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Claims  |
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What is claimed is:
1. A method determining a base sequence of a template DNA comprising the steps of:
(a) hybridizing a primer with a template DNA;
(b) performing a complementary strand extension reaction using a polymerase for extending said hybridized primer or an extended primer produced by repeating said step (b) to following step (d), by incorporating a single chemically modified
nucleotide of four kinds of chemically modified nucleotides to 3'-terminus of said hybridized primer or said extended primer, in the presence of said four kinds of chemically modified nucleotides, wherein said single chemically modified nucleotide is
complementary with a base sequence of said template DNA, and each of said four kinds of chemically modified nucleotides has a chemical modification for preventing a continuous progress of said complementary strand extension reaction, after incorporating
said single chemically modified nucleotide to 3'-terminus of said hybridized primer or said extended primer;
(c) detecting said single chemically modified nucleotide incorporated to 3'-terminus of said hybridized primer or said extended primer, and determining a kind of a base of said single chemically modified nucleotide incorporated to 3'-terminus of
said hybridized primer or said extended primer;
(d) carrying out a chemical reaction for changing a chemical structure of said single chemically modification nucleotide incorporated to 3'-terminus of said hybridized primer or said extended primer, in order to bring about a state at which said
complementary strand extension reaction for extending said hybridized primer or said extended primer can proceed; and
(e) repeating said step (b) to said step (d), and determining said base sequence of said template DNA one base by one base sequentially based on the kinds of bases of said chemically modified nucleotides incorporated to said extended primer by
said complementary strand extension reaction.
2. A method according to claim 1, wherein said chemical reaction in said step (d) is a photochemical reaction.
3. A method according to claim 1, wherein said chemically modified nucleotides are caged nucleotides.
4. A method according to claim 1, wherein each of said chemically modified nucleotides has at least one fluorophore label.
5. A method according to claim 1, wherein each of said chemically modified nucleotides has at least one fluorophore label, and said fluorophore label(s) varies depend on a kind of a base of said chemically modified nucleotide.
6. A method according to claim 1, wherein each of said chemically modified nucleotides has at least one fluorophore label, and said at least one fluorophore label is released from said chemically modified nucleotide by said chemical reaction.
7. A method determining a base sequence of a template DNA comprising the steps of:
(a) hybridizing a primer with a template DNA;
(b) performing a complementary strand extension reaction using a polymerase for extending said hybridized primer or an extended primer produced by repeating said step (b) to following step (d), by incorporating a single chemically modified
nucleotide of four kinds of chemically modified nucleotides to 3'-terminus of said hybridized primer or said extended primer, in a flow of a buffer solution including said four kinds of chemically modified nucleotides, wherein said single chemically
modified nucleotide is complementary with a base sequence of said template DNA, and each of said four kinds of chemically modified nucleotides has a chemical modification for preventing a continuous progress of said complementary strand extension
reaction, after incorporating said single chemically modified nucleotide to 3'-terminus of said hybridized primer or said extended primer;
(c) detecting said single chemically modified nucleotide incorporated to 3'-terminus of said hybridized primer or said extended primer, and determining a kind of a base of said single chemically modified nucleotide incorporated to 3'-terminus of
said hybridized primer or said extended primer;
(d) carrying out a chemical reaction for changing a chemical structure of said single chemically modification nucleotide incorporated to 3'-terminus of said hybridized primer or said extended primer, in order to bring about a state at which said
complementary strand extension reaction for extending said hybridized primer or said extended primer can proceed; and
(e) repeating said step (b) to said step (d), and determining said base sequence of said template DNA one base by one base sequentially based on the kinds of bases of said chemically modified nucleotides incorporated to said extended primer by
said complementary strand extension reaction.
8. A method of determining a base sequence of a template DNA comprising the steps of:
(a) hybridizing a primer with a template DNA;
(b) performing a complementary strand extension reaction using a polymerase for extending said hybridized primer or an extended primer produced by repeating said step (b) to following step (d), by incorporating a single caged nucleotide of four
kinds of caged nucleotides to 3'-terminus of said hybridized primer or said extended primer, in the presence of said four kinds of caged nucleotides, wherein said single caged nucleotide is complementary with a base sequence of said template DNA, and
each of four kinds of caged nucleotides has at least one fluorophore label, and a continuous progress of said complementary strand extension reaction is prevented after incorporating said single caged nucleotide to 3'-terminus of said hybridized primer
or said extended primer;
(c) exciting said fluorophore label(s) included in said single caged nucleotide incorporated to 3'-terminus of said hybridized primer or said extended primer, by laser irradiation to emit fluorescence from said fluorophore label(s), and detecting
fluorescence to determine a kind of a base of said caged nucleotide incorporated to 3'-terminus of said hybridized primer or said extended primer, and determining a kind of a base of said single caged nucleotide incorporated to 3'-terminus of said
hybridized primer or said extended primer;
(d) releasing said fluorophore label(s) from said caged nucleotide incorporated to 3'-terminus of said hybridized primer or said extended primer, in order to bring about a state at which said complementary strand extension reaction for extending
said hybridized primer or said extended primer can proceed; and
(e) repeating said step (b) to said step (d), and determining said base sequence of said template DNA one base by one base sequentially based on the kinds of bases of said caged nucleotides incorporated to said extended primer by said
complementary strand extension reaction.
9. A method determining a base sequence of a template DNA comprising the steps of:
(a) hybridizing a primer with a template DNA;
(b) performing a complementary strand extension reaction using a polymerase for extending said hybridized primer or an extended primer produced by repeating said step (b) to following step (d), by incorporating a single chemically modified
nucleotide of four kinds of chemically modified nucleotides to 3'-terminus of said hybridized primer or said extended primer, in the presence of said four kinds of chemically modified nucleotides, wherein said single chemically modified nucleotide is
complementary with a base sequence of said template DNA, and each of said four kinds of chemically modified nucleotides has a chemical modification for preventing a continuous progress of said complementary strand extension reaction, after incorporating
said single caged nucleotide to 3'-terminus of said hybridized primer or said extended primer;
(c) detecting said single chemically modified nucleotide incorporated to 3'-terminus of said hybridized primer or said extended primer, and determining a kind of a base of said single chemically modified nucleotide incorporated to 3'-terminus of
said hybridized primer or said extended primer;
(d) releasing said chemical modification from said chemically modified nucleotide incorporated to 3'-terminus of said hybridized primer or said extended primer, in order to bring about a state at which said complementary strand extension reaction
for extending said hybridized primer or said extended primer can proceed; and
(e) repeating said step (b) to said step (d), and determining said base sequence of said template DNA one base by one base sequentially based on the kinds of bases of said chemically modified nucleotides incorporated to said extended primer by
said complementary strand extension reaction.
10. A method determining a base sequence of a template DNA comprising the steps of:
(a) hybridizing a primer with a template DNA;
(b) performing a complementary strand extension reaction using a polymerase for extending said hybridized primer or an extended primer produced by repeating said step (b) to following step (d), by incorporating a single chemically modified
nucleotide of four kinds of chemically modified nucleotides to 3'-terminus of said hybridized primer or said extended primer, in a flow of a buffer solution including said four kinds of chemically modified nucleotides, wherein said single chemically
modified nucleotide is complementary with a base sequence of said template DNA, and each of said four kinds of chemically modified nucleotides has a chemical modification for preventing a continuous progress of said complementary strand extension
reaction, after incorporating said single chemically modified nucleotide to 3'-terminus of said hybridized primer or said extended primer;
(c) detecting said single chemically modified nucleotide incorporated to 3'-terminus of said hybridized primer or said extended primer, and determining a kind of a base of said single chemically modified nucleotide incorporated to 3'-terminus of
said hybridized primer or said extended primer;
(d) releasing said chemical modification from said chemically modified nucleotide incorporated to 3'-terminus of said hybridized primer or said extended primer, in order to bring about a state at which said complementary strand extension reaction
for extending said hybridized primer or said extended primer can proceed; and
(e) repeating said step (b) to said step (d), and determining said base sequence of said template DNA one base by one base sequentially based on the kinds of bases of said chemically modified nucleotides incorporated to said extended primer by
said complementary strand extension reaction.
11. A method according to claim 10, wherein said chemically modified nucleotides are caged nucleotides, each having at least one fluorophore label.
12. A method determining a base sequence of a template DNA comprising the steps of:
(a) hybridizing a primer with a template DNA;
(b) performing a complementary strand extension reaction using a polymerase for extending said hybridized primer or an extended primer produced by repeating said step (b) to following step (d), by incorporating a single caged nucleotide of four
kinds of caged nucleotides to 3'-terminus of said hybridized primer or said extended primer, in a flow of a buffer solution including said four kinds of caged nucleotides, wherein said single caged nucleotide is complementary with a base sequence of said
template DNA, and each of said four kinds of caged nucleotides has a at least one fluorophore label a continuous progress of said complementary strand extension reaction is prevented after incorporating said single caged nucleotide to 3'-terminus of said
hybridized primer or said extended primer;
(c) exciting said fluorophore label(s) included in said single caged nucleotide incorporated to 3'-terminus of said hybridized primer or said
extended primer, by laser irradiation to emit fluorescence from said fluorophore label(s), and detecting fluorescence to determine a kind of a base of said caged nucleotide incorporated to 3'-terminus of said hybridized primer or said extended
primer, and determining a kind of a base of said single caged nucleotide incorporated to 3'-terminus of said hybridized primer or said extended primer;
(d) releasing said fluorophore label(s) from said caged nucleotide incorporated to 3'-terminus of said hybridized primer or said extended primer, in order to bring about a state at which said complementary strand extension reaction for extending
said hybridized primer or said extended primer can proceed; and
(e) repeating said step (b) to said step (d), and determining said base sequence of said template DNA one base by one base sequentially based on the kinds of bases of said caged nucleotides incorporated to said extended primer by said
complementary strand extension reaction. |
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Description |
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TECHNICAL FIELD
This invention relates to an apparatus for analysis of DNA, RNA and the like. In particular, it relates to an apparatus effective in determination of the base sequences of DNA and RNA or analysis of restriction fragments and specific fragments.
BACKGROUND ART
Techniques for analysis of DNA, RNA and the like have become important in the medical and biological fields including gene analysis and genetic diagnosis. Both the determination of the base sequences of DNA or RNA and the analysis of restriction
fragments or specific fragments are based on separation carried out on the basis of molecular weight by electrophoresis. In this case, a fragment or a group of fragments is previously labeled with a radioactive or fluorescent label, and a development
pattern of separation on the basis of molecular weight is measured after or during the electrophoresis of the labeled fragment or fragments, whereby the fragment or fragments are analyzed. The need for, in particular, a DNA sequencing apparatus has
recently increased in relation to genome analysis, so that the development of the apparatus is in progress. DNA sequencing using a fluorophore label is explained below. Dideoxy reaction according to the Sanger method is carried out before
electrophoretic separation. An oligonucleotide having a length of 20 bases which is complementary to the known base sequence portion of a sample DNA to be analyzed is synthesized and then labeled with a fluorophore. This oligonucleotide is
complementarily bonded as a primer to about 10.sup.-12 mol of the sample DNA, and the elongation reaction of a complementary strand is carried out with polymerase. In this case, as substrates, there are added four deoxynucleotide triphosphates, i.e.,
deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), as well as dideoxyadenosine triphosphate (ddATP). When ddATP is incorporated by the elongation of the
complementary strand, the complementary strand is not further elongated, so that fragments of various lengths terminated by adenine (A) are prepared. The same reaction as above is independently carried out except for adding each of dideoxycytidine
triphosphate (ddCTP), dideoxyguanosine triphosphate (ddGTP) and dideoxythymidine triphosphate (ddTTP) in place of ddATP. The primers used in these reactions, respectively, have the same base sequence but are labeled with four kinds of fluorophores,
respectively, which can be distinguished from one another by fluorescence separation into spectral components.
The 4 kinds of the reaction products thus obtained are mixed to prepare fragments which are complementary to the sample DNA, have a length up to about 1000 bases, differ in length by steps of 1 base, and have any of the 4 kinds of the fluorophore
labels, depending on the kind of the terminal base. For each of the fluorophore labels, the amount of fragments having each length (number of bases) is about 10.sup.-15 mol. Then, the samples prepared are separated by electrophoresis with a resolving
power of 1 base. In the electrophoresis, there is widely used a slab gel obtained by polymerizing acrylamide between two glass plates about 0.3 mm apart from each other. When the samples are placed 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 samples migrate toward the lower end while being separated. When a position about 30 cm below the upper end is irradiated with laser beams while carrying out the
electrophoresis, the fluorophore-labeled fragments separated pass the laser irradiation position to be subjected to excitation, in the order of increasing length. By measuring the emitted fluorescence while separating it into spectral components with a
plurality of filters, the kinds of terminal bases of all the fragments can be determined in the order of increasing fragment length, from the change with time of the fluorescence intensity of the four kinds of the fluorophores. Since the thus determined
order of the bases is complementary to that of the sample DNA, the base sequence of the sample DNA can be determined.
There have been proposed several novel DNA sequencing methods using no electrophoresis. In a first prior art, in carrying out the elongation reaction of a complementary strand by using polymerase and a sample DNA as template, the 4 kinds of the
substrates are added one by one, and the amount of the substrates incorporated into the complementary strand is determined at each stage by utilizing light absorption or fluorescence, to determine the base sequence of the sample DNA (JP-A-4-505251). In
a second prior art, the elongation reaction of a complementary strand is carried out by using polymerase, a sample DNA as template and the 4 kinds of the substrates labeled with different labels, respectively, after which bases are released one by one
from the 3'-end of the thus synthesized complementary strand with exonuclease, and the labels of the released bases, respectively, are measured in turn to determine the base sequence of the sample DNA (Journal of Biomolecular Structure & Dynamics 7,
301-309 (1989)). In a third prior art, the base sequence of a sample DNA is determined by repeating a cycle consisting of a step of carrying out DNA polymerase reaction by using four dATP derivatives (MdNTPs) which have a detectable label and can be
incorporated into a template DNA as substrates for DNA polymerase to stop DNA strand elongation reaction, owing to the presence of their protecting group, a step of detecting the incorporated MdNTP, and a step of returning this MdNTP to its original
state at which the elongation is possible. In this prior art, the DNA strand elongation is stopped every time the DNA strand is elongated by one base, and the enzyme and the substrates are removed from a system (a solution) containing the template, a
primer and the MdNTPs, after which the MdNTP incorporated is detected, and the protecting group (and the label) of the MdNTP incorporated into the template are removed to return this MdNTP to its original state at which the DNA strand elongation is
possible (Japanese Patent Application No. 2-57978). These proposals, however, set forth mere ideas at present and no practical application thereof has been reported.
DISCLOSURE OF THE INVENTION
In DNA sequencing methods which have been put to practical use, separation on the basis of molecular weight is carried out by electrophoresis. For DNA sequencing, the resolving power for the separation on the basis of molecular weight should be
a length of one base. Usually, the resolving power of the electrophoresis decreases with an increase of the length (number of bases) of DNA fragments to be separated. That is, even if a DNA fragment having a length of 50 bases and that having a length
of 51 bases can be separated from each other on the basis of the length difference of one base, a DNA fragment having a length of 500 bases and that having a length of 501 bases cannot always be separated from each other on the basis of the length
difference of one base. The length (number of bases) at separation limit which indicates the limit of the length (number of bases) within which a resolving power of one base can be attained, is determined by the electrophoresis conditions, i.e., the
composition of a medium for separation, the length of electrophoresis lanes and the intensity of electric field. Various optimizations have been carried out for increasing the length (number of bases) at separation limit, but there is no report that the
limit exceeds 1000 bases (Electrophoresis 13, 495-499 (1992) and Electrophoresis 13, 616-619 (1992)). There has been theoretically explained the fact that the length (number of bases) at separation limit is at most 1000 bases (Electrophoresis 13,
574-582 (1992)). That is, when electrophoresis is employed, the length of a base sequence which can be determined from one kind of sample DNA is at most 1000 bases. On the other hand, in large-scale base sequence determination represented by genome
analysis, the length (number of bases) at separation limit definitely affects the analysis efficiency (Science 254, 59-67 (1991)). For example, a VAC clone (a typical large clone) has a length of about 1 M bases, and hence when its successive portions
each having a length of 1000 bases are analyzed, at least 1000 samples should be analyzed. However, successive fragments each having a length of 1000 bases can be prepared neither too far from nor to near the end of a long DNA. In practice, random
fragments are prepared and then analyzed at random, and the original long DNA base sequence is reconstructed by utilizing the overlap of the base sequences of the fragments with one another. This method is called a shot gun method and is most widely
adopted for genome analysis. It, however, is disadvantageous in that the same base sequence should be repeatedly analyzed because the random fragments should have overlaps which permits the reconstruction.
The degree of this overlapping is called redundancy and increases with a decrease of a length (number of bases) at which base sequence determination can be carried out at a time, i.e., the length (number of bases) at separation limit in
electrophoresis. When the length (number of bases) at separation limit is 1000 bases, the redundancy is about 10. That is, it is necessary to determine base sequences having a total length of 10 times the length of a DNA whose base sequence is to be
determined. Therefore, about 10,000 samples should be analyzed for determining a base sequence having a length of 1 M bases. The number of samples which can be analyzed with a DNA sequencing apparatus is at most 100 per day. Thus, it takes at least
100 days to determine the whole base sequence having a length of 1 M bases with the apparatus. For effectively carrying out genome analysis or genetic diagnosis which will acquire greater importance, there is required a technique for achieving in
several days the determination of a base sequence having a length of 1 M bases.
Another problem in the prior art is that a large number of sample DNAs are necessary for base sequence determination. For carrying out dideoxy reaction by the Sanger method, about 10.sup.-12 mol of each sample DNA is usually necessary.
Therefore, the sample DNAS should be previously purified and amplified by techniques such as cloning and PCR. These techniques require much time and labor and hence constitute a remarkable rate-limiting step in the progress of genome analysis. For
efficiently carrying out genome analysis and genetic diagnosis, there is required a technique which permits base sequence determination by the use of a small amount of sample DNAs, ultimately a single DNA molecule. The proposed DNA sequencing methods,
however, involve the following problems.
In the first prior art, since the 4 kinds of the nucleotide substrates are added one by one, it is necessary to change repeatedly the composition of a solution in which the elongation reaction of a complementary strand is carried out. In
addition, when the kind of base in the solution is changed, washing is necessary for preventing the presence of foreign bases. Therefore, at least 8 runs of solution replacement are needed per cycle. on an average, one base is usually determined by one
cycle, so that the 8 runs of solution replacement considerably lowers the base sequence determination rate. A key problem in the first prior art is that the amount of a signal to be quantified is accumulated with the elongation of the complementary
strand, resulting in difficult measurement of a change in the signal amount which corresponds to the incorporation of one base. Thus, it is difficult to determine a DNA base sequence having a large length (a large number of bases).
In the second prior art, all of the 4 kinds of the bases labeled with 4 kinds of fluorophores, respectively, are incorporated into a complementary strand, but this incorporation is technically difficult. As described hereinafter, fluorophores
which emit a large amount of fluorescence and can be distinguished from one another by separation of the fluorescence into its spectral components have a large molecular size, so that owing to steric hindrance, it is difficult to incorporate a plurality
of fluorophore-labeled nucleotides side by side into the same complementary strand. Another problem in the second prior art is that the release of the bases one by one by the use of endonuclease is difficult to control. When a plurality of bases are
continuously released in a shorter time outside the detection limit, the bases released are detected at the same time, so that the order of the bases released, i.e., the base sequence cannot be determined. For determining the base sequence, the release
of the bases should be intermittent, not continuous, but such control is difficult.
In the third prior art, the MdNTPs not incorporated into the template should be removed from the solution in the step of detecting the MdNTP incorporated into the template and the step of returning this MdNTP to its original state at which the
elongation is possible, and MdNTPs should be added to the solution at the time of starting the next and new cycle. If these operations are neglected, a signal from the MdNTP incorporated into the template is mixed with that from the MdNTPs not
incorporated into the template, so that the objective signal from the MdNTP incorporated into the template cannot be accurately measured. Moreover, if the protecting groups of the MdNTPs not incorporated into the template are also removed and the dNTPS
produced by the removal of the protecttng group are incorporated into the template, the progress of the predetermined cycle is blocked. Therefore, the composition of the solution should be changed at least twice per cycle, and this operation lowers the
base sequence determination rate. Thus, there is the following problem: if even a small amount of the MdNTPs not incorporated into the template remain in the solution owing to the insufficient change of composition of the solution, they cause much noise
in the measurement.
An object of the present invention is to provide method and apparatus for determining a very long DNA base sequence by determining one by one the kinds of the bases of nucleotides incorporated during the elongation reaction of a complementary
strand using polymerase, in order to solve various problems in the proposed DNA sequencing methods using no electrophoresis.
In the present invention, a template DNA single molecule (a sample) is held in the field of view of a fluorescence microscope, a complementary strand is elongated by one base each time while controlling the elongation, and one fluorophore-labeled
base incorporated thereinto is measured as a single molecule. To conduct DNA sequencing, it is sufficient that the kinds of the bases of nucleotides incorporated by the elongation reaction of the complementary strand using polymerase can be monitored
one by one. All nucleotides to be incorporated are chemically modified so as to satisfy the following two conditions at the same time: (1) the elongation reaction does not proceed after the incorporation of one nucleotide, and (2) after the
determination of the kind of the nucleotide incorporated, the incorporation of the next nucleotide is made possible. These two conditions can be fulfilled, for example, by combining caged compounds having fluorophores, respectively, as labels with
nucleotides, respectively. The caged compound refers to a compound which masks the residue concerned in activity of a physiologically active substance with a nitrobenzyl group or the like and releases the modifying group on light irradiation. For
example, the caged compound is a substance having a 2-nitrobenzyl group it introduced thereinto which releases the 2-nitrobenzyl group on ultraviolet irradiation and is widely used in the field of biology (Annu. Rev. Biophys. Biophys. Chem. 18,
239-270 (1989)). Molecular Probes Inc. and the like sell various caged compounds. As shown in FIG. 1, the chemically modified nucleotides used in the present invention are caged compounds obtained by bonding a 2-nitrobenzyl group to a physiologically
active substrate X (a nucleotide), and have the following capability: there is inhibited the inherent activity of the nucleotide without the chemical modification, i.e., the activity to undergo continuous incorporation by the synthetic reaction of a
complementary strand, and the caged substance (2-nitrobenzyl group) is released by irradiation with ultraviolet light of 360 nm or less, whereby the chemically modified nucleotide can be converted to the substrate X or HX, which has its inherent
physiological activity. In FIG. 1, R is H or an alkyl group (e.g. CH.sub.3).
FIGS. 3 to 7 show a process for producing Texas Red-labeled caged dGTP as an example of the chemically modified nucleotide having the above-mentioned capability. In FIG. 3, a derivative of dGTP (FIG. 2) (Science 238, 336-341, 1987), a starting
material for chemically modifying the base, is reacted with a water-soluble carbodiimide (HOOC(CH.sub.2).sub.2 COOH) to obtain a dGTP derivative having a carboxyl group as a linker end. on the other hand, as shown in FIG. 4, 2-nitroacetophenon (Aldrich,
N920-9) is reacted with nitric acid to introduce a nitro group thereinto at the carbon 4 position, and the nitro group is reduced into an amino group. As shown in FIG. 5, the compound on the right side in FIG. 4 is reacted with Texas Red (Molecular
Probes, T-353) to be bonded thereto. Next, as shown in FIG. 6, the compound on the right side in FIG. 5 is reacted with NH.sub.2 --NH.sub.2 and then MnO.sub.2 to convert the acetophenone group of the compound on the right side in FIG. 5 to a diazoethane
group (J. Am. Chem. Soc. 110, 7170-7177, (1988)). The compound on the right side in FIG. 3 and that on the right side in FIG. 6 are reacted to obtain a fluorophore (Texas Red)-labeled caged nucleotide (dGTP), i.e., dGTP having as an introduced group a
2-nitrobenzyl group having Texas Red attached thereto as a label, as shown in FIG. 7. Also for other kinds of bases, fluorophore-labeled caged nucleotides (nucleotides having a fluorophore label-attached caged compound bonded thereto) can be
synthesized.
It has been confirmed by various experiments that a substance labeled in the base portion of a nucleotide like the substance shown in FIG. 7 can be incorporated by the elongation reaction of a complementary strand using polymerase. For example,
it has been confirmed that dideoxynucleotides ddNTPS labeled with various fluorophores, respectively, in their base portions, i.e., terminators for the synthesis of a complementary strand are incorporated by the synthesis of a complementary strand
(Nucleic Acids Res. 20, 2471-2483 (1992)). In the case of fluorophores having a large molecular size, no continuous incorporation of two or more fluorophore-labeled deoxynucleotides occurs because of steric hindrance (Anal. Biochem. 234, 166-174
(1996)). That is, a deoxynucleotide labeled with a relatively large fluorophore as in FIG. 7 can be incorporated by the synthesis of a complementary strand, but the complementary strand is not further elongated after this incorporation. When the
compound shown in FIG. 7 is irradiated with ultraviolet light of 360 nm or less, its chemical structure is changed as shown in FIG. 8 according to the photochemical reaction shown in FIG. 1, so that the caged substance having the fluorophore attached
thereto is released from the base portion of the compound shown in FIG. 7. When the compound is subjected to the same reaction as above after being incorporated into the complementary strand, the steric hindrance is removed, so that the complementary
strand is elongated again. In this case, the linker portion remains in the base as shown in FIG. 8 but it does not affect the elongation of the complementary strand because of its small size.
That is, when the elongation reaction of a complementary strand with polymerase is carried out by using as substrates, Texas Red-labeled caged dGTP shown in FIG. 7 and caged dATP, dCTP and dTTP which have been labeled with different fluorophores,
respectively, the elongation reaction can be controlled so that the bases may be incorporated one by one as described above. Furthermore, the kinds of the bases incorporated can be determined by fluorescence measurement. The fluorescence measurement is
carried out by conducting laser irradiation or the evanescent irradiation described hereinafter after spatially separating the incorporated nucleotide from other suspended nucleotides in order to excite only the incorporated
nucleotide but not the other suspended nucleotides by irradiation with exciting laser beams. The base sequence of a template DNA can be determined by incorporating the fluorophore-labeled caged nucleotides one by one by reaction with polymerase
by repeating the above steps, i.e., (1) the incorporation of one of the fluorophore-labeled caged nucleotides by the use of polymerase, (2) the excitation of the incorporated fluorophore label by laser irradiation, (3) the separation of the emitted
fluorescence into its spectral components and the determination of the kind of the base from the kind of the fluorophore, and (4) the release of the fluorophore-labeled caged substance by the photochemical reaction caused by ultraviolet irradiation.
Next, there is explained below single-molecule measurement in which fluorescence from a fluorophore labeling one nucleotide incorporated is detected. FIG. 9 is a schematic diagram showing an outline of the structure of the principal part of an
apparatus for carrying out the single-molecule measurement. Since impurities, dust and the like extremely interfere with the single-molecule measurement, all operations are carried out in a clean room and close attention is paid to all optical systems.
A cell filled with a buffer solution is located over the objective lens of an inverted fluorescence microscope, and a sample DNA 7 as a template is held in the field of view of the fluorescence microscope in the buffer solution by the technique described
hereinafter. The position of holding the sample DNA is in close vicinity to the inner top surface of the cell, and its distance from the top surface is maintained at 100 nm or less. A prism 4 is located on the outer top surface of the cell. Exciting
laser beams 1 are introduced obliquely from above through the prism 4, perfectly reflected from the inner top surface of the cell, and then conducted obliquely upward through the prism 4. In this case, a slight amount of exciting light called evanescent
waves 13 is infiltrated into the buffer solution in the cell in the vicinity of the top surface. The intensity of the exciting light decreases exponentially with an increase of the distance from the inner top surface of the cell. When Ar laser beams of
515 nm are used, the intensity becomes 1/e at a distance of about 150 nm from the top surface. The above irradiation method is called an evanescent irradiation method. When the evanescent irradiation method is adopted, only a substance present at a
distance of 150 nm or less from the inner surface of the cell is excited, and background light in fluorescence measurement, such as Raman scattering in water can be reduced to the utmost, so that a single molecule can be subjected to fluorescent
measurement (Nature 374, 555-559 (1995)). The fluorescence emitted is monitored as a fluorescence-microscopic image by means of a high-sensitivity two-dimensional camera through the objective lens from under the cell. Since one fluorophore-labeled
nucleotide (fluorophore-labeled caged nucleotide) 9 incorporated by polymerase reaction 10 starting from a primer 8 is fixed on the template DNA 7, the fluorescence is observed as a bright spot in a two-dimensional image.
On the other hand, since non-incorporated, suspended, fluorophore-labeled nucleotides move about in the cell actively and three-dimensionally owing to Brownian motion, each of the fluorophores is not observed as a bright spot in the
two-dimensional image but causes an increase of the whole background light. However, since the space where the fluorophores are excited extends to only 150 nm or less from the inner surface of the cell, the increment of the background light is so small
that the single-molecule measurement of the fluorophore fixed on the template DNA can be carried out. The two-dimensional image is divided in four by a prism located on a light-receiving optical system and passed through different filters to be
detected, and the kind of the fluorophore is determined in a moment, whereby the kind of base of the incorporated nucleotide is determined. This fluorescence selection method is described in detail in JP-A-2-269936. The evanescent irradiation method is
adopted in the same manner as above also for releasing the fluorophore-labeled caged substance of the incorporated nucleotide 9. Ultraviolet pulse laser beams 2 are introduced obliquely from above through the prism 4, perfectly reflected from the top
surface of the cell, and then conducted obliquely upward through the prism 4. The space extending to 150 nm or less from the inner surface of the cell is irradiated with ultraviolet light, and the fluorophore-labeled caged substance of the incorporated
nucleotide is selectively released. In this case, there is a possibility that the fluorophore-labeled caged substances of a very small number of suspended nucleotides accidentally present in the space extending to 150 nm or less from the inner surface
of the cell may also be released and that the resulting chemically non-modified nucleotides may be incorporated by the subsequent polymerase reaction. For eliminating this possibility, the buffer solution in the cell is always allowed to flow in one
direction so that fresh buffer solution may be supplied.
Next, there is explained below a structure for holding a single molecule of the template DNA 7 in the field of view of the fluorescence microscope. Beads (solid carriers) 5 and 6 with a diameter of about 100 nm are attached to the ends,
respectively, of the sample DNA 7 which is used as a template. As to a material for the beads, the beads are made of polystyrene, and the bead 6 is magnetic. The sample DNA 7 is introduced into the buffer solution in the cell, and the nonmagnetic bead
5 attached to the single DNA molecule is captured in the field of view of the microscope in the cell by the use of a laser trap 3 (Science 271, 795-799 (1996)). Optical tweezers using a laser as a typical light source is a technique which is recently
rapidly spreading as a non-contact manipulator (Optics Lett. 11, 288-290 (1986)). In particular, a laser trap using an IR laser is widely utilized because the influence of irradiation is minimum when a sample derived from a living body sample is used
(JP-A-2-91545). When laser beams are condensed in water with a lens, fine particles in water can be captured in the vicinity of the focus of the lens with a restrainting force of approximately 2-6 pN/mW. After completion of the laser trap for the bead
(solid carrier) 5 on one side of the single sample DNA 7, a magnetic field is applied in a direction parallel to the top and under surfaces of the cell to allow the magnetic bead 6 on the other side to generate a static magnetic force 11, whereby the
single DNA molecule 7 is extended (Science 271, 1835-1837 (1996)). The extending force is controlled by varying the intensity of the magnetic field. While maintaining the above state, the stage of the microscope which has the cell fixed thereon is
slowly lowered and it is brought to a position 100 nm or less under the inner top surface of the cell while capturing the single DNA molecule 7.
As explained above, in the present invention, base sequence determination can be carried out by using one kind of sample DNA, and the base sequence of the sample DNA can be determined with only complementary strand synthesis by using polymerase
reaction. No trouble is caused in the measurement even if a polymerase molecule catalyzing the elongation of the complementary strand releases from DNA molecule and another polymerase molecule continues the elongation of the complementary strand. That
is, a base. sequence having a substantially desirable length can be determined by successively supplying active polymerase molecules. In the present invention, the repeated solution replacement, the problem in the first prior art, is not necessary.
Furthermore, in the present invention, since the fluorophore incorporated into the complementary strand is released every cycle, there is no problem of accumulation of the amount of a signal from the fluorophore, so that base sequence determination can
be carried out irrespective of the length (number of bases) of DNA. On the other hand, the incorporation of fluorophores in succession into a complementary strand, the problem in the second prior art, is not necessary in the present invention. In the
second prior art, it is difficult to control the release of bases one by one and the elongation by controlling the activities of the enzymes. By contrast, in the present invention, the elongation is structurally controlled, so that the elongation can be
controlled for each base without controlling the activity of the enzyme. In addition, the change of the composition of the solution, the problem in the third prior art, is not necessary in the present invention. That is, in the present invention, only
a chemically modified nucleotide incorporated into a template can be selectively measured in the presence of chemically modified nucleotides not incorporated into the template, and the chemically modifying substance can be released selectively only from
the nucleotide incorporated into the template. Therefore, the chemically modified nucleotides not incorporated into the template need not be removed from the solution during the cycle.
The rate of the base sequence determination according to the present invention is dependent on the cycle time of the steps explained above. The step of incorporating a molecule of fluorophore-labeled caged nucleotide with polymerase can be
carried out in 0.1 second or less. Various kinds of DNA polymerases are on the market and permit incorporation of nucleotides at a rate of 30 bases per second in the case of the slowest elongation rate. That is, the average time required for the
incorporation of one base is 0.03 second and the above time of 0.1 second is a considerably high estimate. 0.5 second is required for carrying out the steps of exciting the incorporated caged nucleotide by laser irradiation, separating the emitted
fluorescence into its spectral components, and determining the kind of the base from the kind of the fluorophore.
For the single-molecule fluorescence measurement, a television rate, i.e., an exposure time of 0.03 second or less may be employed (Nature 374, 555-559 (1995)), though in the present invention an exposure time of 0.5 second is employed for
high-sensitivity measurement. The laser irradiation time is also 0.5 second and the laser irradiation is synchronized with fluorescence exposure. The determination of kind of the base from the kind of the fluorophore is completed in 0.1 second or less,
and it does not affect the cycle time because it can be carried out simultaneously with the subsequent step. The step of releasing the fluorophore-labeled caged substance by ultraviolet irradiation can be completed in 0.1 second or less. The
fluorophore-labeled caged substance can be released in the order of millisecond by ultraviolet pulse laser irradiation for 10 nanosecond (Annu. Rev. Biophys. Biophys. Chem. 18, 239-270 (1989)). When the steps described above are automatically
repeated by using a computer, the cycle can be repeated once in 1 second even if the time required for controlling a laser and a camera is assumed to be 0.3 second per cycle. Since the kind of one base can be determined per cycle, the rate of the base
sequence determination becomes one base per second or shorter. When the method of the present invention is applied to the determination of the base sequence with a length of 1 M bases of a YAC clone, a typical large clone, the determination can be
completed in 1 M seconds, i.e., about 12 days which is 1 order of magnitude shorter than a time required for carrying out the determination by a conventional method. According to the method of the present invention, a plurality of sample DNAs can be
dealt with in parallel by hold | | |
