JPH04244954A - Apparatus for determining base sequence - Google Patents

Abstract

PURPOSE:To make the automation from migration to the determination of a base sequence possible by storing the fluorescent signals of all of coordinates in a scanning direction and calculating the time change quantities of the respective coordinates within a set time region and calculating coordinates imparting the max. value to set a sample migration rane. CONSTITUTION:A shark-shaped comb 33 is provided at one end of a slab-shaped migration gel and has ten sample charging slots 34. Samples to be charged are charged by the kinds of terminal bases to be labelled with a fluorescent substance FITC. When migration voltage is applied from a migration power supply 8, the samples migrate in a migration direction 14 as migration bands 16 to be separated and reach a measuring part. In the measuring part, the exciting beam of argon laser 18 is applied to the migration bands 16 and the emitted fluorescence is detected by a photomultiplier tube 28 and the signal thereof is amplified and converted to a digital value by an A/D converter 32 to be taken in a microcomputer 31. By this method, the data at respective coordinates points are collected and the time change quantity thereof is calculated to calculate coordinates imparting the max. value to set the same to a migration rane and the automation from migration to the determination of a base sequence becomes possible.

Description

[Detailed description of the invention]

0001

[Industrial Field of Application] The present invention relates to an apparatus for determining the base sequence of nucleic acids such as DNA, and in particular, the present invention relates to an apparatus for determining the base sequence of nucleic acids such as DNA, and in particular, using a shark-shaped comb, for example, to introduce fluorescently labeled nucleic acid fragments into the sample input section of a slab-like gel of a gel electrophoresis apparatus. The terminal bases are loaded into multiple migration lanes arranged close to each other and run simultaneously, and the excitation and
The present invention relates to an online base sequence determining device that detects fluorescence during electrophoresis using a detection system and determines the base sequence using a data processing device.

0002

[Prior Art] In an online base sequencing device using a slab-like gel, nucleic acid fragments that have been treated in advance by the Sanger method have terminal base types of A (adenine), G (guanine), T (thymine), and C. (cytosine) in separate electrophoresis lanes. However, the sample input position is not fixed when using a sample input member such as a shark-shaped comb. Such a DNA base sequencing device is, for example, a Bio/Technology
, Vol. 6, pp. 816-821 (1988)
Ya J. Biochemical and Bio
Physical Methods, Vol. 13
, pp. 315-323 (1986), etc. However, these documents do not mention how to determine at which coordinates of the slab gel an input sample will emerge.

0003

[Problem to be solved by the invention] In an apparatus that determines base sequences using a slab gel, it is necessary to automatically determine which position on the gel on the coordinates orthogonal to the electrophoresis direction is the optimal detection location for the input sample. It is important to determine the
Currently, the user manually sets the settings while looking at the detected signal pattern. An object of the present invention is to automatically determine the lane in which an input sample will run.

0004

[Means for Solving the Problems] The present invention is shown in FIG. 4
Reference numeral 2 denotes a fluorescence time signal storage means, into which the fluorescence signal and scanning data indicating the coordinates of the scanning direction are input, and at least until the lane in which the sample migrates is determined by the lane determination means 52 described later, the fluorescence time signal storage means stores the fluorescence signal in the direction perpendicular to the migration direction ( The fluorescence signals are stored for all measured coordinates (scanning direction). 44 is a time domain setting means for setting a time domain for lane determination;
46 is a time variation calculation means for calculating the time variation of the fluorescence signal for each measurement coordinate in a set time domain, that is, the difference between the maximum value and the minimum value of the fluorescence signal; 48 is the time when the coordinates are changed; Maximum coordinate calculating means for calculating the coordinates giving the point where the amount of variation becomes maximum; 50, slot information setting means in which information indicating the relationship between the sample input slot and the sample is set; and 52, the calculated maximum coordinates are set. lane determining means for determining a lane by associating the slot information with the slot information;
Reference numeral 54 denotes a base sequence determining means for determining the base sequence based on the fluorescence time signal of the determined lane.

[0005]

[Effect] When calculating the amount of time fluctuation for each coordinate within the set time domain, the coordinate where the sample migrates has the maximum amount of time fluctuation compared to the surrounding coordinates, so the maximum value The coordinates that give are the coordinates where the sample migrates,
In other words, it becomes a lane. Therefore, by associating the lane coordinates calculated by the local maximum coordinate calculating means 48 with the slot information known in advance at the time of inputting the sample and set in the slot information setting section 50, the optimal detection coordinates of each sample can be detected. Can be done.

[0006]

Embodiment FIG. 2 shows an embodiment of a base sequencing apparatus to which the present invention is applied. 2 is a slab-like migration gel, and polyacrylamide gel is used. Both ends of the electrophoretic gel 2 are immersed in electrode layers 4 and 6, and the electrode layers 4 and 6 contain an electrolyte. A migration voltage is applied between the electrode layers 4 and 6 by a migration power source 8 .

A shark-shaped comb 33 is provided at one end of the electrophoretic gel 2 for injecting a sample. Ten sample input slots 34 are formed in the shark-shaped comb 33, and samples are input by terminal bases A, G, C, and T using a template. The samples are four types of DNA fragments that have been labeled with FITC, a fluorescent substance, using a known method, and processed using the Sanger method so that each of the bases A, G, T, and C is placed at the end. FITC is excited by an argon laser with a wavelength of 488 nm and
It emits fluorescence with a wavelength of nm. When a migration voltage is applied from the migration power source 8, the sample becomes a migration band 16, migrates through the migration gel 2 in the migration direction 14 over time, and is separated, and reaches the measurement section.

The measuring section includes an excitation system that irradiates excitation light from an argon laser 18 that emits a 488 nm laser beam using a condensing lens 20 and a mirror 21, and if there is a migration band 16 at the location where the excitation light beam hits. Fluorescence emitted from the fluorescent substance in the electrophoretic band 16 is collected by an objective lens 22, and then filtered through a 520 nm interference filter 24 and a condensing lens 26.
A detection system is provided in which the photoelectrons are detected by a photomultiplier tube 28 via an optical fiber bundle 27. The excitation/detection system including a condenser lens 20, a mirror 21, an objective lens 22, an interference filter 24, a condenser lens 26, and an optical fiber bundle 27 has an excitation light beam irradiation position in a direction perpendicular to the migration direction 14 (scanning direction). 29) A scanning stage 30 that mechanically moves to scan the measurement line at a fixed time interval, for example, every 5 seconds.
is provided.

Detection signal (fluorescence signal) of photomultiplier tube 28
is taken into a signal processing microcomputer 31, which is a data processing device, via an amplifier and an A/D converter 32. The microcomputer 31 also receives a signal corresponding to the position of the irradiation part when the excitation light beam irradiates the measurement part on the migration gel 2 as scanning data. In this way, all fluorescent signals obtained by scanning in the scanning direction are taken into the microcomputer 31 together with location information.

Next, the operation will be explained using FIGS. 3 to 6. The sample introduced using the shark-shaped comb 33 is
As shown in FIG. 3, using template a, the terminal bases A, G, C, and T are injected in this order, and using template b, the terminal bases A, G, C, and T are injected in this order. The slots 34 into which each sample is inserted are numbered 1, 2, 3, . . . from the left side when viewed from the optical system in FIG. No samples are placed in the 5th and 10th slots. Due to the nature of the shark-shaped comb, the sample migrates in close contact with the comb. Furthermore, since the position of the shark-shaped comb 33 is arbitrary, the position of the slot 34 is not fixed.

[0011] The sample is run on the gel 2, and the scanning stage 3
An example of a fluorescence pattern obtained by scanning 0 is shown in FIG. The horizontal axis is the coordinate (perpendicular to the electrophoresis direction), in this case, 512 measurement points for a 200 mm scanning area,
That is, data sampling points are given. The vertical axis represents the intensity of detected fluorescence, and the depth represents the passage of time.

The signal 41 that appears first is the signal of excess primer, and is connected to the adjacent lane. As time passes, the influence of the excess primer signal tail becomes less and the sample signal becomes clearer. The region where the signal 42 of the DNA fragment is most clear is determined by the electrophoresis conditions, so the time region 4 for lane determination is added to this region in advance.
Set 3. For example, as a running gel, the thickness is 0.
When using a polyacrylamide gel with a diameter of 25 mm, a width of 260 mm, and a length of 373 mm, and detecting fluorescence at about 300 mm from the top end, when electrophoresis is performed under constant power electrophoresis at 20 W, it takes 2 hours to 2 hours after the start of electrophoresis. A time range up to minutes is appropriate.

FIG. 5 shows the difference between the maximum value and the minimum value of the fluorescence time signal at each coordinate point (0 to 512) in the time domain 43, that is, the amount of time variation, calculated and plotted against the coordinates. The coordinates that give the local maximum value in FIG. 5 are calculated. In this case, if the left end of the scan is 0, the coordinates of the maximum value are 100, 140
, 180, 220, 300, 340, 380 and 420
It is. The coordinates giving this maximum are A, G, C, T of template a in FIG. 3, A, G, G of template b,
These are the optimal detection coordinates for each sample of C and T. After the lane is determined, the base sequence is determined using the fluorescence time signal on that lane.

[0014]

[Effects of the Invention] In the present invention, the amount of time variation for each coordinate within a set time domain is calculated, the coordinate at which this amount of time variation gives the maximum value is found, and this is set as the lane for the sample to migrate. Therefore, even if the shape and position of the comb for introducing samples is uncertain, as long as the order of the introduced samples is known, the optimal detection coordinates will be automatically determined, making it possible to automate everything from electrophoresis after sample introduction to base sequence determination. .

[Brief explanation of the drawing]

FIG. 1 is a block diagram illustrating the present invention.

FIG. 2 is a perspective view of essential parts showing one embodiment.

FIG. 3 is a diagram showing a sample input order based on a template.

FIG. 4 is a diagram showing an example of detected fluorescent signals.

FIG. 5 is a diagram showing calculated fluorescence time fluctuation amounts.

FIG. 6 is a flowchart showing an operation procedure.

[Explanation of symbols]

42 Fluorescence time signal storage means 44
Time domain setting means 46 Time variation calculation means 48 Local maximum coordinate calculation means 50 Slot information setting means 52
Lane determination means

Claims (1)

[Claims] Claim 1: In an apparatus that performs gel electrophoresis on fluorescently labeled nucleic acid fragments and sequentially detects fluorescence during the electrophoresis to determine the development pattern and determine the base sequence, at least the lane in which the sample is electrophoresed by the lane determining means described below. A fluorescence time signal storage means for storing fluorescence signals for all measurement coordinates in a direction orthogonal to the electrophoresis direction until the time is determined, a time region setting means for setting a time region for lane determination, and a time region setting means for setting a time region for lane determination; A time variation calculation means that calculates the time variation of the fluorescence signal for each measurement coordinate in the region, that is, the difference between the maximum value and the minimum value of the fluorescence signal, and the point at which the time variation becomes maximum when the coordinates are changed. A maximum coordinate calculation means that calculates the coordinates that give lane determining means for determining the
1. A base sequence determination device comprising: base sequence determining means for determining a base sequence based on a fluorescence time signal of a determined lane.