An electrophoresis pattern reading system of fluorescent type is comprised of a detachable migration unit comprising a gel functioning as a base for electrophoresis and a gel-supporting body for supporting the gel; an electrophoresis unit, to which the migration unit is mounted, for performing electrophoresis by applying migrating voltage to the gel to which a sample labeled with a fluorescent substance is added; and a reading unit including an instrumentation subunit for reading an electrophoresis pattern, to which the migration unit is mounted after electrophoresis and which receives fluorescence emitted from the fluorescent substance of the sample on the gel upon application of light to the gel. For example, a plurality of the migration units and the electrophoresis units are provided, and each of the plural migration units are mounted to one reading unit in order after electrophoresis has been performed with the respective electrophoresis units for a long period of time, thereby reading the electrophoresis pattern in a short period of time.
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19. A method for reading an electrophoresis pattern of fluorescence-detection type, comprising:
the step of pouring a sample into a gel in a migration unit, the step of mounting the migration unit in an electrophoresis unit, thereafter the step of performing electrophoresis of the sample, the step of thereafter removing the migration unit with the electrophoresed sample from the electrophoresis unit, the step of mounting the removed migration unit with the electrophoresed sample in a reading unit, and the step of reading the electrophoresis pattern of the electrophoresed sample in the migration unit by the reading unit.
24. A method for reading an electrophoresis pattern of fluorescence-detection type, comprising:
(a) pouring a plurality of samples one each into a like plurality of gels one each into in a like plurality of migration units; (b) mounting each migration unit one each in a like plurality of electrophoresis units; (c) thereafter performing electrophoresis of each sample; (d) thereafter removing one migration unit with its electrophoresed sample from a first one of the plurality of electrophoresis units; (e) mounting the removed migration unit with its electrophoresed sample in a reading unit; (f) reading the electrophoresis pattern of the electrophoresis electrophoresed sample in the migration unit by the reading unit; and (g) repeating the steps (d) through (f) for each remaining electrophoresed sample, and using the same reading unit for each reading step.
1. An electrophoresis pattern reading system of fluorescence-detection type, useful for analyzing a gel-based sample, the sample being labeled with a fluorescent substance that fluoresces upon application of light thereto, comprising:
a detachable migration unit comprising a gel functioning as a base for a sample to be analyzed by electrophoresis and a gel-supporting body for supporting the gel; an electrophoresis unit, to which the migration unit is detachably mounted, for performing electrophoresis by applying migrating voltage to the gel to which the sample labeled with a fluorescent substance is added; and a reading unit physically separate from the electrophoresis unit for reading an electrophoresis pattern, the reading unit including means for detachably mounting the migration unit detached from and apart from the electrophoresis unit after electrophoresis, and said reading unit having means for passing light to the detachably mounted migration unit and for receiving fluorescence emitted from the fluorescent substance of the sample on the gel upon application of the light.
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25. An electrophoresis pattern reading system of fluorescence-detection type as claimed in
a sample holder for detachably holding the sample apart from any electrophoresis unit and further defining the sample plane; a light source emitting the light of the first wavelength to the sample in a light source direction angularly intersecting the sample plane; a scanner for scanning by moving the light emitted by the light source and the sample holder relative to each other in a first direction, which is a direction nearly parallel or nearly perpendicular to the direction of electrophoresis, in the sample plane and in a second direction in the sample plane at a perpendicular angle with respect to the first direction, to irradiate the fluorescent substance, to excite the fluorescent substance and to produce fluorescence at the second wavelength; the light source direction being at an angle of incidence with respect to the sample plane sufficient to transmit the light to the fluorescent substance; a collector for collecting the fluorescence at the second wavelength along an emittance direction at an angle of emittance relative to the sample plane, and including a spectral filter for rejecting light of the first wavelength and passing light of the second wavelength; the light source forming the light source direction and the collector forming the emittance direction to intersect each other at a scanning location on the sample; and a sensor for sensing the fluorescence at the second wavelength after the fluorescence is collected and filtered by said collector, over a period of time for each of a plurality of the scanning locations, to produce a correlated electrical analog signal having an analog value proportional to quantity of the fluorescence at the second wavelength received during the
period of time. 31. An electrophoresis pattern reader according to claim 30, including an analog to digital converter for converting the analog value of the analog signal to a digital value; and means for outputting a scan of the digital values correlated to the scanning of the sample. 32. An electrophoresis pattern reader according to claim 30, wherein said scanner provides the first direction parallel to a direction of electrophoresis in the electrophoresis pattern and provides the second direction substantially perpendicular to the first direction. 33. An electrophoresis pattern reader according to claim 30, further including a mirror for reflecting fluorescence of the second wavelength to said collector. 34. An electrophoresis pattern reader according to claim 30, wherein said light source is a laser. 35. An electrophoresis pattern reader according to claim 31, wherein said scanner includes a vibrating mirror, said collector includes a collector lens and a condenser; wherein said sensor is a photomultiplier for photoelectrical conversion; and further including an amplifier between said sensor and said analog to digital converter. 36. An electrophoresis pattern reader according to claim 30, further including means for changing scanning speed by varying an exciting light spot size at the scanning location through changing the angle of incidence of the exciting light to change the ratio of exciting light spot size on the electrophoresis pattern in said first direction and said second direction. 37. An electrophoresis pattern reader according to claim 30, wherein said scanner moves the light emitted by the light source in only one scanning plane as one dimensional scanning in the first direction and relatively moves the light source and the sample in the second direction while maintaining substantially constant the angle of incidence defined by the angle of intersection between the light source plane and the sample plane; said sensor spatially separating light from the surface of the sample of the fluorescence from scattered light from the light source and the sample plane; said sensor producing the analog signal with spatially separate electrical components separated according to the spatially separate light components; and means for extracting only the electrical component of the fluorescence from other electrical components on the basis of the spatially separate electrical components for improving a signal to noise ratio. 38. An electrophoresis pattern reader according to claim 37, wherein the angle of intersection between the light source plane and the sample plane is sufficiently smaller than 90° to physically separate scattered/reflected light to be spaced from an optical axis of the sensor wherein the greatest intensity of received fluorescence is detected, as viewed at the sensor. 39. An electrophoresis pattern reader according to claim 38, wherein the scattered/reflected light produces intensity peaks at said sensor physically spaced from intensity peaks of the received fluorescence, disposed parallel to the sample plane. |
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The present invention relates to an electrophoresis pattern reading system of a fluorescence type and, more particularly, to a pattern reader for electrophoresis of fluorescence type, in which detachable migration units are mounted to a plurality of electrophoresis units which are electrophoresed simultaneously with each other, and an electrophoresis pattern is read by a common reader unit, thereby efficiently implementing electrophoresis and reading the electrophoresis pattern.
The pattern reader for electrophoresis of the fluorescence type has the advantage that it does not require dangerous and expensive radioisotopes.
Generally speaking, electrophoresis analysis methods using fluorescence method had been used for analysis of various genetic structures including DNA sequencing (determination of a sequence of bases of the gene), mass spectrometry of proteins such as amino acids and analysis of polymer structures. Such an electrophoresis analysis method involves implementing electrophoresis using a sample of fragments labeled with a fluorescent substance and a distribution pattern developed by electrophoresis is analyzed to thereby analyze the samples.
Description will be made of a DNA sequencing device as a representative example of an electrophoresis pattern reader.
In the DNA sequencing using the DNA sequencing device, a sample of a DNA whose structure is determined is first cut into fragments with a restriction enzyme by controlling reactivity against a chemical reaction specific to a site of each a base and labeling them with a fluorescent substance. The fragments are different in length from each other and have each a particular base selected from four kinds of bases labeled at their cut ends, consisting of adenine (A), cytosine (C), guanine (G) and thymine (T). As the fragmented DNA sample can be separated by electrophoresis in accordance with the length of the fragment, each fragment is separated by means of electrophoresis and radiated with laser light to excite the fluorescent substance labeled on each of the fragments. A measurement of the distribution in intensity of the fluorescence emitted from the fluorescent substance permits the reading of a sequence of bases, thereby determining the structure of the DNA.
FIG. 13 is a view showing an example of the distribution of DNA fragments obtained by electrophoresis. As the distance of migration varies with lengths of DNA fragments (the difference of their molecular weights), the fragments having the same molecular weights gather together as time passes, and in an electrophoresis pattern 70, as shown in FIG. 13, bands 66 are formed so as to correspond to the molecular weights of the DNA fragments. As a whole, the electrophoresis pattern is provided such that the bands 66 are formed in lanes 71, 72, 73 and 74. It is to be noted herein that, as there is the difference in molecular weight by one base or more among the bases A, G, C and T of the fragments, the distances of migration for all fragments are different from the other. Hence, it can be theoretically concluded that the bands 66 in the lanes 71 to 74 are not disposed transversely in a row with each other. For DNA pluralfrom theand a mirror 45. In other words, in instances where the main scan direction is perpendicular to the direction of electrophoresis, the cylindrical lens 44 in the convex form in section is disposed in the direction nearly parallel to the direction of electrophoresis. Likewise, as shown in FIG. 4e, it is possible to change the reading speed by varying the pixel size for reading with a ratio of the longitudinal light spot size to the transverse light spot size by changing the angle of incidence of the exciting light 42, the location of the cylindrical lens 44, and so on. Further, where it is acceptable if the resolving power in the second scanning direction would be lower than that in the first scanning direction, the data processing for the read pixel data is performed by thinning out the pixel data in the transverse direction which is the second scanning direction, namely, by thinning out the scanning position by one or more pixel data. This permits the processing of the pixel data at a high speed, which follows.
Description will be made of variants of examples.
In the above description on the examples, the scanning method is adopted in which laser beams are scanned for reading using the vibration mirror 22 in the instrumentation subunit 7. There may also be used the method using a polygonal mirror or the scanning method in which the direction of the axis of light is changed by using the optical characteristics such as refraction, interference and so on. The use of an image sensor or an array sensor as the optical sensor for detecting the fluorescence emitted for scanning by the exciting light applied to the migration segment permits part of the reading scanning to be performed electronically, thereby simplifying a scanning mechanism by scanning laser beams for reading by scanning.
FIG. 7 is a diagrammatic representation of an outline of the construction showing the essential portion in which a one-dimensional sensor of the image sensor of a semiconductor device as the optical sensor for the light-receiving section for detecting fluorescence from the migration segment. As shown in FIG. 7, fluorescence 13 emitted upon receipt of light for excitement from the light source 21 is condensed by the condenser 46 and led to an image sensor 48 of a static induction transistor type after transmitting through the optical filter 47, thereby converting the fluorescence into electrical signals. The image sensor 48 of the static induction transistor type is suitable for receiving a slight degree of fluorescence caused by a noise by a dark current as low as several orders. Further, a one-dimensional image sensor of a CCD sensor cooled may provide a light-receiving section of a likewise high sensitivity.
As described hereinabove, by using the one-dimensional image sensor as the optical sensor of the light-receiving section to be used for reading the fluorescence, the scanning in the main scanning direction can be executed electronically, thereby simplifying the scanning mechanism for reading. The use of the one-dimensional sensor can allow the scattered light 13a of the exciting light emitted on the glass surface of the migration section 5 to separate the component transmitted through the optical filter 47 from the component of the fluorescence 13 from the fluorescent substance in a physical position, thereby extracting only the component of the fluorescence 13 effectively and improving a ratio of the signal for detecting the signal for the fluorescence intensity pattern to noise. This specifically can improve the limit of detecting the fluorescent signal by one order or more.
FIG. 8 is a diagrammatical representation for describing the reading method for reading the electrophoresis pattern by using the electrophoresis pattern reading system of fluorescent type according to an embodiment of the present invention. As shown in FIG. 8, this embodiment uses the electrophoresis pattern reading system of fluorescent type composed of plural electrophoresis units 1a, 1b, . . . , 1n and one reading unit 6 (instrumentation subunit 7). The migration sections 5 are mounted to the plural electrophoresis units 1a, 1b, . . . , 1n, and each of the migration sections 5 is subjected to electrophoresis for an analyzing sample. After electrophoresis, the migration sections 5 are removed from the electrophoresis units 1a, 1b, . . . , 1n and they are mounted in order to the instrumentation subunit 7 of the reading unit 6 for reading the electrophoresis pattern. Although not shown in FIG. 8, the data processor unit (8; FIG. 1) is connected to the instrumentation subunit 7 of the reading unit 7, so that the data processor unit processes data in order for analysis of the sample from the electrophoresis pattern read. In this case, a conventional electrophoresis device can be used as an electrophoresis unit and the electrophoresed gel is mounted to the reading unit 6 and the pattern of bands on the gel can be read.
As described hereinabove, electrophoresis starts up after preparing for a sample to be analyzed and setting it to each of the migration sections 5 which in turn are mounted to the electrophoresis units 1a, 1b, . . . , 1n. Electrophoresis requires about 5 to 8 hours. After electrophoresis has been finished, the migration section 5 is removed from the electrophoresis unit and mounted to the instrumentation subunit 7 of the reading unit 6 to measure the distribution of the fluorescent substance. The migration section 5 is placed on the reading base 7c, the lid 7a is closed, and the switch of the display-operating panel 7b is pressed to generate laser output from the window 7d for reading and to scan the migration section 5 set. The reading time required is about 0.5 hours for reading a 300 mm×400 mm region.
Therefore, each researcher can implement electrophoresis by exclusively using the electrophoresis unit and read the electrophoresis pattern using the common reading unit. This arrangement can effectively use each of the units without occupying the expensive electrophoresis system of fluorescent type as a whole for a long period of time.
The embodiments according to the present invention as described hereinabove including the variants and applications can be summarized as follows:
(1) The electrophoresis pattern reading system of fluorescent type is comprised of a separate combination of the electrophoresis unit and the reading unit. Electrophoresis is performed by using the migration unit comprised of the gel functioning as a base for electrophoresis and the gel-supporting body for supporting the gel and mounting the migration unit to the electrophoresis unit. After electrophoresis has been finished, the migration unit is removed from the electrophoresis unit and mounted to the reading unit. In the reading unit, the gel in the migration unit is irradiated with light for exciting the fluorescent substance and the fluorescence emitted from the fluorescent substance of the sample on the gel is received to read the electrophoresis pattern. The electrophoresis unit is provided with the power supply for applying the migrating voltage for electrophoresis to the gel into which the sample labeled with the fluorescent substance is poured.
(2) One reading unit can be provided with a plurality of the electrophoresis units. In other words, the electrophoresis units can be provided in the number more than the reading units.
(3) In reading the electrophoresis pattern by using the electrophoresis pattern reading system of fluorescent type, the migration unit comprised of the gel-supporting body for supporting the gel is mounted to the electrophoresis unit and, after electrophoresis has been finished, the migration unit is removed from the electrophoresis unit and mounted to the reading unit, thereby reading the electrophoresis pattern.
(4) The plural migration units electrophoresed by plural different electrophoresis units are mounted in order to the common reading unit and each of the electrophoresis patterns of the gel in the migration unit is read.
(5) The reading unit is provided with a spot light source for generating light for exciting fluorescence of the fluorescent substance, a scanning section (a vibration mirror, a lens and so on) for scanning the light from the spot light source in the direction nearly parallel to the direction electrophoresis by radiation upon the gel, and a reading section having a light-receiving subsection for receiving the fluorescence from the fluorescent substance.
(6) The light-receiving subsection is composed of a light-receiving unit of a one-dimensional image sensor. The direction in which the light is received is nearly parallel to the direction of electrophoresis.
(7) When the direction nearly parallel to the direction of electrophoresis is set as a first direction and the direction perpendicular to the direction of electrophoresis is set as a second direction, the electrophoresis pattern is read by making the pixel size for reading the gel after electrophoresis longer in the first direction than in the second direction.
(8) When the direction nearly parallel to the direction of electrophoresis is set as a first direction and the direction perpendicular to the direction of electrophoresis is set as a second direction, data of a pattern of distribution of the fluorescent substance is obtained by reading the electrophoresis pattern while thinning out the distance equivalent of one pixel or more in the second direction.
(9) In irradiating the gel with light from the spot light source, light is used for the reading unit, whose width in the direction perpendicular to the direction of electrophoresis is extended by equal times to ten and several times on the basis of the reading width of the irradiated area in the direction parallel to the direction of electrophoresis.
(10) The light-receiving subsection is composed of a light-receiving unit of the image sensor of a static induction transistor type, and the direction in which the light is received is nearly parallel to the direction of electrophoresis.
(12) The data from the reading unit may be processed after being transmitted to the data processor unit provided in a location away through a data communication path.
Although the present invention has been specifically described by way of examples, it should be noted that the present invention is understood to be not restricted to the examples and to include variations and modifications within the scope of the invention without departing therefrom.
As have been described hereinabove, the present invention permits an efficient provision of image of distribution of the fluorescent substance without occupying the expensive electrophoresis system of fluorescent type as a whole in analyzing the structure of DNAs by the fluorescence method, for example, by allowing each researcher to use the electrophoresis unit for exclusive purposes and to share the common reading unit with other researchers.
Koga, Keigi, Fujimiya, Hitoshi, Nakajima, Shigeo, Nasu, Hisanori
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