JPH0618523A - Method and device for deciding base sequence of dna using directional orientation of dna - Google Patents

Abstract

PURPOSE:To decide all base sequences of a plurality of long DNAs by arranging the DNAs, with the DNAs being oriented in a fixed direction from the upstream side to the downstream side (or from the downstream side to the upstream side) of genes, and successively cutting the DNAs into pieces from their ends by using a laser beam, etc., and then, deciding the base sequence of each piece. CONSTITUTION:In order to realize the title directional orientation, a DNA is first elongated by using an electric field and one end of the DNA is fixed to the end of an electrode 2. When the DNA is then cut off at a part near its end by using a restriction enzyme, only such a cut piece that has a fixed directional property can remain on the electrode 2. Therefore, when the DNA having directional orientation thus obtained is successively cut into pieces from its end with a laser beam and the sequence of the cut pieces is decided by a prior art method, the base sequence of the original long DNA can be founds by connecting the sequences of the cut piece. Since the base sequence of the DNA can be successively decided when this method is used, the time and labor of the conventional method required for estimating total sequence from the sequences of randomly cut pieces are not required.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a DNA base sequence determination method and apparatus.

[0002]

2. Description of the Related Art A conventional DNA base sequencer is capable of analyzing several hundred to one thousand base pairs at a time. However, since the DNA handled in biochemistry is generally much longer than this, it is usual to first cut it with a restriction enzyme and then analyze it with a nucleotide sequencer.

[0003]

The problem of such a technique is that the obtained sequence was located in the original long DNA in order to analyze the DNA fragmented by the restriction enzyme. I don't know if there is one. In the current technology, various fragments produced using many kinds of restriction enzymes are analyzed, and the entire sequence is deduced from the obtained analysis results. It has drawbacks such as the difficulty of the analysis itself using 2) and the difficulty of estimating the entire nucleotide sequence from fragmentary information. In particular, the DNA to be analyzed
When becomes longer, the labor required for analysis becomes geometrically larger. An object of the present invention is to sequentially cut long DNA,
By determining the base sequence of each fragment,
It is to provide a means for determining the entire base sequence of the original DNA.

[0004]

Means for Solving the Problems DN by a high frequency and high electric field
It is known that A can be extended and that one end of the extended DNA can be fixed to the electrode edge by the dielectrophoretic force. However, since the base sequence of DNA cannot be recognized by such a method, it is possible to obtain a gene in which the upstream side of the gene is fixed to the electrode and a gene in which the downstream side is fixed with a probability of 50% each. Therefore, a fragment obtained by sequentially cutting the thus extended and fixed DNA from one end with a laser or the like becomes a mixture of two kinds of base sequences. On the other hand, in the present invention, a molecule having a base recognizing property is first used to align the orientation in one direction. That is, for example, all of the DNA oriented by the electric field has a shape in which the downstream side of the gene is in contact with the electrode edge. By sequentially cutting the DNA arranged in this manner from the end not in contact with the electrode, it becomes possible to analyze the genes in sequence from the upstream side.

[0005]

1 and 2 show an embodiment of the present invention. In this embodiment, the electrodes 2 and 2'opposed on the insulating substrate 1 are used. When a DNA solution is introduced here and a high frequency voltage is applied between the electrodes, the DNA is elongated and oriented, and due to the effect of dielectrophoresis, one end of the DNA is attracted to the electrodes until it contacts the electrodes. At this time, the end of the DNA that is in contact with the electrode is adsorbed by the electrode, and the DNA does not leave from here even if the voltage is removed. FIG. 3 schematically shows the DNA thus adsorbed in contact with the electrode 2. In the DNA oriented in this way, there are 50% of each of two types, one in which the upstream side of the gene is in contact with the electrode and the other in which the downstream side is in contact with the electrode. In FIG. 3, the direction of DNA is represented by arrows and alphabets from the upstream side to the downstream side of the gene. Even if such DNA is cut from the end not in contact with the electrode, 2
A unique analysis is not possible because a mixture of fragments of the type is obtained. However, as described in claim 2 of the present invention, the DNA fixed at one end to the electrode edge is treated with a restriction enzyme (here, between regions A and B) that recognizes a certain base sequence on the DNA. It is written that it is cut by the above method), but in the case where B is in contact with the electrode edge, only a part is cut off, while A is cut off.
Most of those that touch the electrode edge are cut off. Therefore, after such treatment, if the DNA is sequentially cut off from the end not in contact with the electrode by using a laser beam or the like, fragments are successively obtained in the order of B → C → D → ... By analyzing and connecting these fragments, the entire base sequence of the original DNA can be determined. The restriction enzyme used here is preferably one that cuts the original DNA into a long fragment and a short fragment, and a restriction enzyme that just cuts from the middle cannot be used.

[0006]

EFFECTS OF THE INVENTION According to the present method, it is possible to directly analyze the base sequences of DNA in sequence without the effort of estimating the original DNA sequence from the base sequences of random DNA fragments. it can.

[Brief description of drawings]

FIG. 1 is a diagram showing an embodiment of the present invention.

FIG. 2 is a diagram showing a side surface of the embodiment shown in FIG.

FIG. 3 is a diagram for explaining an operation example of the present invention.

[Explanation of symbols]

1: Insulating substrate. 2, 2 ': electrodes. 3: DNA molecule. 4: An arrow and an alphabetic symbol for indicating the direction of the base sequence of the gene in the DNA molecule. 5: restriction enzyme cleavage site.

Claims (11)

[Claims] 1. A DNA fragment obtained by arranging DNA in one direction and sequentially cutting out from one end of the DNA is analyzed, and the results are connected to obtain the original D fragment.
A method and apparatus for determining a DNA base sequence, characterized by determining the entire base sequence of NA. 2. The method for determining a DNA base sequence and the apparatus according to claim 1, wherein DNA having one end fixed on an electrode by using an electric field recognizes a base sequence other than at the center of its length. A method and apparatus for determining a DNA base sequence, characterized by obtaining a unidirectionally arranged DNA by cleaving with an enzyme. 3. The method and apparatus for determining a DNA base sequence according to claim 1, wherein a site other than the center of the length of DNA is modified with a base recognizing molecule, and this site is on the substrate. D is applied to a specific part and an electric field is applied.
A method and apparatus for determining a DNA base sequence, characterized in that a DNA arranged in one direction is obtained by extending the entire NA molecule. 4. The method and apparatus for determining a DNA base sequence according to claim 3, wherein the specific portion on the substrate is an electrode edge. 5. The DNA base sequence determination method and apparatus according to claim 3, wherein the specific portion on the substrate is a pattern drawn in advance on the substrate.
A base sequence determination method and the same apparatus. 6. The method for determining a DNA base sequence and the apparatus according to claim 1, wherein only both ends of the DNA extended by an electric field are immobilized on a substrate and then cleaved with a restriction enzyme,
A method and apparatus for determining a DNA base sequence, characterized by obtaining DNA arranged in one direction by utilizing electric force and flow generated by reapplying an electric field. 7. The method and apparatus for determining a DNA base sequence according to claim 6, wherein at least one end is fixed on an electrode edge when fixing the DNA. . 8. The DNA base sequence determination method and apparatus according to claim 6, wherein at the time of immobilizing DNA, at least one end is immobilized by utilizing intermolecular bonds. Sequencing method and apparatus. 9. The DNA base sequence determination method and apparatus according to claim 6, wherein at the time of fixing DNA, at least one end is fixed on a pattern drawn on a substrate. Determination method and device. 10. The DNA base sequence determination method and the apparatus according to claim 6, wherein the DNA is fixed by light irradiation. 11. The method and apparatus for determining a DNA base sequence according to claim 1 or 2, wherein the DNA is cleaved using a laser beam.