JPH04330281A - Detection of base sequence of dna - Google Patents

Abstract

PURPOSE:To provide a process for safely, easily and quickly detecting the base sequence of even a small amount of DNA. CONSTITUTION:Among four kinds of bases constituting a DNA, a molecule 2 containing adenine 8 is fixed on an exploratory needle 1 of an interatomic force microscope and the needle is brought close to a single-stranded DNA 3 fixed on a substrate 4 and scanned with an accuracy of atomic level while measuring the interatomic force. A strong force is applied to the needle 1 exclusively when the adenine 8 contained in the molecule 2 fixed to the exploratory needle 1 is brought close to the thymine base 6 in the DNA. The base sequence of the single-stranded DNA 3 can be determined by repeating the similar operation using molecules containing the other three kinds of bases.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a method for detecting DNA base sequences, which is useful in the fields of molecular biology, medicine, forensic medicine, agriculture, forestry and fisheries, and the pharmaceutical industry.

0002

[Prior Art] In recent years, research in biotechnology has focused on manipulating the genes of organisms to produce organisms with better and more useful properties, and to produce organisms that more efficiently produce useful substances. It is being actively carried out. In these biotechnology fields, it is important to know the base sequence of DNA (deoxyribonucleic acid), which influences the characteristics of genes, and several methods for detecting the base sequence of DNA have been proposed.

A DNA base sequence detection method that has been frequently used in recent years will be explained with reference to FIGS. 2 to 4. FIG. 2 is a conceptual diagram showing how double-stranded DNA is synthesized from single-stranded DNA using DNA polymerase and four types of nucleotides that constitute DNA. As shown in Figure 2 (a), the DNA molecule to be investigated is treated with alkali to create single-stranded DNA.
Make it A9. Next, as shown in FIG. 2(b), a primer 10 containing radioactive 32P is attached to this single-stranded DNA 9. In this solution, four types of deoxyribonucleoside triphosphate 11, which are the constituent molecules of DNA (the general name of this molecule is nucleotide, contain adenine as a base (A), thymine (T), and cytosine) are added to this solution. (C), guanine-containing (G)) and DN
When A polymerase is present, complementary base pairs (adenine to thymine and cytosine to guanine form hydrogen bonds specifically on single-stranded DNA 9), as shown in Figure 2(c). ) and double-stranded DNA 12 is completed.

Now, let us consider the case where a small amount of dideoxyribonucleoside triphosphate, in which the hydroxyl group at the 3' position of deoxyribose, which is a constituent molecule of a nucleotide, is substituted with hydrogen is present. When such a modified nucleotide is incorporated into DNA, the next nucleotide cannot be added, and the reaction stops there. Therefore, as shown in Figure 3, when a small amount of modified nucleotide 13 having adenine as a base is mixed and reacted for a certain period of time, the modified nucleotide 13 having adenine will be randomly distributed in the chain like a monomer when synthesizing a random polymer. As a result, a variety of double-stranded DNAs with different lengths of terminal bases, such as adenine, are created. FIG. 3 is a model diagram illustrating a method for producing DNAs of various lengths with adenine ends, which illustrate this situation.

[0005] In FIG. 3, 14, 15, 16, and 17 each represent a nucleotide hydrogen-bonded with a base constituting the single-stranded DNA 9, and the same reference numerals as in FIG. The explanation will be omitted. In FIG. 3, (a) shows each component before the reaction, and (b) shows a model of one of various double-stranded DNAs in which the terminal base obtained by the reaction is adenine, (c) shows three types of models of various double-stranded DNAs with different lengths of adenine bases at the ends.

Similarly, the same operation was repeated for the other three types of bases to obtain adenine, cytosine, and
DNA of various lengths is created with thymine and guanine at the ends. As shown in Fig. 4, by electrophoresing the four types of DNA solutions 18, 19, 20, and 21 prepared in this way in four lanes, a pattern 22 as shown in Fig. 4 was observed by autoradiography. Because you can
The base sequence of the original DNA can be detected. Note that the arrow 23 in FIG. 4 indicates the direction of the electric field for electrophoresis.

[0007]

[Problem to be solved by the invention] The above-mentioned conventional DNA
This method of detecting base sequences is widely used, and several thousand base sequences can be read in a day. However, it includes problems as described below. (1) A large amount of DNA is required. (2) Since radioactive 32P is used, special facilities are required and there is also a risk of exposure to radiation. (3) The half-life of 32P is as short as 14 days, and reagents must be constantly prepared. (4) Since the operation is divided into several stages, it is a tedious and patient job.

[0008] Therefore, it is an industrially important issue to provide a method that can safely, simply, and quickly detect base sequences even with a small amount of DNA. An object of the present invention is to provide a method that can safely, simply, and quickly detect the base sequence of even a small amount of DNA.

[0009]

[Means for Solving the Problems] In order to achieve the above object, the DNA base sequence detection method of the present invention uses any one of four types of molecules that interact specifically with each of the four types of bases that constitute DNA. Prepare three or four types of probes, one type of which is fixed, and use these probes as the probes of an atomic force microscope. be brought close to the double-stranded DNA,
It consists of scanning with atomic-level precision while measuring forces.

[0010] Furthermore, in the above structure, it is preferable that the four types of molecules that specifically interact with each other are molecules containing bases constituting DNA. Furthermore, in the configuration,
It is preferable that the four types of molecules that specifically interact are molecules containing bases that constitute RNA (ribonucleic acid). According to the method of the present invention, the four types of bases that make up DNA can be directly identified through chemical interaction, thereby solving the conventional problems. Details are shown below.

FIG. 1 is a schematic diagram showing the principle of the detection method of the present invention. As shown in FIG. 1, a molecule 2 containing the base adenine 8, among the four types of bases that make up DNA, is fixed on the probe 1 of an atomic force microscope (AFM), and the probe 1 is attached to a substrate 4. It approaches the single-stranded DNA 3 immobilized on top, and scans with atomic-level precision while measuring the force generated as a result of the interaction. In addition, the present invention uses three or four types of molecules in which the probe of an atomic force microscope has one of four types of molecules fixed thereon, each of which interacts specifically with the four types of bases that make up DNA. It is unique in that it uses a probe, but in other respects its structure and operating principles are basically the same as ordinary atomic force microscopes. Therefore, a detailed explanation of its structure etc. will be omitted.

Adenine 8 in molecule 2 fixed on probe 1
When on thymine 6, a strong force due to hydrogen bonding acts, which is not seen in the case of the other bases adenine 8, guanine 7, and cytosine 5. By examining the location where this force acts, the position of thymine 6 in DNA 3 can be determined. The position of probe 1 in (a) in the figure is when adenine 8 fixed on probe 1 is located on guanine 7 in DNA 3. It shows. The position of probe 1 in (b) in the figure is when adenine 8 fixed on probe 1 is located on thymine 6 in DNA 3. In this case, the probe is placed at a position where a strong force due to hydrogen bonding is exerted. 1
It shows that there is.

[0013] Then, by repeating these operations using three types of probes to which molecules containing thymine, molecules containing cytosine, and molecules containing guanine are immobilized, adenine and guanine in the single-stranded DNA 3 are isolated. , the position of cytosine is known. From the above, single-stranded DN
The base sequence of A can be detected. Furthermore, knowing the base sequence of single-stranded DNA means that adenine only binds specifically to thymine, and guanine only binds specifically to cytosine.
This means that the base sequence of the original double-stranded DNA can be determined.

By the way, since DNA contains four types of bases: thymine, adenine, cytosine, and guanine, in principle, if the positions of these three types of bases can be detected, the DNA base sequence can be determined. Therefore, although four types of probes are used in the above, any three of the four types of probes each have one type of molecule containing thymine, adenine, cytosine, or guanine fixed thereon. DNA base sequences can also be detected using .

Furthermore, it is not necessary to limit molecules containing thymine, adenine, cytosine, and guanine to be fixed on the probe;
Any material may be used as long as it specifically interacts with the bases constituting DNA. For example, specific interactions include:
Examples include, but are not limited to, atomic forces such as hydrogen bonds and interionic forces. For example, R.N.
Various molecules such as molecules containing the bases constituting A or derivatives thereof can be used. For RNA, R
The four types of bases that make up NA are uracil, adenine, cytosine, and guanine. Of the bases that make up DNA, uracil specifically binds to adenine, and other bases have the same specificity as in the case of DNA described above. Make a target join.

[0016] In the present invention, since positions containing DNA bases are detected on a molecule-by-molecule basis as described above, the probe of the atomic force microscope is positioned on the single-stranded DNA to be detected.
A: Scan the substrate on which the sample is fixed with atomic-level precision. Atomic force microscopes are capable of such operations, but for example, in the method of the present invention,
Using a tapered probe with a width of about 4 μm at the root, a 1 μm square area is scanned in three-dimensional X-, Y-, and Z-axis directions with an accuracy of about 0.01 to 0.1 angstroms. and record interactions such as atomic forces.

In order to scan with such precision, normally in an atomic force microscope, the stage on which the sample is placed is composed of a piezoelectric element whose length can be expanded or contracted by applying a voltage. Scanning is normally performed by controlling and moving this piezoelectric element in three-dimensional directions. The measurement of the atomic force uses a sensor to detect the atomic force, so that the atomic force is always constant. A system that detects by moving the sample and probe closer or farther apart, such as by moving the sample and probe farther apart, so that the atomic force always remains at a constant value, and recording this movement as an electrical signal. It becomes.

[0018] Below, we will explain how to immobilize a single-stranded DNA molecule onto a substrate and how to immobilize molecules that interact specifically with the four types of bases that make up DNA onto the AFM tip. Explain an example.

1: Method for immobilizing single-stranded DNA on a substrate 1
-1: Physical adsorption method A single-stranded DNA solution (10 to 50 μg/ml) is dropped onto a well-washed highly flat graphite, glass substrate, gold-coated glass substrate, or mica, or Substrates are immersed in this DNA solution to immobilize single-stranded DNA to these substrates. Thereafter, these substrates are thoroughly washed with pure water and used as measurement substrates.

1-2: Chemical bonding method As the substrate, a substrate with a relatively flat surface with hydroxyl groups, such as a silicon substrate with an oxide film, a graphite substrate with an oxidized surface, a substrate made of mica, or a glass substrate, is used. It will be done.

First, approximately 10 μg of single-stranded DNA is added to 50 ml of hydrochloric acid solution (5-50%). Next, the substrate was placed in this solution and allowed to react at around room temperature for several hours, resulting in the formation of single-stranded DNA.
are fixed to these boards. Thereafter, these substrates are thoroughly rinsed with distilled water, and then immersed in clean distilled water again to be cleaned and used as measurement substrates.

1-3: Chemical bonding method As the substrate, a substrate with a relatively flat surface containing hydroxyl groups, such as a silicon substrate with an oxide film, a graphite substrate with an oxidized surface, a substrate made of mica, or a glass substrate, is used. It will be done.

First, a silane coupling agent [CH3 -C6 H
4-OOC-(CH2)n-SiCl3] was dissolved at 1% by weight in an organic solvent (hexadecane 80%, chloroform 12%, carbon tetrachloride 8%) to prepare a reaction solution,
A substrate is placed in this reaction solution, and the reaction is allowed to proceed for about 2 hours in a nitrogen atmosphere.

Next, this substrate is immersed in two chloroform solutions for 15 minutes each, and then washed with water. In addition, in this reaction, the value of n in the chemical formula of the silane coupling agent can be from 0 to 25, but if it is from 10 to 20, the DNA
This is preferable because it can prevent the reactivity of the bases constituting the compounds from being affected by steric hindrance or the like. In succession,
This substrate is made of several percent lithium aluminum hydride (
20 at room temperature in an ether solution containing LiAlH4).
Minute reaction monkey.

Finally, this substrate was placed in 50 ml of hydrochloric acid solution (5-50%) in which about 10 μg of single-stranded DNA was dissolved, and after reacting for several hours at around room temperature, it was thoroughly washed with distilled water and Fix the double-stranded DNA.

1-4: Chemical bonding method As the substrate, a substrate with a relatively flat surface containing hydroxyl groups, such as a silicon substrate with an oxide film, a graphite substrate with an oxidized surface, a substrate made of mica, or a glass substrate, is used. It will be done.

First, the terminal has a vinyl group (-CH=CH2
) trichlorosilane coupling agent (CH2 = CH
-(CH2)n-SiCl3) in an organic solvent (hexadecane 80%, chloroform 12%, carbon tetrachloride 8%)
A reaction solution is obtained by dissolving 1% by weight in the solution, a substrate is placed in this reaction solution, and the reaction is carried out in a nitrogen atmosphere for about 2 hours. Next, this substrate is immersed in two chloroform solutions for 15 minutes each, and then washed with water. In addition, in this reaction, the value of n in the chemical formula of the silane coupling agent is 0 to 25.
However, as in the case described above, the case of 10 to 20 is convenient.

Next, this substrate was reacted with a tetrahydrofuran solution (1M) in which diborane was dissolved for 1 minute at room temperature in an argon atmosphere, and then a mixed solution of hydrogen peroxide and sodium hydroxide (hydrogen peroxide 30%, hydroxide Sodium 0.1
M) for 1 minute. Finally, add this board to approximately 1
Hydrochloric acid solution containing 0 μg of single-stranded DNA (5-50%)
After pouring into 50 ml and reacting at around room temperature for several hours, the single-stranded DNA is fixed by washing thoroughly with distilled water.

2: Method of fixing molecules to AFM probe 2-1
: Method of fixing the four types of bases that make up DNA onto the AFM probe The AFM probe used is made of silicon nitride or silicon oxide. In the case of silicon nitride, the probe is oxidized by any one of alkali treatment with sodium hydroxide, treatment with hot nitric acid, treatment with hot sulfuric acid, and heat treatment in an oxygen atmosphere to add hydroxyl groups to the surface.

[0030] As molecules containing bases that make up DNA, four types of nucleotides that make up DNA are used. Nucleotides can be immobilized on the probe using the same method as in any of 1-2 to 1-4. However, the above-mentioned AFM probe is used instead of the substrate, and nucleotides are used instead of single-stranded DNA.

2-2: Method of fixing four types of bases constituting RNA onto the AFM probe The AFM probe used is made of silicon nitride or silicon oxide. In the case of silicon nitride, the probe is oxidized by any one of alkali treatment with sodium hydroxide, treatment with hot nitric acid, treatment with hot sulfuric acid, and heat treatment in an oxygen atmosphere to add hydroxyl groups to the surface.

[0032] As molecules containing bases constituting RNA, four types of nucleotides constituting RNA are used. Nucleotides can be immobilized on the probe using the same method as in 1-2 to 1-4. However, the above-mentioned AFM probe is used instead of the substrate, and nucleotides constituting RNA are used instead of single-stranded DNA.

[0033]

[Operation] The method for detecting DNA base sequences of the present invention is based on the method for detecting DNA base sequences in which one of the four types of molecules that interact specifically with each of the four types of bases constituting DNA is immobilized.
Prepare one or four types of probes, use these probes as probes for an atomic force microscope, and bring each probe close to the single-stranded DNA fixed on the substrate using the atomic force microscope. Since it scans with atomic level precision while measuring force, it is safe, the process is simple and quick, and there is no need to use radioactive elements.In addition, in principle, one D
Since the measurement can be performed as long as there is NA, it is possible to provide a method that can detect the base sequence even with a much smaller amount of DNA than conventional methods. Furthermore, using three or four types of microprobes, similar operations can be used to perform routine work.
Since the base sequence of NA can be detected, the base sequence of DNA can be determined easily and quickly with less effort than conventional methods.

In addition, in the present invention, by adopting a preferred embodiment in which the four types of molecules that interact specifically are molecules containing bases that constitute DNA, it is possible to fix the sample to the substrate and attach it to the probe. D molecules can be immobilized using almost the same procedure, and since the interactions are known in advance, D
The base sequence of NA can be determined. Furthermore, in the present invention,
By adopting a preferred embodiment in which the four types of molecules that specifically interact are molecules containing bases that constitute RNA, the immobilization of the sample on the substrate and the immobilization of the molecules on the probe are almost the same operations. Furthermore, since the interactions are known in advance, the DNA base sequence can be determined more easily.

[0035]

【Example】

Example 1 The base sequence of Escherichia coli DNA that had been cut and extracted by 30 base pairs was detected by the method of the present invention. Details are shown below. First, 20μ/mg of E. coli DNA consisting of 30 base pairs
15mM sodium chloride and 1
．． It was dissolved in a mixed solution of 5mM sodium citrate. After soaking this solution in a boiling water bath for 10 minutes,
The E. coli DNA was quenched with ice water to turn it into single-stranded DNA.

[0036] This single-stranded DNA was dispersed and immobilized on a graphite substrate using the method 1-1 described above. Further, the nucleotides constituting the DNA were immobilized on the tip of the AFM using the method 2-1 described above (among them, the method 1-3 was particularly applied). First, a probe on which a nucleotide containing adenine is immobilized is brought close to the substrate surface on which single-stranded DNA is immobilized, and the force between the probe and the substrate surface is kept constant. Atomic level precision (accuracy of 0.1 angstroms) while adjusting the distance of
This probe was scanned. Here, the scanning range of the probe is 10
0x100 nm2, and the shape of the group of curves drawn by the locus of the probe was examined.

When the shapes of the curve groups at various locations on the substrate surface were investigated, shapes resembling rod-shaped objects with a radius of several nanometers were observed at several locations on a plane. Moreover, this rod had protrusions in seven places. These were interpreted as follows. First, the method for observing the substrate surface described above is basically the same as the method for observing the shape of a solid surface using conventional AFM. Therefore,
Since the shape of the group of curves drawn by the trajectory of the probe represents the shape of the substrate surface, the rod-shaped objects were interpreted to represent single-stranded DNA.

However, the apparent shape of the DNA in the portion where thymine is present is slightly different from the shape shown by conventional AFM. In other words, a force due to hydrogen bonding acts between the adenine fixed on the tip and thymine in DNA, and this force is sufficiently larger than the force acting between the tip and other bases or the substrate surface, so that the probe is When the needle reaches the thymine in the DNA, it tries to move far away from the DNA in order to keep the interatomic force constant. Therefore, protrusions that cannot be seen with conventional AFM are observed on the curve group representing DNA. Therefore, it was determined that thymine was located at this protrusion. From the above, the positions of seven thymines on DNA were determined.

Next, using three types of probes to which nucleotides containing either thymine, guanine, or cytosine were fixed, similar operations were performed to determine the positions of adenine, cytosine, and guanine on the DNA, respectively. It was investigated. A base sequence of E. coli DNA consisting of 30 base pairs was detected from the base positions in the DNA investigated using the above four types of probes.

[0040] It was also possible to detect the base sequence of DNA using only three types of probes to which nucleotides containing either thymine, guanine, or cytosine were fixed. In other words, when single-stranded DNA was examined using each of these three types of probes, many protrusions were observed on the curve group representing the DNA, and this revealed the positions of adenine, cytosine, and guanine on the DNA. Do you get it. By the way, no matter which of the above-mentioned probes were used, there were locations where no protrusions appeared on the group of curves representing DNA. Therefore,
It was determined that thymine was present at this location. Moreover, based on the size of this place, it was possible to determine how many thymines existed here. From the above, E. coli D consisting of 30 base pairs
I was able to detect the NA base sequence.

Example 2 Single-stranded DNA was immobilized on a mica substrate using the method 1-3 described above, and 3.
A base sequence of E. coli DNA consisting of 0 base pairs could be detected.

Example 3 Single-stranded DNA was immobilized on a mica substrate using the method 1-3 described above, and the method 2-1 described above (in particular, method 1-2 was applied). The nucleotides constituting the DNA were immobilized on the tip of the AFM.

Other than that, the same method as in Example 1 was used,
A base sequence of E. coli DNA consisting of 30 base pairs could be detected.

Example 4 Nucleotides constituting RNA were subjected to AFM using the method 2-2 described above (particularly applying method 1-3).
The base sequence of Escherichia coli DNA consisting of 30 base pairs could be detected using the same method as in Example 1 except for fixing it on the probe.

Example 5 Single-stranded DNA was immobilized on a mica substrate using the method 1-3 described above, and method 2-2 described above (among them, method 1-3 was particularly applied). The nucleotides constituting the RNA were immobilized on the AFM tip. Otherwise, E. coli DNA consisting of 30 base pairs was prepared by the same method as in Example 1.
The base sequence of A could be detected.

Example 6 Single-stranded DNA was immobilized on a mica substrate using the method 1-3 described above, and the method 2-2 described above (among them, method 1-2 was particularly applied). The nucleotides constituting the RNA were immobilized on the tip of the AFM.

Other than that, the same method as in Example 1 was used,
A base sequence of E. coli DNA consisting of 30 base pairs could be detected. Rapid determination of DNA base sequences is extremely important in the fields of molecular biology, medicine, forensic medicine, agriculture, forestry and fisheries, and the pharmaceutical industry. In particular, treatment of genetic diseases and
When conducting breeding of plants and producing useful substances using A operations, reading the DNA base sequence is necessary.
It is thought that this technology will become increasingly important in the future as a basic technology for implementing these methods. The present invention is simpler than conventional methods and requires only a small amount of DNA, so it can be effectively applied to these fields.

Furthermore, a "human genome analysis project" is currently being planned to determine the entire human DNA sequence. In this case, the number of human DNA base pairs to be read is extremely large, 2.8 billion, and there is a strong desire to develop a means to read the base sequence quickly and accurately. The present invention can be expected to be a powerful means for this genome analysis. In the examples, four types of nucleotides constituting DNA or RNA were immobilized on the AFM probe, but it is not necessary to limit them to these molecules. Needless to say, anything is good as long as it has a positive effect.

[0049]

Effects of the Invention The DNA base sequence detection method of the present invention is safe, has simple steps, and can quickly detect the base sequence even with a small amount of DNA. In addition, in the present invention, 4 that specifically interacts with
By adopting a preferable embodiment in which the type of molecule is a molecule containing a base constituting DNA, it is possible to immobilize a sample on a substrate and immobilize a molecule on a probe using almost the same operation, and
Since the interaction is known in advance, the DNA base sequence can be detected more easily.

[0050] In addition, in a preferred embodiment of the present invention, the four types of molecules that interact specifically are molecules containing bases constituting RNA, so that the sample can be immobilized on the substrate and attached to the probe. The molecules of can be immobilized by the same operation,
Also, since the interactions are known in advance, it is easier to interact with DNA.
It is possible to determine the nucleotide sequence of

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram showing the principle of detecting the base sequence of single-stranded DNA according to the present invention.

FIG. 2 is a conceptual diagram showing how double-stranded DNA is synthesized from single-stranded DNA.

FIG. 3 is a model diagram showing methods for producing DNAs of various lengths with adenine at the end.

[Figure 4] 4 observed by autoradiography
It is a figure which shows the electrophoresis pattern of the mixed solution of different types of DNA.

[Explanation of symbols]

1 AFM probe 2 Molecules containing bases that make up DNA 3
Single-stranded DNA 4 Substrate 5 Cytosine 6 Thymine 7 Guanine 8 Adenine 9 Single-stranded DNA whose base sequence is to be investigated 10
Primer 11 Four types of nucleotides that make up DNA 12
Double-stranded DNA 13 Dideoxynucleoside triphosphate with adenine as a base 14 Nucleotides hydrogen-bonded with bases constituting single-stranded DNA 9 15 Nucleotides hydrogen-bonded with bases constituting single-stranded DNA 9 16 Constituting single-stranded DNA 9 A nucleotide that is hydrogen-bonded with a base. 17 A nucleotide hydrogen-bonded with a base that constitutes single-stranded DNA9. 18 DNA of various lengths with adenine at the end19
DNA of various lengths with thymine at the end 20 DNA of various lengths with cytosine at the end 21 DNA of various lengths with guanine at the end 22 Four types of mixed solutions observed by autoradiography 18, 19,
Electrophoresis patterns 20 and 21 23 Direction of electrophoresis electric field

Claims (3)

[Claims] [Claim 1] Three or four types of probes are prepared on which any one of four types of molecules that interact specifically with the four types of bases constituting DNA are immobilized, and these probes are immobilized. The probe is the probe of an atomic force microscope, and each probe is brought close to the single-stranded DNA fixed on the substrate by the atomic force microscope, and the probe is scanned with atomic-level precision while measuring the force. A DNA base sequence detection method comprising the steps of: Claim 2: Claim 1, wherein the four types of molecules that specifically interact are molecules containing bases that constitute DNA.
The DNA base sequence detection method described above. Claim 3: Claim 1, wherein the four types of molecules that specifically interact are molecules containing bases constituting RNA.
The DNA base sequence detection method described above.