamide is polymerized between two glass plates separated
`by an interval of approximately 0.3 mm. When the sample is injected at the upper end of
`the slab gel, and an electric field is applied to the upper and lower ends of the slab gel, the
`sample migrates toward the lower end while separating. When a position approximately 30
`cm from the upper end is irradiated by a laser while conducting electrophoresis, separated
`fluorescently labeled fragments are excited as they pass the position of laser irradiation, in
`order from the shorter to the longer. When the emitted fluorescence is measured while
`being spectrally separated with the use of multiple filters, the terminal base types of all of
`the fragments can be determined, in order, from the shorter fragments to the longer, based
`on the change over time in the fluorescent intensity of the four types of fluorophore. With
`the order of these base types being in a complementary relationship with the sample DNA,
`the base sequence of the sample DNA can be determined.
` A number of new DNA base sequence determination methods have been proposed
`which do not employ electrophoresis. With a first prior art, when a complementary-chain
`elongation reaction is conducted by polymerase using the sample DNA as a template, four
`types of base are added, in order, one at a time, and the base quantity incorporated into the
`complementary chain at each step is quantified by photoabsorption or fluorescence,
`whereupon the base sequence of the sample DNA is determined (TOKKAI [Unexamined
`Patent Application] No. H4-505251/1992, gazette). With a second prior art, using the
`sample DNA as a template, and employing four types of base labeled mutually differently,
`a complementary-chain elongation reaction is conducted by polymerase, after which one
`base at a time is released from the 3ƍ end of the complementary chain synthesized by
`exonuclease, and the labels of the released bases are measured, in order, to determine the
`base sequence of the sample DNA (Journal of Biomolecular Structure & Dynamics 7, 301 –
`309 (1989)). With a third prior art, the base sequence of sample DNA is determined by
`repeating a cycle of steps, namely a step for conducting a DNA polymerase reaction using
`four types of dNTP derivative (MdNTP) having labels that can be detected, and that,
`incorporated into a template DNA as DNA polymerase substrate, can stop a DNA chain
`elongation reaction by the presence of a protective group, a step for then detecting the
`MdNTP incorporated, and a step for returning the MdNTP to an elongatable state. With this
`prior art, DNA chain elongation is stopped at the point in time when one base is elongated,
`oxygen and the substrate are removed from the system (solution) wherein the template,
`primer, and MdNTP are present, the MdNTP incorporated is detected, the protective group
`(and label) of the MdNTP incorporated into the template are released, and a condition is
`induced wherein DNA chain elongation is possible (Pat. Applic. No. H2-57978/1990).
`However, these proposals are at present in the idea stage, and there are no reports of them
`having been made practicable.
`
`Disclosure of Invention
`
` Currently, insofar as practical methods of determining DNA base sequences are
`concerned, separation by molecular weight is conducted by electrophoresis. For DNA base
`sequence determination, a resolving power of 1 base length is required for separation by
`
`Exhibit No. 1011
`Page 4
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`WO 98/33939 PCT/JP97/00239
`3
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`molecular weight. The resolving power of electrophoresis ordinarily declines as the lengths
`of the bases to be separated become longer. That is, even though one base length can be
`separated between a base length of 50 bases and one of 51 bases, that does not mean that
`one base length can be separated between a base length of 500 bases and one of 501 bases.
`How long of a base length can be resolved by 1 base length, that is, the limit of base length
`separation, is determined by the electrophoresis conditions, that is, the composition of the
`separation medium, the electrophoresis path length, and the electric field intensity. In order
`to increase the limit of base length separation, various optimizations have been
`implemented to date, but there are no reports of a limit exceeding a length of 1000 bases
`(Electrophoresis 13, 495 – 499 (1992), Electrophoresis 13, 616 – 619 (1992)). The
`maximum of 1000 bases for the limit of base length separation has been theoretically
`explained (Electrophoresis 13, 574 – 582 (1992)). That is, insofar as electrophoresis is
`employed, the maximum length at which the base sequence can be determined from one
`sample DNA is a length of 1000 bases. With large-scale base sequence determination
`typified by genome analysis, on the other hand, the limit of base length separation holds the
`key to analytical efficiency (Science 254, 59 – 67 (1991)). The YAC clone, which is a
`typical large clone, for example, has a base length of approximately 1M bases, wherefore, a
`minimum of 1000 samples must be analyzed in order to analyze each base length of 1000
`bases. However, it is not possible to prepare fragments one by one having a base length of
`1000 bases, in order, from the end of a long DNA, without either excess or insufficiency. In
`practice, random fragments are prepared, these are randomly analyzed, and the base
`sequence of the original long DNA is reconfigured using overlaps in the base sequences of
`the fragments. This method, called the shotgun method, is the most widely used method
`today in genome analysis. However, in order to impart overlaps to random fragments so as
`to enable reconfiguration, it is necessary to repeatedly analyze the same base sequence,
`over and over, which is a problem.
`
`The degree of this overlapping, which is called redundancy, increases as the base length
`wherewith one-time base sequence determination is possible becomes smaller, that is, as
`the limit of base length separation in electrophoresis becomes smaller. For a limit of base
`length separation of 1000 bases, a redundancy of approximately 10 is required. That is, it is
`necessary to conduct base sequence determination for a length that is 10 times that of the
`DNA for which the base sequence is to be determined. As a consequence, one must analyze
`approximately 10,000 samples in order to determine the base sequence for a base length of
`1M bases. The number of samples that can be analyzed in one day by one DNA base
`sequence determination device is 100 at most. To determine the base sequence of the entire
`base length of 1M bases with one apparatus would require 100 days. In order to practically
`conduct genome analysis, or the genetic diagnostics the importance whereof will increase
`in future, requires art whereby base sequences 1M bases long can be determined in a few
`days.
` Another problem with the prior art is the necessity therewith of having many DNA
`samples in order to determine base sequences. In order to conduct the dideoxy reactions
`with the Sanger method, a DNA sample of approximately 10í12 mol is ordinarily needed for
`one sample. For that reason, it is necessary to refine and amplify sample DNA beforehand,
`using techniques such as cloning or PCR. Such procedures require time and effort, and
`
`Exhibit No. 1011
`Page 5
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`WO 98/33939 PCT/JP97/00239
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`constitute a large rate-limiting process which impedes the progress of genome analysis. In
`order to advance genome analysis and genetic diagnostics efficiently, art is wanted
`wherewith base sequences can be determined from a small quantity of sample DNA, and,
`ultimately, even from a single DNA molecule. The DNA base sequence determination
`methods proposed thus far which do not use electrophoresis are, however, subject to the
`following shortcomings.
`
`In the first prior art example, four types of nucleotide substrate are added, in order, one
`at a time, making it necessary to repeatedly alter the composition of the solution wherein
`the complementary-chain elongation reaction is being conducted. When changing the base
`type in a solution, moreover, cleaning is necessary in order to prevent the admixture of
`another type of base. As a consequence, at least eight solution replacements are required for
`every cycle. Because, ordinarily, one base is determined each cycle, on average, eight
`solution replacements will considerably reduce the speed of base sequence determination.
`The most fundamental problem with the first prior art is that the quantity of signals to be
`quantified accumulates as the complementary chain lengthens, making it extremely difficult
`to measure the changes in signal quantity corresponding to the incorporation of one base. In
`other words, determining the base sequences of DNA having long base length is very
`difficult.
` With the second prior art, four types of base labeled with four types of fluorophore are
`incorporated into the complementary chain, but this is technically very difficult. As will be
`discussed subsequently, because the fluorescence quantity is large, or because the
`molecular size of the fluorophores that can mutually be distinguished by fluorescent
`spectral separation, is large, it is very difficult, due to steric hindrance, for a plurality of
`fluorescently labeled nucleotides to be incorporated next to each other in the same
`complementary chain. Another problem with the second prior art is that it is very difficult
`to control the release of one base at a time using exonuclease. When a plurality of bases has
`been released continuously within a range equal to or less than the detection limitation time,
`the released bases are all detected simultaneously, so the order of base release, which is to
`say the base sequence, cannot be determined. In order to determine the base sequence, it is
`necessary for the release of each base to occur at some interval of time, and not
`continuously, but effecting such control is extremely difficult.
` With the third prior art example, it is necessary, in the step for detecting the MdNTP
`incorporated into the template, and in the step for returning the MdNTP to an elongatable
`condition, to remove MdNTP not incorporated into the template from the solution, and also
`to add MdNTP into the solution at the point in time when the next, new, cycle starts. When
`this is neglected, the signals from the MdNTP incorporated into the template and the
`signals from the MdNTP not incorporated into the template become mixed, whereupon the
`signals from the MdNTP incorporated into the template targeted cannot be accurately
`measured. Furthermore, when the protective groups of MdNTP not incorporated into the
`template are also released, and dNTP from which the protective group has been released is
`incorporated into the template, the prescribed cycle ceases to advance. As a consequence, a
`minimum of two solution content changes will be necessary for each cycle, which causes
`the speed of base sequence determination to be retarded. Thus, when the change in solution
`content is incomplete, and the slightest amount of MdNTP not incorporated into the
`
`Exhibit No. 1011
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`WO 98/33939 PCT/JP97/00239
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`template remains in the solution, that causes a lot of noise when conducting measurements,
`which is a problem.
` An object of the present invention is to provide a method and an apparatus for
`determining the base sequences of very long DNA [chains] by determining the types of
`bases of nucleotides incorporated when conducting complementary-chain elongation
`reactions by polymerase, in order, one at a time, in order to resolve the problems with the
`DNA base sequence determination methods proposed to date which do not employ
`electrophoresis.
` With the present invention, a single template DNA molecule (sample) is held within the
`field of view of a fluorescence microscope, complementary-chain elongation is conducted,
`under control, one base at a time, and each fluorescently labeled base incorporated is
`subjected to single molecular measurement. In performing DNA base sequencing, it is only
`necessary to monitor, one by one, the types of bases of nucleotides incorporated in the
`complementary-chain elongation reaction by polymerase. There are two conditions, namely
`(1) that the elongation reaction not advance after one nucleotide has been incorporated, and
`(2), that, after determining the base type of the one nucleotide incorporated, incorporation
`of the next single nucleotide be made possible, that all nucleotides to be incorporated must
`be chemically modified so as to simultaneously satisfy. These two conditions can be
`achieved by combining nucleotides, on the one hand, and caged compounds having
`fluorophores as labels, on the other, for example. Caged compounds are compounds that
`mask residues contributing to the activity of physiologically active substances by the
`nitrobenzyl group or the like, and cause the dissociation of modifying groups by photo-
`irradiation. For example, a caged compound is a substance wherein the 2-nitrobenzyl group
`has been introduced, which 2-nitrobenzyl group is released by UV irradiation. This
`procedure is widely used in the field of biology (Annu. Rev. Biophys. Biophys. Chem. 18,
`239 – 270 (1989)). Many different kinds of caged compound are sold by companies such as
`Molecular Probes, Inc. The chemically modified nucleotides used in the present invention,
`as diagrammed in Fig. 1, are caged compounds wherein a 2-nitrobenzyl group is bonded to
`a physiologically active substrate X (a nucleotide), and have the capability to suppress the
`inherent activity of chemically modified nucleotides, that is, of the activity of being
`continually incorporated by complementary chain synthesis reactions, the ability to release
`the caged substance (2-nitrobenzyl group) when subjected to UV irradiation at 360 nm or
`below, and to convert [the chemically modified nucleotides] to a substrate X or HX
`exhibiting inherent physiological activity. In Fig. 1, R is H or an alkyl group (such as CH3)
`or the like.
`
`The method of manufacturing Texas Red-labeled caged dGTP, as an example of a
`chemically modified nucleotide having the capabilities noted above, is diagrammed in Figs.
`3 to 7. In Fig. 3, water-soluble carbodimide (HOOC (CH2)2 COOH) is reacted with a
`derivative of dGTP (Fig. 2) that is a starting substance when chemically modifying a base
`(Science 238, 336 – 341, 1987), to obtain a dGTP derivative having a carboxyl group as a
`linker end. As diagrammed in Fig. 4, on the other hand, nitric acid is reacted with 2-
`nitroacetophenon (Aldrich, N920-9), a nitro group is inducted at the carbon 4 position, and
`that nitro group is reduced to convert it to an amino group. As diagrammed in Fig. 5, the
`compound on the left side in Fig. 4 is reacted with Texas Red (Molecular [sic] Probes, T-
`
`Exhibit No. 1011
`Page 7
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`WO 98/33939 PCT/JP97/00239
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`353), causing it to bond. Then, as diagrammed in Fig. 6, NH2íNH2 is reacted with the
`compound on the right side in Fig. 5, MnO2 is further reacted therewith, and the
`acetophenone of the compound on the left side in Fig. 6 is converted to a diazoethane group
`(J. Am Chem. Soc. 110, 7170 – 7177, (1998)). The compounds on the right side in Figs. 3
`and 6 are reacted, whereupon, as diagrammed in Fig. 7, a fluorescently labeled (Texas Red)
`caged nucleotide (dGTP) is obtained that is dGTP wherein a 2-nitrobenzyl group to which
`Texas Red has been bound as a label has been introduced. For other base types also,
`fluorescently labeled caged nucleotides (nucleotides to which are bound caged compounds
`to which a fluorescent label has been attached) can be synthesized.
` As seen in the substance diagrammed in Fig. 7, substances labeled at the position of the
`base of the nucleotide can be incorporated by polymerase-based complementary-chain
`elongation reactions, as has been confirmed by various experiments. It has been confirmed,
`for example, that dideoxy nucleotide ddNTPs, the positions of the bases whereof are
`labeled by various fluorophores, that is, that the terminators of the complementary chain
`synthesis, are incorporated by complementary chain synthesis (Nucleic Acids Respectively.
`20, 2471 – 2483 (1992)). When the molecular size of the fluorophore is large, steric
`hindrance occurs, so two or more fluorescently labeled deoxynucleotides are not
`incorporated continuously (Anal. Biochem. 234, 166 – 174 (1996)). That is, a
`deoxynucleotide labeled by a comparatively large fluorophore, as diagrammed in Fig. 7,
`can be incorporated by complementary chain synthesis, but, once it has been incorporated,
`all further complementary chain elongation will cease. When the compound diagrammed in
`Fig. 7 is subjected to UV irradiation at 360 nm or below, the chemical structure changes
`according to the photochemical reaction diagrammed in Fig. 1, as diagrammed in Fig. 8,
`and caged substances with a fluorophore attached are released from the positions of the
`bases of the compound diagrammed in Fig. 7. If the like reaction is brought about with a
`complementary chain incorporated, steric hindrance will be eliminated, and the
`complementary chain will again be elongated. At that time, the linker portion remains in the
`base, as diagrammed in Fig. 8, but, because the size of that linker portion is small,
`complementary-chain elongation is not affected.
`
`That is, if polymerase complementary-chain elongation reactions are conducted with
`the Texas Red-labeled caged dGTP diagrammed in Fig. 7, and caged dATP, dCTP, and
`dTTP are labeled with different fluorophores, as substrates, the one-base-at-a-tie elongation
`reaction described above can be controlled, and the type of base incorporated can also be
`identified by fluorescence measurement. With fluorescence measurement, excitation laser
`irradiation is conducted, and, in order to excite only the incorporated nucleotides while not
`exciting the other suspended nucleotides, laser irradiation is conducted after spatially
`separating the two, or the evanescent irradiation described subsequently is used. By
`repeating the steps described above, that is, (1) incorporating one fluorescently labeled
`caged nucleotide using polymerase, (2) exciting the incorporated fluorescent label
`incorporated by laser irradiation, (3) spectrally separating the emitted fluorescence and
`determining the type of base from the type of fluorophore, and (4) releasing the
`fluorescently labeled caged substance by a photochemical reaction through UV irradiation,
`the fluorescently labeled caged nucleotides can be incorporated one by one, using
`polymerase, whereupon the base sequence of the template DNA is determined.
`
`Exhibit No. 1011
`Page 8
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` Next, the single molecule measurement that detects fluorescence from the fluorophore
`labeling one incorporated nucleotide is described. Fig. 9 is a simplified diagram of the
`configuration of the main components of an apparatus for conducting this single molecule
`measurement. Because single molecule measurement is extremely abhorrent of all
`impurities and contamination and such like, all procedures are conducted in a clean room,
`paying particularly close attention to all optical systems. A cell filled with a buffer solution
`is placed above the objective lens of an inverted fluorescence microscope, and the sample
`DNA 7 that is to be the template is held, by the technique described below, in the buffer
`solution, inside the field of view of the fluorescence microscope. The position wherein the
`sample DNA is held is brought close to the upper surface inside the cell, and the distance
`from that upper surface is maintained at 100 nm or less. A prism 4 is disposed at the upper
`surface outside the cell. An excitation laser beam 1 is introduced from the upper right,
`obliquely through the prism 4, perfectly reflected by the upper surface inside the cell, and
`conducted toward the upper left, obliquely through the prism 4 once again. When this is
`done, exciting light called evanescent waves 13 slightly infiltrates the buffer solution in the
`cell in the vicinity of the upper surface thereof. The intensity of this exciting light
`diminishes exponentially as the distance from the upper surface inside the cell increases.
`When a 515 nm Ar laser is used, the intensity will be 1/e at a position separated
`approximately 150 nm from the upper surface. This irradiation method is called the
`evanescent irradiation method. If this evanescent irradiation method is employed, only
`substances present within 150 nm of the surface inside the cell will be excited, background
`light from fluorescence measurement, such as water Raman scattering, can be reduced to
`almost nothing, and single molecule fluorescence measurement can be performed (Nature
`374, 555 – 559 (1995)). The emitted fluorescence is monitored as a fluorescence
`microscopic image by a supersensitive two-dimensional camera through the objective lens
`from below the cell. One fluorescently labeled nucleotide (fluorescently labeled caged
`nucleotide) 9 incorporated by a polymerase reaction 10, with a primer 8 as the origin, is
`fixed in the template DNA 7, wherefore the fluorescence is observed as a bright spot in a
`two-dimensional image.
` On the other hand, because suspended fluorescently labeled nucleotides that are not
`incorporated move vigorously about the cell three-dimensionally due to Brownian motion,
`the fluorophores are not observed one by one in the two-dimensional image as bright spots,
`but appear as an overall increase in background light. Because the space wherein the
`fluorophores are excited is limited to the minute range of 150 nm or less from the surface
`within the cell, the amount of increase in background light is small, and single molecule
`observation of the fluorophores fixed to the template DNA can be accomplished. The two-
`dimensional image is divided into four by a prism disposed above a light-receiving optical
`system, and passed through different filters, whereupon the type of fluorophore is identified
`at the moment of detection, and, thereby, the type of incorporated nucleotide base is also
`determined. This fluorescence selection method is described in detail in TOKKAI H2-
`269936/1990, gazette. The evanescent irradiation method is also used in like manner when
`releasing the fluorescently labeled caged substance of the incorporated nucleotide 9. The
`UV pulse laser beam 2 is conducted obliquely from upper right through the prism 4,
`perfectly reflected by the upper surface of the cell, and then conducted obliquely toward the
`
`Exhibit No. 1011
`Page 9
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`8
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`upper left, again through the prism 4. The range within 150 nm from the surface inside the
`cell is UV-irradiated, and the fluorescently labeled caged substance of the incorporated
`nucleotide is selectively released. At that time, it is possible that the fluorescently labeled
`caged substance of an extremely small number of suspended nucleotides that happen to be
`within the range of 150 nm or less from the surface within the cell will also be released, and
`that non-chemically modified nucleotides that are generated will be incorporated in the
`subsequent polymerase reaction. In order to reduce that possibility to zero, the buffer
`solution in the cell is continually made to flow in one direction so that fresh buffer solution
`is constantly supplied.
` Next, the configuration for holding a single molecule of template DNA 7 within the
`field of view of the fluorescence microscope is described. To the two ends of the