JPH06289018A - Device for determining base arrangement of nucleic acid - Google Patents

Abstract

PURPOSE:To eliminate the need for a marker by reducing measuring time and then improving reading accuracy. CONSTITUTION:A mass analyzer 30 is used, a sample plate where a DNA fragment group sample for each end base is spread is laid out at an ion source, and then laser beams are applied, thus ionizing the sample. Mass spectrum data for each of four types of end base groups from the mass analyzer are read and stored at storage parts 32A, 32G, 32C, and 32T and then the peak value is read in the order of the number of masses from four types of mass spectrum data by a base arrangement determination part 34, thus determining the base arrangement of the sample DNA.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a device called a DNA sequencer, for analyzing a base sequence of DNA.

[0002]

2. Description of the Related Art In a conventional DNA sequencer, a fluorescent primer (a primer chemically bound to a fluorescent substance as a marker) or a primer labeled with a radioisotope is used, and the end bases are A (adenine) and G by a Sanger reaction. (Guanine), C (cytosine), and T (thymine), respectively, are prepared. Using it as a sample, put it into the sample input part of the slab-like gel of the polyacrylamide gel electrophoretic apparatus for each end base, and simultaneously perform electrophoresis, and detect the DNA fragment by online or offline method to determine the DNA base sequence. ing.

[0003]

The gel electrophoresis apparatus requires a long time for electrophoresis. For example, reading about 400 bases requires 4 to 8 hours for migration. In addition, the accuracy of sequence determination increases when the number of bases increases, which causes poor accuracy due to gel electrophoresis and reverses the order of detection between lanes with different terminal bases due to different migration speeds depending on the gel location. There is also a problem that becomes difficult.

In addition, a marker such as a fluorophore, a radioisotope, a chemiluminescent substance, etc. for detecting a DNA fragment and a detector are combined with each other, so that a large amount of sample is required or a long sequence is read. The problem is that it is difficult. Therefore, it is an object of the present invention to provide a nucleic acid base sequence determination device that shortens the time required for measurement, improves the base sequence reading accuracy, and does not require a marker.

[0005]

In the present invention, instead of separating DNA fragment groups by electrophoresis, a sample of DNA fragment groups prepared for each terminal base is analyzed by a mass spectrometer for each terminal base, and four types of DNA fragments are analyzed. It is intended to determine the base sequence of DNA by obtaining mass spectrum data for each end base, collecting the data, and reading the peaks in order of mass number.

Therefore, the present invention provides DN for each terminal base.
An ion source for arranging a sample plate coated with the A fragment group sample and irradiating the sample at the measurement position with a laser beam to ionize the sample, an analysis unit for separating ions generated by the ion source by mass number, And a mass spectrometer equipped with a detector for detecting the separated ions, and a storage unit for loading the mass spectrum data for each of the four types of terminal bases from the mass spectrometer and storing the data for each terminal base, and the storage unit And a base sequence determination unit that determines the base sequence of the sample DNA by reading peaks in order of mass number from four types of mass spectrum data for each end base.

In a preferred embodiment of the present invention, when the base sequence determination part has a mass number interval part smaller than the mass number corresponding to one base unit in the mass spectrum containing four kinds of terminal bases, the sample is heterogeneous. It further includes a heterozygous determination processing unit that processes as DNA having a junction.

[0008]

The solution of the DNA fragment group sample for each end base prepared from the template DNA using the Sanger reaction is applied to the sample plate at different positions for each end base. The sample plate is placed on the ion source of the mass spectrometer and irradiated with a laser beam to generate DNA fragment ions. In the ionization by the laser beam, the DNA fragment contained in the sample is ionized as it is without being decomposed. In this way, D
Obtain mass spectra of NA fragments. Each DNA
Although a primer is attached to the fragment, the mass number of the primer is known and the mass number of each base is also known, so the peak position of the mass spectrum is also determined. The base sequence can be determined by collecting the mass spectra of the four types of terminal bases and reading them sequentially from the peak of the next mass number of the primer.

A sample of each DNA fragment group whose terminal base is A, G, C or T is prepared from the template DNA by Sanger reaction. For example, it is assumed that the template DNA is [primer corresponding part] + AAGCTAAGTTCAGG. In the Sanger reaction, the DN in the upper part of the following equation
A is created.

As a result, A obtained by the Sanger reaction
There are the following three fragments with ends. (Primer) + TTCGA (Primer) + TTCGATTCA (Primer) + TTCGATTCAA Similarly, the following five fragments have a T-terminus. (Primer) + T (Primer) + TT (Primer) + TTCGAT (Primer) + TTCGATT (Primer) + TTCGATTCAAGT The following two fragments have a G-terminal. (Primer) + TTCG (Primer) + TTCGATTCAAG The following four fragments have C-terminals. (Primer) + TTC (Primer) + TTCGATTC (Primer) + TTCGATTCAAGTC (Primer) + TTCGATTCAAGTCC

The mass spectrum of each of the DNA fragment groups for each end base as a sample is as shown in FIG. With these four mass spectrum data,
From the next peak of the primer, the mass number (molecular weight) is read in that order, and the base sequence TTCGAATTCAAGTCC is determined. It shows the complementary nucleotide sequences of these template DNAs.

[0012]

EXAMPLE FIG. 2A shows a mass spectrometer in one example. In FIG. 2 (A), 2 is an ion source, and a sample plate 4 to which a DNA fragment group sample solution prepared by the Sanger method is applied for each end base is arranged. The sample plate 4 is shown in FIG.
Is a long metal plate, and the DNA fragment group sample solution 6 for each end base is applied along the longitudinal direction of the metal plate. The sample solution 6 is arranged on the sample plate 4 for each of the types A, G, C, and T of the terminal bases, and a set of four coated samples constitutes one set.

The sample plate 4 is the ion source 2 of (A).
Is inserted in the direction perpendicular to the paper surface, is sent in the direction of the arrow in (B), and is sequentially ionized one by one for each terminal base for measurement. A nitrogen laser 8 is arranged outside the ion source 2 in order to ionize the sample applied to the sample plate 4 by the ion source 2 and arranged at the ionization position.
The laser beam 10 from is emitted to the sample placed at the ionization position of the ion source 2 through the laser beam introduction terminal 12. In order to extract the sample ions generated at the ionization position toward the detector, the extraction electrode 12 is arranged on the detector side in the ion source, and the extraction electrode 12 has a high voltage of, for example, −30 KeV with respect to the sample plate 4. Is being applied. 16 is a detector, and the detector 16 and the ion source 2
A drift region 18 in which the extracted ions fly toward the detector 16 is arranged between them. This mass spectrometer is a time-of-flight (TOF) mass spectrometer, and the time from ionization to reaching the detector 16 corresponds to the mass number, and the smaller the mass number, the shorter the ion. By reaching in time, the DNA fragment groups are separated according to their mass numbers. Reference numeral 26 denotes an exhaust means for exhausting the mass spectrometer, which is equipped with a turbo molecular pump as a main exhaust means.

A photodiode 20 is arranged to detect the timing of irradiation of the sample with the laser beam 10 and use it as a trigger signal for starting the flight of ions. Digital oscilloscope 2 to display the mass spectrum
2 is provided, and the digital oscilloscope 22 takes in the output signal of the detector 16 using the detection signal of the photodiode 20 as a trigger signal and displays it. Detector 16
The detection signal of is also taken into the computer 24 and stored therein, and is used for base sequence determination.

The sample plate 4 is sequentially fed in the direction perpendicular to the paper surface of FIG. 2A, and the coated samples for each end base are sequentially analyzed. The mass spectrometer used in the present invention is not limited to the one illustrated in FIG. 2, but the details of the mass spectrometer itself shown in FIG. 2 are described in Analytical Chemistry, Vol.
21, pp.1027-1039 (1992).

In order to determine the DNA base sequence from the mass spectrum data that has been taken in by the computer 24,
It comprises the means shown in FIG. Four data storage units 32A, 32G, 32C, and 32T that store the mass spectrum data for each end base from the mass spectrometer 30 are provided. The base sequence determination unit 34 determines the base sequence of the DNA by grouping the mass spectrum data of the four storage units and arranging them in order from the peak with the smallest mass number. The determined base sequence is displayed on the display unit 36.

When the DNA fragment group sample is prepared by the PCR method and the template DNA contains a heterozygote, the DNA fragment group sample contains two kinds of sequences. At this time, in the case of one kind of sequence, the peaks of the mass spectrum obtained by collecting four mass spectrum data for each end base are arranged at the mass number intervals corresponding to one base unit, but the heterojunction is included. In the case of, two peaks coexist at close positions. Therefore, when the heterojunction determination processing unit 38 is further provided, when the peak interval of the mass spectrum obtained by gathering the four mass spectrum data includes a part having a narrower interval than the mass number interval corresponding to one base unit, , And determine that two types of sequences are included.

Of the blocks surrounded by the computer 24 in FIG. 3, the storage units 32A, 32G, 32C and 32T are R.
This is realized by setting respective areas in a storage device such as an AM or a hard disk device, and the base sequence determination unit 34 and the heterojunction determination processing unit 38 are realized by software using a CPU and a memory device.

The operation of one embodiment will be described with reference to FIG. A sample plate coated with a DNA fragment group sample solution for each end base is installed as an ion source, and after evacuation, the sample at the ionization position is irradiated with the laser beam 10 to obtain a mass spectrum of the sample. For example, first, mass analysis of the A-terminal fragment group is performed, and the data is loaded into a computer. Similarly, after repeating the G-terminal fragment group sample, the C-terminal fragment group sample, and the T-terminal fragment group sample in sequence, the four types of mass spectrum data are collected together and the peaks are arranged in order of the mass numbers to determine the DNA base sequence. Get and display it.

This operation is repeated as many times as there are samples of four kinds of samples. Although a time-of-flight mass spectrometer is used as a mass spectrometer in the embodiments, the mass spectrometer is not necessarily limited to this, and a mass spectrometer that separates ions by a magnetic field may be used.

[0021]

INDUSTRIAL APPLICABILITY In the present invention, DNA prepared by adjusting the terminal bases
The fragment group samples were separated by a mass spectrometer, and the base sequence was determined by collecting the mass spectrum data for the four types of end base-specific samples.
The result can be obtained in a very short time such as several seconds. Moreover, compared to gel electrophoresis, there is no need to attach markers such as fluorophores and radioisotopes, and there is no smileing in the case of gel electrophoresis, and it is possible to determine the base sequence with higher accuracy than gel electrophoresis. it can.

[Brief description of drawings]

FIG. 1 is a diagram showing a mass spectrum of a DNA fragment group sample according to end bases.

FIG. 2 is a diagram showing an example, (A) is a schematic configuration diagram showing a mass spectrometer, and (B) is an enlarged plan view showing a sample plate used in the example.

FIG. 3 is a block diagram showing functions of a computer in the embodiment.

FIG. 4 is a flowchart showing the operation of the embodiment.

[Explanation of symbols]

2 Ion source 4 Sample plate 6 Coated DNA fragment group sample by end base 8 Nitrogen laser 10 Laser beam 16 Detector 20 Photodiode 24 Computer 32A, 32G, 32C, 32T Mass spectrum data storage section for each end base 34 Base sequence Determining unit 36 Display unit 38 Heterojunction determination processing unit

Claims (2)

[Claims] 1. An ion source for arranging a sample plate coated with a DNA fragment sample for each of four types of terminal bases that has undergone the Sanger reaction, and irradiating the sample at the measurement position with a laser beam to ionize the sample. A mass spectrometer equipped with an analysis unit for separating ions generated by an ion source by mass number, and a detector for detecting the separated ions, and mass spectrum data for each of four types of end bases from the mass spectrometer. A storage unit that captures and stores each end base, and a base sequence determination unit that determines a base sequence of sample DNA by reading peaks in order of mass number from four types of mass spectrum data for each end base of the storage unit An apparatus for determining a base sequence of a nucleic acid, characterized in that 2. The base sequence determination unit, when a mass spectrum obtained as a result of arranging four kinds of mass spectrum data in order of mass number has a mass number interval smaller than the mass number corresponding to one base unit, D whose sample has a heterojunction
The nucleic acid base sequence determination device according to claim 1, further comprising a heterozygous determination processing unit that processes as NA.