JPH06508927A - Method and apparatus for electrophoretic separation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method and apparatus for electrophoretic separation field of invention This invention is particularly applicable, but not limited to, DNA sequencing. The present invention relates to a method and apparatus for electrophoretic separation.

Background of the invention Currently used DNA sequencing methods generally involve sequencing hundreds of base pairs. It takes several hours of operation time to obtain useful electrophoresis results. This means that conventional radiation Applied Biosys also uses sex-labeled electrophoretic slab gel sequencing methods. Thames and Millipore (Applied Bios7rtems and Mi Use a primer labeled with a fluorescent substance, such as the one commercially available from llipore. The same applies to modern automated DNA sequencers used. It takes time transfer the DNA fragments to the desired size through a gel medium such as polyacrylamide or agar This is to move it enough to provide resolution. The required resolution is This is necessary to generate the band as separate from the n+1th band. Ru. Obviously, if the band fractionation is to be reliable, the width of the band should be It must be less than this width of separation. The characteristic width of the band is due to various reasons. Therefore, it is generally limited to the thickness of the gel, so in practice it is 0.5-1.5m. m thick gels are frequently used, thus handling maximum values of n which can exceed 1000. It may be necessary to use very long gels to achieve the resolution required for Ru.

Capillary gel electrophoresis has recently been proposed to speed up DNA sequence analysis. This is known as pneumophoresis. However, this proposal has some practical limitations. There are limitations, one of which is the adverse effect that increased electric field strength can have on resolution.

This invention particularly relates to the problem of realizing a small-scale DNA sequencing system, In particular, but not limited to, systems using fluorescent gel imaging. Ru. The real problem lies in the imaging aspect of such small-scale systems. However, it is difficult to downsize the sequencing gel system and to process small amounts of gel. This is the point where the load (loxd) is applied.

invention According to certain aspects of the invention, the gel thickness is in the range of 25 to 250 microns. An electrophoretic gel is run between two transparent plates that are fixed together in a defined manner. n) A method of electrophoretic separation is provided.

More specifically, the thickness of the gel is preferably on the order of 50 microns; The expression "on the order of 0 microns" is used in the accompanying claims and herein generally. Refers to gel thickness in the range of 25 to 100 microns.

By subtracting the gel thickness from the usual 0.5 to 1.5 mm thickness, The gel run time required to achieve resolution is substantially reduced because the thickness The subtraction results in finer bands for a given imaging system and is typically This is because the required resolution can be achieved with a shorter gel than that of a gel. fruit In use, the length of the gel may be as short as about 60 mm. [Table of approximately 5QmmJ Currently, in the appended claims and herein, the range generally ranges from 40 to 100 mm. Refers to the length of the gel.

Conveniently, the width of the gel track may also vary, for example from 2 to 10 times the thickness of the gel. It can be reduced to several times the thickness of the tile.

Therefore, in practice, gel track widths of approximately 200 mm are typically used. obtain. ``The expression approximately 200 mmJ is used in the accompanying claims and herein in general. refers to a width in the range of 100 to 500 mm.

Since the width of the gel track is reduced, several tracks of electrophoresis can be performed between two transparent plates. It is possible to run the gel.

Therefore, according to another aspect of the invention, one of them is the width of the track, the width of the track. grooved with rectangular cross-section slots that define track depth and track spacing. , an electrophoretic gel is run in parallel tracks defined between two transparent plates. An electrophoretic separation method is provided.

According to yet another aspect of the invention, the Contains two transparent plates for defining an electrophoresis gel, for receiving and running an electrophoresis gel; one plate is flat, the other is grooved with slots of rectangular cross section, the width of the track equipment for electrophoretic separation, defining track depth, track spacing, and track spacing. location is provided.

This invention can be applied to the electrolysis of a wide range of substances, including proteins, carbohydrates, and DNA fragments. Applicable to pneumophoretic separation (-dimensional), especially for DNA sequencing .

If the scale of the DNA sequencing system is determined by the multiplication factor X, then It is possible to consider in more detail how the various parameters interact. where X is less than 1, preferably X is less than 0.1.

The length of the gel is 5, the width is W1, and the thickness is t. This is the average voltage V for a time T. It is run in stream I. The electric field in the gel is V/L, and the power dissipated is V1/Lt per area.

For example, in the case of fluorescent gel imaging, area A of the gel is illuminated with blue light. child This is a cooled COD device used to visualize fluorescent bands within the gel. factor m up to the area AD which is the area of the detector or other detector (component coupling device) is optically reduced.

Scaling down the sequencing system with X increases the overall speed and Substantial positive impact on efficiency.

If the gel thickness is reduced proportionally to the length, the resolution is almost, but not completely, preserved. held. In practice, somewhat larger gels are needed to give the highest resolution possible. is necessary.

The electric field required to move the band from one side of the gel to the other remains unchanged. can run the gel X times faster. The current is per unit width of the gel. Since the power dissipation is multiplied by X, the power dissipation in the gel is scatter is reduced. Therefore, if the electric field (volt/m) is maintained, then The gel can be run for X times, or for X2 times that time if the voltage V is maintained. It is possible.

If the size is reduced by a factor of 10, the running time can theoretically be reduced by a factor of 100.

Next, regarding gel imaging, the scale factor of X is the intensity of the irradiated light. The intensity is changed by 1/X2, and the fluorescence output from each fluorescent DNA fragment corresponds to It means to change. The required reduction ratio is also scaled by X, so The light transmission efficiency is improved by 1/X2, and the overall detection signal is improved by 1/X4.

Unfortunately, things aren't actually this good. This is because the gel load also This is because it must be reduced again. Current gels are already near the limit of their capacity. overload widens the band and degrades resolution. Game To maintain the concentration of fluorescent fragments in the sample, the dose must be changed by X3. Therefore, the net sensitivity improvement is only 1/X.

So, in summary, by multiplying all scales by By scaling the photonucleotide sequencing system, what used to take Detection sensitivity scaled by 1/X can be achieved with a migration time of X to X2 times that of is obtained. This brings the overall efficiency to the scale of 1/X3. in practice You don't get much, but it's still a lot.

The following discussion concerns the case of a DNA sequencing system that uses fluorescent gel imaging. However, the numbers shown only indicate the order of magnitude of the dimensions. It should be considered as a limitation and not as a limitation.

First, assume that the COD is 1000 pixels from one side of the gel to the other, each 20 microns. 1:1 from gel to COD, which is the most efficient arrangement for light transfer. For imaging, the gel has an active width of 20 mm. The gel has 80 tracks, each They may be 250 microns apart. The track is 200 microns wide, between The gap may be 50 microns.

Since the CCD has 20 micron pixels, the band width is less than 40 microns. This is not necessary, since COD generally limits resolution to this level. be. 50 micron to keep gel resolution high and minimize length. Gel thickness is required. With a length of 60mm, the performance will be perfect. cormorant. Compared to the current 60 cm gel, this gel has one-tenth the voltage (200 cm). (200 V instead of 0), and can be run with currents of only a few milliamps. can be used to determine base sequences at much lower costs and with much safer power supplies. The process can be completed in 20-30 minutes instead of 3-5 hours.

Description of examples Based on the foregoing description, the following is an example of this development for use in DNA sequencing. Experiments have shown that this is the best example of brightness.

The device may be made of suitable materials such as glass, Pyrex, or 2 pieces of transparent, low-fluorescence plastic sheet material, approximately 20mm x 60mm. Contains 1 board. One board is flat and the other board has track positions and spacing. The defining cross section is grooved with rectangular slots. The grooves are formed in the plate by molding. They may be etched, etched, or machined. Good too. On the surface of the grooved plate, the grooves become deeper and deeper, making it easier to load the gel. It becomes a funnel with only one side to help. These grooves are several mm deep at the top edge. , the depth is reduced to a predetermined depth of 50 microns a few mm from the top of the gel. It becomes The plate contains 80 parallel grooves, each 200 microns wide, with The gap is 50 microns.

Two plates, one flat and the other grooved, are fixed together (no spacers are used). ), a suitable electrophoretic gel is introduced in the usual manner. DNA fragments go into solution and, in some cases, with a micropipette or microsyringe. Poured at the top of each truck under bot control.

DNA is collected before electrophoresis or in a sandwiched gel plate. Either by centrifugation (gel plates are small enough that this is feasible) Thus, it is pushed to the top of the gel. The gel is then run in the usual manner, but with 2 It uses a voltage of 00v and a current of only a few milliamps. The bottom of the gel, For example, 10 mm is irradiated by irradiating the edge of the gel plate, The resulting fluorescence is transmitted to a slow-scan COD detector cooled by a coupling lens. , which is currently being implemented.

The bands are imaged and the data reduced essentially as it is currently done. The same, but better defined gel geometry in this way allows each track to be Establishing the location of a truck is now faster and more reliable.

By using such equipment, gels can be run in 20-30 minutes.

This invention uses two transparent plates with grooves on one side to reduce the width of the track. As illustrated in reference, track width is a critical factor in reducing gel run time. It will be understood that this is not the case. What is important in order to shorten the electrophoresis time is , is to reduce the gel thickness.

Therefore, as defined and explained above, defining the reduced gel thickness It is also possible to implement the invention using two flat transparent plates. therefore, On the one hand, the specific value of the grooves on the transparent plate limits the position of the track on which the gel migrates. belongs to. However, it is essential to limit the track in this way. Kedana (1゜ Copy of amendment and submission of translation ability (Article 184-8 of the Patent Law) 1994 January 7-

Claims (12)

[Claims] 1. A method of electrophoretic separation, in which a combined size of 25 to 250 microns is fixed. An electrophoretic gel is run between two transparent plates that define the thickness of the gel in the range of Method. 2. Claims in which the thickness of the gel (as defined above) is of the order of 50 microns. The method described in 1. 3. An apparatus for electrophoretic separation in which two transparent plates are fixed together and For electrophoretic gels with thicknesses in the range of 25 to 250 microns between A device that both defines one track. 4. The plates have a gel thickness of the order of 50 microns between them (as defined earlier). 4. The apparatus of claim 3, wherein: 5. A method of electrophoretic separation in which parallel tracks defined between two transparent plates are used. Electrophoresis gel is run on a plate, one of which has a slot with a rectangular cross section. grooves to define track width, track depth, and track spacing. How to determine. 6. Apparatus for electrophoretic separation, which are fixed together and have parallel tracks between them. includes two transparent plates for receiving and running an electrophoresis gel, defining a , one plate is flat, the other is grooved with slots of rectangular cross section, and the track A device that defines track width, track depth, and track spacing. 7. The method according to any one of claims 1 to 6, which is applied to DNA base sequencing. law or device. 8. According to claim 7, the method is applied to DNA base sequencing using fluorescent gel imaging. method or apparatus. 9. The gel is run between two transparent plates, one of which is grooved with a slot of rectangular cross section. and between the plates (as defined above) each of the order of 50 microns. Claim 1 defining a plurality of spaced apart parallel tracks having a depth of 9. The method or apparatus according to any one of 8. 10. The tracks are each approximately 200 microns wide (as both specified above). 10. The method or apparatus of claim 9, wherein the method or apparatus has a length of approximately 60 mm. 11. Any of claims 1 to 10, wherein the gel is run at a voltage of the order of 200V. The method or apparatus described in any of the above. 12. Any one of claims 1 to 11, wherein a CCD is used for image detection. The method or apparatus described.