JPH0737981B2 - Nucleic acid base sequencing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention uses the Maxam-Gilbert method or Sa
Regarding the method when making a decision by the ngar method.

(B) Conventional technology As a method for determining the nucleotide sequence of nucleic acid, Maxam-Gilbert
Method (Methods in Enzymyology, vol65pp499〜561, Acadewi
c Press, 1980] and Sangar method [Proceeding of Nationa
l Academy of Science USAvol 74 P 5463 ~ (1977
Year)] is 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, the nucleic acid labeled with a radioisotope in advance has been used for the detection of the migration pattern by the radiation detection, but recently, a safer fluorescence labeling method has been proposed. For example, Japanese Patent Laid-Open Publication No. Sho 60-220860 describes that a nucleic acid fragment is labeled with another type of fluorescent agent depending on whether the end of the nucleic acid fragment is adenine, guacin, cytosine, or thymine. A method for sequencing at one time by electrophoresis 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 In a method for determining a base sequence, in which a fragment of a nucleic acid base is labeled with a fluorescent substance and the base sequence is determined from the fluorescent spectrum from the fluorescent substance, (1) the label (2) The peak wavelength (λ ₁ ) closest to the wavelength having the strongest fluorescence of the received fluorescence spectrum (spectrum (a)) is stored. The process of finding the stored spectrum (denoted as spectrum (b)) having (3) The process of processing the spectrum (b) (intensity at λ ₁ is A ₁ ) according to the following equation However, A (λ ₁ ) is the intensity at λ ₁ of the spectrum (a) (4) The process of (5) and (4) in which the spectrum other than the spectrum used in the above operation is processed in the same manner from the remaining spectrum. Repeatedly, for all previously stored fluorescence spectra, The process of deriving (m is 1 to 4) (6) However, the present invention processes the detected fluorescence spectrum in the above-described calculation process to determine the fluorescence, that is, the nucleic acid. The terminal base of the fragment is determined.

(F) Examples The present invention will be described based on Examples. FIG. 1 shows an apparatus for carrying out the method of the 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. After labeling, the three types of samples are placed in the grooves on the top of the electrophoresis gel and run.

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. Here, since the lens of 6 transmits both excitation light and 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 travels straight through the beam splitter 5 to be split by the prism 7 for spectroscopy (split in the vertical direction in the figure), and each wavelength component of the array type sensor 8 The position of the sample 11 is expanded in the Y direction in the X direction. Here, it is necessary for the array type sensor of 8 to have sufficient sensitivity to visible light. For example, Matsushita Electric MN8210W (diode array, 398 × 496 element), Toshiba TCD205C (charge coupled device, 400
X 500 elements), Hamamatsu Photonics Micro Channel Plate F1551, 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 output from such an array element is processed by 9 electronic arithmetic circuits. In addition, 10 is an electrode for migration.
An example of the signal processing process by the arithmetic circuit 9 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, _let λx be the wavelength that gives the peak of the received fluorescent light in (a) and its intensity be AX. First, as a procedure, the one closest to λx is searched from λ ₁ , λ ₂ , λ ₃ and λ ₄ . This is assumed to be λ ₂ . At this time, if the intensity at λ ₂ in (a) is A (λ ₂ ), then make. The result is shown in FIG.

Then do the same for (c) as before. Since λ that gives the peak of (c) is λxx, the one closest to λxx is searched from λ ₁ , λ ₃ , and λ ₄ , and if it is λ ₁ , its height (λ ₁ ) and the spectrum of The ratio of To obtain (d).

Hereafter, the same operation results in 4 spectra ...
Component is decomposed, and when A (λm) / Am is above a predetermined threshold for each component, there is a fluorescent substance (that is, there is a base corresponding to the fluorescence at the end).
To consider.

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.

[Brief description of drawings]

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

Claims (1)

[Claims] 1. A method for determining a nucleotide sequence in which a fragment of a nucleic acid base is labeled with a fluorescent substance, and a base sequence is determined from a fluorescent spectrum from the fluorescent substance. (1) Each labeled fluorescent substance (2) The stored spectrum having the peak wavelength (designated as λ ₁ ) closest to the wavelength having the strongest fluorescence of the received fluorescence spectrum (designated as spectrum (a)) (2) Process of finding spectrum (b)) (3) process of spectrum (b) (intensity at λ ₁ is A ₁ ) according to the following formula Where A (λ ₁ ) is the intensity (4) of the spectrum (a) at λ ₁ (4) The process (5) (4) in which the spectrum other than the spectrum used in the above operation is similarly processed from the remaining spectrum Repeatedly, for all previously stored fluorescence spectra, The process of deriving (m is 1 to 4) (6) A method for determining a nucleic acid base sequence, which comprises the step of determining that a fluorescent substance is present when the threshold value is above a predetermined threshold.