JPH0795033B2 - Gel electrophoresis equipment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention uses the Maxam-Gilbevt method or the Sang method to determine the nucleotide sequence of a nucleic acid.
The present invention relates to a gel electrophoresis apparatus used for determination by the ar method, which is characterized in that a nucleic acid fragment is fluorescently labeled in advance, and the nucleic acid fragment during migration is detected by the fluorescence.

(B) Conventional technology As a method for determining the nucleotide sequence of nucleic acid, Maxam-Gilbert
Method (Methods in Enzymology, vol65 pp499 ~ 561, Acadewi
c Press, 1980] and the Sangar method [Proceeding of National
Academy of Science USAvol74 pp5463 (1977))
It has been known. In these methods, a nucleic acid fragment is subjected to gel electrophoresis at the final stage of sequencing, and the migration pattern is read to determine the sequence. Until now,
Although the detection of this migration pattern has been performed by radiation detection by using a nucleic acid labeled with a radioisotope in advance, a safer fluorescent labeling method has recently been proposed. For example, Japanese Laid-Open Patent Publication Sho 60-
The 220860 is labeled with another type of fluorescent agent depending on whether the end of the nucleic acid fragment is adenine, guacin, cytosine, or thymine, so that sequencing can be performed at a time by electrophoresis using a single column. The method of doing is disclosed.

(C) Problems to be Solved by the Invention However, in the above-mentioned prior art, unless the migration distance is set sufficiently long, pattern separation becomes insufficient and an error occurs in the sequence determination result. Making the migration distance sufficiently long means that it takes a lot of time to perform the sequencing, which is extremely disadvantageous especially in the case of a nucleic acid having a long sequence.

(D) Means for solving the problem When the migration distance is shortened, it becomes difficult to distinguish the contents of the band due to the overlapping of the fluorescence spectra. In order to prevent this, in the present invention, the fluorescent light is dispersed, and the two-dimensional photoelectric conversion element array that simultaneously detects the wavelength component of the dispersed light and the position components corresponding to a plurality of lanes is measured as a continuous spectrum and is measured. The first feature is that the continuous spectrum is calculated and decomposed into components to determine the presence or absence of fluorescent light, and the second feature is that the array type photoelectric conversion element is cooled.

As the array type photoelectric conversion element used in the present invention, a usual charge coupled element or a diode array is preferable. In particular, it is possible to use a two-dimensional array as the element and simultaneously determine the sequence of a large number of nucleic acids. Further, a prism or a grating is preferable as a means for decomposing the fluorescent radiation into a continuous spectrum.

For cooling the photoelectric conversion element, for example, liquid nitrogen is used.
It is possible to cool to 195 ° C.

For the calculation of the fluorescence spectrum, for example, an ordinary personal computer can be used.

(E) Action In the present invention, the spectral spectrum is used to decompose the wavelength spectrum of the fluorescent radiation, the photoelectric conversion element array continuously receives the light, and the arithmetic processing means processes the received spectrum to determine the fluorescent light.
The arithmetic processing means processes the received light signal by the following calculation procedure.

<Calculation procedure> Nucleic acid bases (adenine, guanine, cytosine,
The fluorescent spectra of the four types of fluorescent substances labeled with each of the four types of thymine) are stored and stored with the peak wavelength closest to the wavelength of the received fluorescent spectrum having the strongest fluorescence. Multiply the spectrum by a constant so that the peak values are equal, and subtract this spectrum. From the remaining spectrum, subtract the spectrum other than the spectrum used in the previous operation by the same operation.

This process is repeated and the constant A (λ
m) / Am (m is 1 to 4) is derived. The fluorescent substance is determined to be "present" when the constant exceeds a predetermined threshold.

(F) Embodiment FIG. 1 shows the structure of an embodiment of the present invention.

1 is a gel for electrophoresis, and polyacrylamide is usually used. This is a vertical type, and is electrophoresed from top to bottom, and two electrophoretic power supplies are applied to the top and bottom. Currently, there are three types of samples, and each sample is processed in advance by the known Maxam-Gilbert method or Sanger method,
Furthermore, it is assumed that each of the terminal bases is labeled with four appropriate types of fluorescent agents. Such fluorescent agents include, for example, FI
TC (emission peak to 515 nm), NBD-F (540 nm) Texas Red
(612nm), MRITC (577nm), etc. The three types of samples after labeling are placed only on the upper part of the electrophoretic gel and electrophoresed.

The zone of nucleic acid fragments in the run is fluorescently measured when it reaches a row. An argon laser 3 emits a 488 nm beam to excite the fluorescent agent. This light is spread in a thin plane shape by the cylindrical lens of 4, reflected by the beam splitter of 5, and condensed by the lens of 6 in the electrophoretic gel to illuminate the running sample. Since the lens of 6 transmits both the excitation light and the fluorescence light, it is preferable that the lens has little chromatic aberration and is bright, and a lens for a single-lens reflex camera is suitable. Fluorescent light emitted from the sample also passes through the lens 6 and goes straight through the beam splitter 5 to be split by the splitting prism 7 (split in the vertical direction in the figure).
The position of the sample 11 is expanded in the X direction of the array type sensor having eight wavelength components. 8 here
Array type sensor, it is necessary to have sufficient sensitivity to visible light. For example, Matsushita Electric MN8210W (diode array, 398 × 496 element), Toshiba TCD205C (charge coupled device, 400 ×)
500 elements), Hamamatsu Photonics Micro Channel Plate F
1551, 1552, 1094, 1208 etc. are suitable. Although not shown in the figure, if these elements are cooled, for example, by cooling with liquid nitrogen, the thermal noise can be reduced to about 1/4 as compared with room temperature. Further, in general, the S / N can be further improved by increasing the charge storage time (light receiving time) of the array element. That is, even with weak light, the photocurrent can be increased by increasing the storage time, so the S / N
(Which is proportional to the photocurrent squared).

The signal generated from such an array element is processed by 9 electronic arithmetic circuits. In addition, 10 is an electrode for migration. An example of a signal processing process in the arithmetic circuit 10 is shown in FIG. (A) is the received fluorescence spectrum. (B)
Are four known fluorescence spectra stored in advance, and ,,, respectively correspond to the fluorescent materials 1, 2, 3, and 4. These are the peak wavelengths λ ₁ , λ ₂ , λ ₃ ,
Although λ ₄ is different, the skirts of the spectrum overlap. Now, the wavelength lambda _X which gives a peak of the received fluorescence light, the intensity A _X of (a). First, as a procedure, the one closest to λ _X is searched from λ ₁ , λ ₂ , λ ₃ , and λ ₄ . It is assumed that this is λ ₂ . At this time, (a)
If the intensity of λ _{2 at} A is λ ₂ , then make. The result is shown in FIG.

Next, the same processing as that described above is performed on (c). Since λ giving the peak of (c) is λ _XX , the one closest to λ _XX is searched from λ ₁ , λ ₃ , and λ ₄ , and if it is λ ₁ , its height A (λ ₁ ) is , The spectrum ratio of To obtain (d).

Hereafter, the same operation results in 4 spectra ...
Component is decomposed into A (λm) / Am for each component
When (m is 1 to 4) is greater than or equal to a predetermined threshold value, it is considered that there is a fluorescent substance (that is, there is a base corresponding to the fluorescent substance at the end).

Thus, even from a nucleic acid fragment that has migrated only a short distance, the terminal bases can be detected by signal processing, so that the entire base sequence can be determined in a short time by a well-known base sequence determination method.

(G) Effect According to the present invention, even if the nucleic acid fragment during migration is not sufficiently separated, the terminal base of the fragment can be clearly known by signal processing.
In other words, complete sequencing can be done in a short time.

Also, if the photoelectric conversion element is cooled, thermal noise (proportional to absolute temperature) can be reduced, so that it can have higher discrimination ability.

[Brief description of drawings]

FIG. 1 is an apparatus showing an embodiment of the present invention, and FIG. 2 is a diagram explaining a signal processing process. 1 ... Electrophoresis gel, 8 ... Array type sensor 9 ... Electronic arithmetic circuit

Claims (1)

[Claims] 1. A gel electrophoresis apparatus for determining a nucleobase sequence, a plurality of lanes for migrating a nucleobase labeled with a fluorescent substance, and a means for spectrally radiating fluorescence emission light from nucleobase fragments in the plurality of lanes. And the wavelength component of the dispersed light and the position components corresponding to a plurality of lanes are simultaneously detected.
A gel electrophoresis apparatus comprising a two-dimensional photoelectric conversion element array and a circuit for processing the electrical output of the two-dimensional photoelectric conversion element array.