JPH07306136A - DNA base sequence determination device and method - Google Patents

Abstract

(57) [Abstract] [PROBLEMS] To provide a novel DNA sequencer capable of easily performing stable measurement against variations in the amount of fluorescent sample while the performance of the fluorescence detector remains unchanged. A vertically-held flat plate gel electrophoretic means having a large number of migration paths for DNA fragments, the migration path of the electrophoresis apparatus being perpendicular to the migration path from the lateral direction of the electrophoresis apparatus. In the DNA base sequence determination device, a laser light irradiation means for irradiating a laser beam for photoexcitation, and a fluorescence detection means for detecting fluorescence generated from a DNA fragment irradiated by the laser light and converting the fluorescence into an electric signal, The laser light irradiating means further includes means for dividing the laser beam from the laser light source into two laser beams having different outputs and making the laser beams incident on different positions in the vertical direction of the migration path, and the fluorescence detecting means is 2 It consists of a three-dimensional camera,
A DNA nucleotide sequencer having a means for synthesizing data measured by the two-dimensional camera.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a DNA base sequencer. More specifically, the present invention uses D
The present invention relates to an apparatus capable of efficiently and quickly determining the base sequence of NA.

[0002]

2. Description of the Related Art Gel electrophoresis is widely used as a method for determining the base sequence of DNA or the like.

Conventionally, during electrophoresis, a sample was labeled with a radioisotope and analyzed, but this method has a drawback that it takes time and labor. Furthermore, from the viewpoint of radioactivity control, maximum safety and control are always required, and analysis can be performed only in a special facility. For this reason,
Recently, a method of labeling a sample with a phosphor has been studied.

In the method using light, a DNA fragment labeled with a fluorescent dye is made to migrate in a gel. A photoexcitation part and a photodetector are provided for each migration path 15 to 20 cm below the migration start part. The DNA fragments passing through here are sequentially measured. For example, DNA of various lengths having different terminal base species is replicated by an operation by an enzymatic reaction (dideoxy method) using a DNA chain whose sequence is to be determined as a template, and these are labeled with a fluorescent substance. That is, a fluorescent substance-labeled adenine (A) fragment group, cytosine (C) fragment group, guanine (G) fragment group, and thymine (T) fragment group are obtained. These fragment groups are mixed, injected into separate migration lane grooves of the electrophoresis gel, and a voltage is applied. Since DNA is a chain polymer having a negative charge, it moves in the gel at a speed inversely proportional to the molecular weight. Since shorter (smaller molecular weight) DNA chains move faster and longer (larger molecular weight) DNA chains move slowly, DNA can be fractionated by molecular weight.

In Japanese Patent Laid-Open No. 63-21556, a line on the gel of the electrophoretic device irradiated with a laser and the arrangement direction of the photodiode array are perpendicular to the electrophoretic direction of the DNA fragments in the electrophoretic device. The DNA nucleotide sequencer having the above-mentioned structure is disclosed.

FIG. 4 is a schematic diagram for explaining the structure of the apparatus. The electrophoresis plate 74 has an electrophoresis gel (for example, polyacrylamide) sandwiched between two glass plates. The overall thickness of the electrophoretic plate is about 10 mm, but the thickness of the gel electrolyte layer itself is less than about 1 mm. An upper end of the comb-shaped gel electrolyte layer exists at a position slightly lower than the upper end of the migration plate 74. DN labeled with a fluorescent substance in the comb-shaped groove 75
Inject the A fragment.

In the apparatus shown in FIG. 4, the laser light emitted from the light source 70 is reflected by the mirror 72, and a certain point in the gel of the electrophoretic plate 74 is horizontally irradiated with the laser light. When the fluorescently labeled DNA fragment 76 that has migrated in the gel passes through this irradiation region, fluorescence is sequentially emitted from this DNA fragment. At this time, the type of terminal base can be identified from the horizontal position of fluorescence emission, the fragment length can be identified from the difference in migration time from the migration start, and the analyte can be identified by the emission wavelength.
The fluorescence from each migration path is imaged by the light receiving portion 82 of the image intensifier 80 by the lens 78. This signal is amplified, converted into an electric signal by the photodiode array 84, and measured. The sequence of each DNA fragment is calculated by computer processing the measurement results, and the target DN is obtained.
The base sequence of A is determined.

The dyes currently used for labeling DNA in the determination of base sequences are, for example, fluorescein isothiocyanate (FITC), eosin isothiocyanate (EITC), tetramethylrhodamine isothiocyanate (TMRITC) and substituted rhodamine isothiocyanate. (XRITC) and the like.

As described above, in the fluorescent DNA base sequence determination, DNA labeled with a fluorescent dye is irradiated with laser light and the fluorescence at that time is detected to determine the base sequence of the DNA. The amount of fluorescence detected varies depending on the amount. The fluorescence intensity also changes depending on the arrangement of the sample. For this reason, the detected fluorescence light amount varies greatly depending on the sample lot. Since a conventional fluorescence-type DNA base sequencer simultaneously measures many samples in one measurement, it is desirable that the range of detectable light amount is wide. In the fluorescence detection means currently used, the measurement range is determined by the noise component of the fluorescence detector and the saturated exposure amount.

Therefore, when a fluorescence detector having a predetermined measurement range is used, in the case of a sample whose fluorescence amount is too strong, the data may peak out because it exceeds the measurement threshold value.
On the other hand, if the fluorescence amount is too weak, the peak may be too small to be legible. In such a case, it is necessary to set the measurement threshold of the fluorescence detector again, but if the threshold is raised, the weak fluorescence is sacrificed, and if the threshold is lowered, the strong fluorescence is sacrificed.

[0011]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a novel DNA base sequencer capable of easily performing stable measurement against variations in the amount of fluorescent sample while the performance of the fluorescence detector remains unchanged. Is to provide.

[0012]

In order to solve the above-mentioned problems, according to the present invention, a vertically-held flat plate gel electrophoresis means having a large number of migration paths for DNA fragments, and the electrophoresis of the electrophoresis apparatus are provided. Laser light irradiation means for photoexcitation, which irradiates a laser beam so that the path is orthogonal to the migration path from the lateral direction of the electrophoretic device, and DNA irradiated by the laser light.
In a DNA base sequence determination device comprising fluorescence detection means for detecting fluorescence generated from a fragment and converting it into an electric signal, the laser light irradiation means comprises two laser beams each having a different output from a laser beam from a laser light source. Further comprising means for making the light incident on different positions in the vertical direction of the migration path, the fluorescence detecting means comprising a two-dimensional image sensor, and means for synthesizing data measured by the two-dimensional image sensor. Provided is a DNA nucleotide sequencer characterized by having.

[0013]

As described above, in the present invention, the laser beam emitted from the laser light source is split into a high-power laser beam and a low-power laser beam in front of the electrophoretic plate, and each split beam is directed in the vertical direction of the electrophoretic plate. To make them enter different positions. In the present invention, a low output laser beam is used for measuring a large fluorescence amount, and a high output laser beam is used for measuring a weak fluorescence amount. Therefore, even if a sample with a large fluorescence amount is measured, the output of the laser beam is low, so that the measured waveform does not peak out of the threshold value. On the other hand, in the case of measuring a sample having a weak fluorescence amount, the output of the laser beam is high, so that the measurement waveform becomes large and the reading becomes easy. These two types of fluorescence images are detected, and the two data are combined into fluorescence detection data. As a result, the range of detectable light amount is several times, and efficient and highly reliable measurement is possible.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in more detail below with reference to the drawings.

FIG. 1 is a partial schematic perspective view showing a schematic configuration of the DNA base sequence determination apparatus of the present invention. DN of the present invention
In the A base sequencer, a laser beam 1 emitted from a laser light source (not shown) is split by a laser beam splitter 3 into two laser beams 5 and 7 having different outputs. For example, one laser beam 5 passes through the splitter 3, and the other laser beam 7 is reflected upward by the splitter 3 and further horizontally reflected by the mirror 9. Therefore, the laser beam 5 and the laser beam 7 are incident on the gel electrolyte layer 76 of the electrophoretic plate 74 at two different positions in the vertical direction, and the gel electrolyte layer 76
Penetrate through.

The output of the laser beam 1 emitted from the laser light source (not shown) is not particularly limited. Generally, a laser beam having an output in the range of 3 mW to 10 mW is preferably used. When the laser beam 1 is split into two laser beams by the beam splitter 3, the output of each split laser beam is not particularly limited. However, it must be divided at an appropriate ratio necessary and sufficient for measuring the large fluorescence amount and the weak fluorescence amount. This division ratio is, for example, the fluorescence detector 11 used.
Regulated by the dynamic range of. Generally, if the split ratio is 1: 9, satisfactory measurement can be performed. Therefore, for example, the output of the original laser beam 1 is 1
If it is 0 mW, the output of the laser beam 7 is set to 1 mW,
The output of the laser beam 5 is preferably 9 mW.

In order to split the original laser beam into two laser beams having an output of a predetermined ratio by the beam splitter 3, for example, with respect to incident light of a plane beam splitter having a reflectance of 10% and a transmittance of 90%. It may be arranged at an angle of 45 °.

The vertical distance between the incident positions of the two split laser beams is not particularly limited. However, the fluorescence detector 11
When a two-dimensional camera is used for the above, in order to be able to sufficiently distinguish the upper and lower fluorescences, several cm, for example, 3c
It is desirable to have a spacing of m.

As described above, the DNA sample injected into the groove 75 above the migration plate is migrated downward by electrophoresis. When the DNA fragment 76 is irradiated with the split laser beam 7 having an output of 1 mW, if this fragment emits a large amount of fluorescence, a measurement waveform indicated by reference numeral 13 is obtained. A DNA fragment that emits only a weak amount of fluorescence does not emit fluorescence that can be detected by the detector 11 even when this output is irradiated with the 1 mW split laser beam 7, and therefore is not displayed in the measurement waveform of reference numeral 13. .

On the other hand, when the electrophoresed DNA fragment 76 is irradiated with the split laser beam 5 having an output of 9 mW, a measurement waveform as indicated by reference numeral 15 is obtained. Output 1m
The large fluorescence amount D measured by the W split laser beam 7
The waveform of the NA fragment peaks out with the split laser beam 5 having an output of 9 mW, and the upper waveform is cut off. However, since the DNA fragment having a weak fluorescence amount which was not measured by the split laser beam 7 having an output of 1 mW produces a sufficient fluorescence amount by the split laser beam 5 having an output of 9 mW, the detector 11
Will be detected and the measured waveform will be displayed.

By synthesizing the two types of measurement waveforms thus obtained, the final measurement result indicated by reference numeral 17 is obtained. In this way, the detection limit of the fluorescence detector can be expanded several times by changing the output of the laser beam while the performance of the fluorescence detector remains unchanged.

The two-dimensional camera used as the fluorescence detector in the present invention is of a video camera type using a charge-coupled device (CCD), and is commercially available from Delft High-Tech Co., Ltd. under a model number such as XX1560. There is. Although not shown, a circuit and the like for processing a signal obtained by this camera are connected.

Next, a method of synthesizing data will be described.
The fluorescence amount measurement data obtained by the laser beam with an output of 1 mW is A data ((a) in FIG. 2), and the fluorescence amount measurement data obtained by the laser beam with an output of 9 mW is B data ((b) in FIG. 2). And I _{A on} the vertical axis of the waveform diagram of FIG. 2A is the brightness value of peak 1 detected by the detector 11, AT is the threshold value of A data, and data exceeding this level is always saturated data in B data. It is a level that becomes. I _B in the vertical axis of the waveform view of FIG. 2 (b) is an estimated luminance value would be detected if the detector 11 is not saturated, SP is the saturation data points of the detector 11. Since the brightness relationship between A data and B data is proportional, I _B =
An equation for α I _A (where α is a constant) is obtained.

When data is switched at the threshold (AT), αI
_A> I _B '[where, I _B' is I _B data on the assumption that no saturation is obtained by the following equation, I _B
′ = NI _B0 ± N ^1/2 n (where N = integrated number, I _B0 = true I _B data, n = noise component)]. For αI _A <I _B ', thereby a step is generated in curve switching point threshold. Therefore, the variation of I _B ′ is NI _B0 −N ^1/2 n ≦ IB _B ≦ NI _B0 + N ^1/2
n, and the variation of αI _A is, α (NI _A0 -N ^1/2
n) ≦ I _A ≦ α (NI _A0 + N ^1/2 n) (where I _A0 is the true I _A data). The above α I _A > I _B ′
To satisfy, the maximum value of αI _A > the maximum value of I _B ′,
α (NI _A0 −N ^1/2 n)> NI _B0 + N ^1/2 n must be satisfied. Therefore, α> (NI _B0 + N ^1/2 n) / (NI
_A0 -N ^1/2 n) = a I _B data maximum value / I _A data minimum value. Therefore, α is obtained by the maximum value of I _B data and the minimum value of I _A data when a constant light amount is input.

FIG. 3 is a flow chart of data synthesis. Data processing begins at block 20. First, block 22 fetches data. At decision block 24, it is determined whether the A data threshold level (AT, see FIG. 2A) has been exceeded. If so, in block 26, the waveform (peak 1) data that exceeds AT is differentiated to obtain the peak apex. At the same time, the brightness I _A at that time and the appearance time t _{1 of the} apex are obtained. Next, at block 28, the mobility V ₁ of peak 1 is obtained from the above t ₁ . V ₁ is obtained by the following relational expression. V ₁ = 1 / t ₁ , where 1 represents the migration distance. Then, in block 30, the peak apex time t _{2 at} which peak 1 appears in the B data is calculated. t ₂ is calculated by the following relational expression. t ₂ = (l + 30) / V ₁ . “30” in the above equation is the distance between the low power laser beam for A data and the high power laser beam for B data (unit:
mm) is shown. Then, at block 32, I _B =
I _A obtained in α and block 26 in relation of .alpha. I _A
I _B is calculated by adding the value of The t ₂ as the vertex, obtains a normal distribution waveform of height I _B, is coupled with B data at the position of t ₂ 'and t _2''. After that, in block 34, the data is output as synthesis end data, and in block 36, the process ends. On the other hand, when it is determined in the decision block 24 that the AT has not been exceeded, the blocks 26, 28, 30 and 32 are bypassed, the block 34 is entered, the data is output as it is as synthesis end data, and the process is ended in the block 36. To do.

[0026]

As described above, according to the present invention, the laser beam emitted from the laser light source is divided into a high output laser beam and a low output laser beam before the electrophoretic plate,
The divided beams are made to enter different positions in the vertical direction of the migration plate. In the present invention, a low output laser beam is used for measuring a large fluorescence amount, and a high output laser beam is used for measuring a weak fluorescence amount. Therefore, even if a sample with a large fluorescence amount is measured, the output of the laser beam is low, so that the measured waveform does not peak out of the threshold value. On the other hand, in the case of measuring a sample having a weak fluorescence amount, the output of the laser beam is high, so that the measurement waveform becomes large and the reading becomes easy. These two types of fluorescence images are detected, and the two data are combined into fluorescence detection data. As a result, the range of detectable light amount is several times, and efficient and highly reliable measurement is possible.

[Brief description of drawings]

FIG. 1 is a partial schematic perspective view showing a schematic configuration of a DNA base sequence determination device of the present invention.

FIG. 2 is a schematic enlarged view of a measurement waveform in a relationship between luminance and time of data obtained by the device shown in FIG. 1, (a) is a fluorescence amount measurement waveform obtained by a laser beam with an output of 1 mW. And (b) is a fluorescence amount measurement waveform obtained by a laser beam with an output of 9 mW.

FIG. 3 is a flowchart showing a combining process of A data and B data shown in FIG.

FIG. 4 D disclosed in JP-A-63-21556
It is a typical block diagram of a NA base sequence determination apparatus.

[Explanation of symbols]

 1 original laser beam 3 beam splitter 5 high power split laser beam 7 low power split laser beam 9 reflection mirror 11 fluorescence detector

Claims (7)

[Claims] 1. Having multiple migration paths for DNA fragments,
Plate-type gel electrophoretic means held vertically, and laser light irradiation means for photoexcitation for irradiating laser light on the electrophoretic path of the electrophoretic device so as to be orthogonal to the electrophoretic path from the lateral direction of the electrophoretic device. A DNA base sequence determination device comprising fluorescence detection means for detecting fluorescence generated from a DNA fragment irradiated by laser light and converting it into an electric signal, wherein the laser light irradiation means respectively emit laser beams from different laser light sources. Further, there is provided means for splitting into two laser beams having an output and making the laser beams incident on different positions in the vertical direction of the migration path, and the fluorescence detecting means comprises a two-dimensional camera, and data measured by the two-dimensional camera. And a means for synthesizing DNA. 2. The DNA sequencer according to claim 1, wherein the laser beam splitting means is a beam splitter, and splits the original laser beam into two laser beams having an output ratio of 1: 9. 3. A split laser beam having a relatively low output is made to enter the migration path from a position near the top of the migration plate, and a split laser beam having a relatively high output is migrated from the same position as the original laser beam. D of Claim 2
NA base sequencer. 4. The DNA sequencing device according to claim 3, wherein the incident positions of the two split laser beams on the migration path are vertically spaced by 30 mm. 5. The two-dimensional camera is a charge coupled device (CCD).
The DNA nucleotide sequencer according to claim 1, which is a camera using 6. A laser beam having a predetermined output emitted from a laser light source is split by a beam splitter into two laser beams having a predetermined output ratio, and a split laser beam having a relatively low output is DNA. The first fluorescence generated when the fragment is incident and the second fluorescence generated when the split laser beam having a relatively high power is incident on the DNA fragment are measured, and the first fluorescence data and The base sequence of the DNA is determined by converting the second fluorescence data into a waveform and synthesizing the first fluorescence measurement waveform and the second fluorescence measurement waveform into a single waveform. DNA base sequencing method. 7. The first fluorescence data and the second fluorescence data are taken, it is determined whether or not the first fluorescence data exceeds a first threshold value, and if it is, a first peak that exceeds the threshold value. Is differentiated to obtain the peak of this first peak, the luminance I _A corresponding to the peak and the peak appearance time t ₁ , and t ₁
The mobility V ₁ of the first peak is obtained from the above, the peak apex time t _{2 at} which the first peak appears in the second fluorescence data is calculated, and the above I _A is multiplied by a predetermined constant to obtain I _B (fluorescence detection). (Estimated brightness that would be detected if the device is not saturated), and a normal distribution waveform of height I _B with t ₂ as the apex,
The method for determining a DNA base sequence according to claim 6, wherein one waveform is obtained by synthesizing this with the waveform of the second fluorescence data.