JPH01209351A - Base sequence determining apparatus - Google Patents

Abstract

PURPOSE:To save labor for measurement, by arranging a labelling agent with a different fluorescence wavelength and a spectroscopic means with a different spectroscopic wavelength to reduce lanes of migration gel. CONSTITUTION:When a terminal base is A or G, a DNA fragment of the A is labelled by SF-526 (methyl derivative at 4,5-position of SF 505) or the like and a DNA fragment of the G by SF 505 or the like respectively different in the peak value of an excitation spectrum and a fluorescence spectrum. The DNA fragments thus labelled are mixed to be injected into a sample injection slot 1 to perform an electrophoresis and separation thereof in a polyacrylic amide migration gel 5. The migration gel is irradiated with an argon laser 11 to measure fluorescence with light receivers 13 and 14 which comprise optical fiber bundles 15 and 16, fluorescence selecting filters 17 and 18 with different max. wavelength of transmission light and photomultipliers 19 and 20. This enables measurement of DNA fragments with one lane of migration gel thereby saving labor for measurement.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to the process of determining the base sequence of a nucleic acid by Sanger's method, in which a primer is labeled in advance with a labeling dye such as a fluorescent substance or a phosphorescent substance. This relates to loading, in which the final stage of sequence reading from gel electrophoresis is carried out by a spectroscopic method using light emitted from the S dye.

(Prior Art) As an apparatus for measuring such a migration pattern, for example, Japanese Patent Application No. 62-80390 is known. In such a device I)
The NA fragment was fractionated according to the terminal bases A (adenine, G (guanine), T (thymine), and C (cytosynph), and after performing X gas phoresis using four lanes on an electrophoresis gel, each lane was scanned. Therefore, it was necessary to perform spectroscopic measurements.

(Problem to be solved) In the process of determining the base sequence of a nucleic acid, the terminal base (r)4
Performing electrophoresis of aVtC-fractionated DNA fragments using four lanes is complicated and also limits the number of specimens that can be processed. For this reason, the present application provides a base sequencing device that is capable of electrophoresing DNA fragments using one or two lanes and reading such slab-like electrophoresis gels, regardless of the terminal base line. (Means for Solving the Problems) The base sequencing device according to the present invention uses a plurality of labeling agents with different fluorescence wavelengths as labeling dyes, and a light receiving device is equipped with a spectroscopic means and a plurality of photodetectors each having a different spectral wavelength. (Function) In the base sequencing method using the device according to the present invention, a plurality of labeling agents with different fluorescence wavelengths are used as labeling dyes for nucleic acid fragments.I) The NA fragment is Each terminal base is labeled with a different labeling agent and run using the same lane on the electrophoresis gel.

The I)NA fragment separated on the slab gel is excited by a source incident from the normal direction of the gel surface, and emits fluorescence derived from a unique labeling agent corresponding to the terminal base.

This firefly-yt is detected by a plurality of light receiving devices set in advance to transmit light corresponding to a plurality of phosphorizing agents, and the type of terminal base is determined and the amount of fluorescence thereof is read. As a result, the pDNA sequence is determined. It shows.

In order to simplify the explanation, the present invention shows an example apparatus in which the terminal bases of the DNA fragment are two types (A and G).

The peak of the fluorescence spectrum at m is Vi520r1m''t's
Ru. The terminal base is G (lJ D
The NA fragment is 9 (carboxyethyl)-
3-byroxy-6-oxo-68-xanthe
It is labeled with ne (SF 5o5). SF'-
The excitation spectral peak of 505 is 4860 m, and the fluorescence spectral peak is 505 nm. The thus labeled DNA fragments are mixed and injected into sample injection slot 1 in FIG. 2.3 is an electrode tank to which a migration voltage is applied from a power source 4 for migration.

The sample is placed in the polyacrylamide gel/L15 at arrow 6.
The DNA fragments are separated into individual DNA fragments.

7.8.9.10 is the DNA fragment expanded in this way Argon laser 11 as an excitation light source that generates excitation light
Excitation light 12, which is a laser beam, is irradiated onto the migration gel. The argon laser 11 emits light of 488r1m.

Reference numerals 13 and 14 are light receiving devices, which include an optical fiber east 15, 16, a fluorescence selection filter/L' filter 17, 18, and a photomultiplier tube 19 (@output D1) and 20 (tgt output D1), respectively.
2) A bundle of 0 light 7 eyelids 15 and 16 made up of 9
One end surface is arranged to form a thin rectangular surface with a shape corresponding to one end surface of the electrophoretic gel/I 15, and this end surface is disposed opposite to one end surface of the electrophoretic gel 5.

The other ends of the original fiber bundles 15 and 16 are bundled into a small bundle. The fluorescence selection filter 17Vi has a maximum wavelength of transmitted light at 526 nm, and the m hanger D1 has a terminal base A? ) Allotted for detecting DNA fragments.

Similarly, the fluorescence selection filter 18 has a maximum wavelength of transmitted light at 505 nm, and the detector D2 is assigned to detect the DNA fragment of the terminal base G. The fluorescent derivative attached to the DNA fragment 10 is excited by the excitation light 12 and emits fluorescent light with a unique wavelength, and the light is repeatedly totally reflected on the surface of the electrophoretic gel 5, and the optical fiber bundle disposed on the end face. From 15.16 to fluorescence selection filter 17.18, V@output Dt.

It reaches D2.

The signal converted into an electrical signal by the detector is sent to the amplifier 21.22
The data is time-divided by the ta9 multiplexer 23 and sampled by the CPU. The CPU determines which detector has the larger signal, and identifies the component corresponding to the detector giving the largest signal as the terminal base of the DNA fragment being measured.

In addition, the fluorescent light intensity is measured using the signal of the detector determined to be large, and the amount of Eri DNA1ffT fragment is measured. As the area is traversed, flood determination is carried out on each section in turn.

Although the above is a simplified explanation, there are actually four types of bases that make up DNA'4, so I) N for each of the two types of terminal bases.
The present invention can be carried out by dividing into A fragments and using two electrophoresis lanes. In this case, for example, a polygon mirror may be used to scan the excitation light into two lanes. Furthermore, it is more preferable to use a device having four light receiving devices.

In this case, four fluorescent markers 9 are used, for example a derivative of the aforementioned 5F-505.

The receiving end faces of the single optical fibers 15 and 16 can also have other shapes. FIG. 3 is a diagram showing another embodiment of the light receiving end of the optical fiber bundle. At =9 shown in FIG. 3, the light-receiving end faces of the original fiber bundles 26 to 29 of each light-receiving device can be arranged in a mosaic pattern on the end face 26 of the electrophoretic gel.

The device according to the present invention can be used in a base sequencing method using a phosphorescent substance as a labeling agent instead of a fluorescent substance.The present invention can also be applied to an off-line measuring device.

(Effects) According to the apparatus of the present invention, DNA fragments can be measured using one lane of the electrophoresis gel, reducing the time and effort required for measurement.

In an apparatus equipped with four light receiving devices, a mechanism for scanning excitation light onto each lane is omitted. 44 half-different infants

[Brief explanation of the drawing]

FIG. 1 shows an embodiment in which the present invention is applied to an online base sequencing device, FIG. 2 shows the data processing circuit of the device shown in FIG. 1, and FIG. It is a figure which shows another embodiment. In the figure 5 Migrating gel 11 Argon laser 13.14 Light receiving device 15.16 Optical fiber east 17, 18 Fluorescence selection filter 21.22 Amplifier 23 Multiplexer 25 Migrating gel end faces 26 to 29 Optical fiber bundle patent applicant Shimadzu Corporation 11 ．． :.

Claims (1)

[Claims] (1) A mechanism for injecting an excitation light beam into a slab-like migration gel in which nucleic acid fragments labeled with a labeling dye have been electrophoretically developed or are being developed from the normal direction of the surface of the slab-like migration gel; In a base sequencing apparatus equipped with a light receiving device that receives fluorescence emitted by a labeling dye of a nucleic acid fragment developed in a gel at an end surface of a migration gel, a plurality of labeling agents having different fluorescent wavelengths are used as the labeling dye. In addition, a base sequence determining device characterized in that the light receiving device is constituted by spectroscopic means each having a different spectral wavelength and a plurality of photodetectors.