WO1993000986A1 - A method and an apparatus for electrophoretic separation - Google Patents
A method and an apparatus for electrophoretic separation Download PDFInfo
- Publication number
- WO1993000986A1 WO1993000986A1 PCT/GB1992/001238 GB9201238W WO9300986A1 WO 1993000986 A1 WO1993000986 A1 WO 1993000986A1 GB 9201238 W GB9201238 W GB 9201238W WO 9300986 A1 WO9300986 A1 WO 9300986A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gel
- microns
- define
- track
- electrophoretic
- Prior art date
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44782—Apparatus specially adapted therefor of a plurality of samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
Definitions
- This invention relates to a method of and apparatus for electrophoretic separation which have particular, but not exclusive, application in DNA sequencing.
- the time is required to move the DNA fragments through a gel medium such as polyacrylamide or agar far enough to give the necessary resolution.
- the resolution required is that needed to produce the n th band as being separated from the n+1 th band.
- the width of a band must be less than this separation if the bands are to be distinguished reliably.
- the characteristic width of a band is generally limited to the thickness of the gel for a variety of reasons, so in practice gels of thickness of 0.5-1.5 mm are frequently used, necessitating the use of very long gels in order to achieve the resolution necessary to work with a maximum value of n that can be excess of 1000.
- the present invention is concerned, inter alia, with the problem of realising a scaled down DNA sequencing system, especially but not exclusively a system employing fluoresecent gel imaging.
- a scaled down DNA sequencing system especially but not exclusively a system employing fluoresecent gel imaging.
- the imaging aspects of such a scaled down system are not a problem; the difficulties arise in miniaturising the sequencing gel system and in loading the gel with scaled down volumes.
- a method of electrophoretic separation in which an electrophoretic gel is run between two transparent plates which are secured together to define a gel thickness in the range 25 to 250 microns.
- a gel thickness of the order of 50 microns is preferred, where the term "of the order of 50 microns" is used herein and in the appended claims to indicate a gel thickness generally in the range 25 to 100 microns.
- the length of the gel may be as short as about 60 mm.
- the term "about 60 mm" is used herein and in the appended claims to indicate a gel length generally in the range 40 to 100 mm.
- the width of gel tracks is also reduced to a small multiple of the gel thickness, say between 2 and 10 times the gel thickness.
- a typical gel track width of about 200 mm may be employed.
- the term "about 200 mm” is used herein and in the appended claims to indicate a width generally in the range 100 to 500 mm.
- a method of electrophoretic separation in which an electrophoretic gel is run in parallel tracks defined between two transparent plates one of which is grooved with slots of rectangular cross- section to define the track widths, track depths and track spacings.
- apparatus for electrophoretic separation which comprises two transparent plates secured together to define parallel tracks between them for receiving and running an electrophoretic gel, one plate being flat and the other being grooved with slots of rectangular cross- section to define the track widths, track depths and track spacings .
- the invention is applicable to electrophoretic separation (one-dimensional) of a wide range of materials, including proteins, carbohydrates and DNA fragments, and finds particular application in DNA sequencing.
- an area A of the gel is illuminated with blue light. This is optically demagnified by a factor m to an area A which is the area of a cooled CCD (charge coupled device) detector or other detector used to visualise the fluorescent bands in the gel.
- CCD charge coupled device
- a scale factor of X means that the illuminating light intensity
- the gel then has an active width of 20 mm assuming 1:1 imaging from the gel to the CCD, which is the most efficient arrangement for light transfer.
- the gel may have 80 tracks, each 250 microns apart.
- the tracks may be 200 microns wide and have 50 micron gaps between them.
- band widths need not be any smaller than 40 microns, since the CCD will limit resolution to about this level.
- a gel thickness of 50 microns will be necessary to keep the resolution of the gel high and minimise the length.
- a length of 60 mm would allow satisfactory performance. Compared with a current 60 cm gel, this gel can be run at one tenth of the voltage (200 V . rather than 2000 V), and a current of only a few illiamps giving a very low cost, much safer power supply, and sequencing can be completed in 20-30 minutes rather than 3-5 hours.
- the apparatus comprises two plates approximately 20 mm x 60 mm of suitable material, eg. glass, pyrex (trade mark) or suitable transparent, low fluorescent plastics sheet material.
- One plate is flat and the other plate is grooved with slots of rectangular cross-section to define the track positions and spacings.
- the grooves may be moulded, etched or machined in the plate.
- the grooves are made progressively deeper to provide a one-sided funnel to assist with gel loading. These grooves should be several mm deep at the top edge, reducing in depth to the prescribed 50 micron depth a few mm from the top of the gel.
- the plate includes 80 parallel grooves, each 200 microns wide with 50 micron gaps between them.
- the two plates, one flat and the other grooved are clamped together (no spacers are used) and a suitable electrophoretic gel is cast in the usual way.
- DNA fragments are put into a solution that is loaded into the top of each track using a micro-pipette or micro-syringe, possibly with robotic control.
- the DNA is forced onto the top of the gel either by a pre-electrophoresis running stage or by centrifugation of the gel plate sandwich (the gel plates are small enough for this to be practicable).
- the gel is then run in the usual way, but using a voltage of 200V and current of only a few milliamps.
- the bottom 10 mm, say, of the gel is illuminated by the edge illumination of the gel plates and the emitted fluorescence is transferred by coupling lenses to the cooled, slow-scan CCD detector, as in current practice.
- gels can be run in 20-30 minutes.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP92914399A EP0594673A1 (en) | 1991-07-12 | 1992-07-08 | A method and an apparatus for electrophoretic separation |
JP5502103A JPH06508927A (en) | 1991-07-12 | 1992-07-08 | Method and apparatus for electrophoretic separation |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9115073.0 | 1991-07-12 | ||
GB919115073A GB9115073D0 (en) | 1991-07-12 | 1991-07-12 | Improvements in electrophoretic separation |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1993000986A1 true WO1993000986A1 (en) | 1993-01-21 |
Family
ID=10698263
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1992/001238 WO1993000986A1 (en) | 1991-07-12 | 1992-07-08 | A method and an apparatus for electrophoretic separation |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0594673A1 (en) |
JP (1) | JPH06508927A (en) |
GB (1) | GB9115073D0 (en) |
WO (1) | WO1993000986A1 (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996013717A1 (en) * | 1994-11-01 | 1996-05-09 | Visible Genetics Inc. | Microgels for use in medical diagnosis and methods of making and using same |
WO1996018891A1 (en) * | 1994-12-15 | 1996-06-20 | University College London | Gel-matrix electrophoresis |
WO1996042012A1 (en) * | 1995-06-08 | 1996-12-27 | Visible Genetics Inc. | Nanofabricated separation matrix for analysis of biopolymers and methods of making and using same |
US5599434A (en) * | 1995-12-12 | 1997-02-04 | Visible Genetics Inc. | Electrophoresis gels and gel holders having adhesive affixed fiber spacers and method of making same |
US5618398A (en) * | 1995-12-12 | 1997-04-08 | Visible Genetics Inc. | Electrophoresis gels and gel holders having fiber spacers and method of making same |
WO1997037216A1 (en) * | 1996-03-29 | 1997-10-09 | Medical Research Council | Composite body and method of use |
WO1997043630A1 (en) * | 1996-05-17 | 1997-11-20 | Purdue Research Foundation | Miniaturized disposable gels for dna analysis |
US6110339A (en) * | 1995-06-08 | 2000-08-29 | Visible Genetics Inc. | Nanofabricated separation matrix for analysis of biopolymers and methods of making and using same |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2944127A1 (en) * | 1978-11-13 | 1980-05-22 | Desaga Nachf Erich Fecht Gmbh | Electrophoretic investigation system - by contacting carrier medium face ends with buffer solution |
US4790919A (en) * | 1984-06-28 | 1988-12-13 | E. I. Du Pont De Nemours And Company | Process for preparation of electrophoresis gel material |
-
1991
- 1991-07-12 GB GB919115073A patent/GB9115073D0/en active Pending
-
1992
- 1992-07-08 EP EP92914399A patent/EP0594673A1/en not_active Withdrawn
- 1992-07-08 JP JP5502103A patent/JPH06508927A/en active Pending
- 1992-07-08 WO PCT/GB1992/001238 patent/WO1993000986A1/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2944127A1 (en) * | 1978-11-13 | 1980-05-22 | Desaga Nachf Erich Fecht Gmbh | Electrophoretic investigation system - by contacting carrier medium face ends with buffer solution |
US4790919A (en) * | 1984-06-28 | 1988-12-13 | E. I. Du Pont De Nemours And Company | Process for preparation of electrophoresis gel material |
Non-Patent Citations (1)
Title |
---|
DETECTION AND QUANTIFICATION OF DNA IN ELECTRO- PHORESIS GELS AND BLOTS, 1991, vol. 4, VCH, Weinheim, Douglas M. Gersten et al, see page 57 - page 61 * |
Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5885431A (en) * | 1994-11-01 | 1999-03-23 | Visible Genetics Inc. | Microgels for use in medical diagnosis and methods of making and using same |
WO1996013717A1 (en) * | 1994-11-01 | 1996-05-09 | Visible Genetics Inc. | Microgels for use in medical diagnosis and methods of making and using same |
US5627022A (en) * | 1994-11-01 | 1997-05-06 | Visible Genetics Inc. | Microgels for use in medical diagnosis and holders useful in fabricating same |
AU691062B2 (en) * | 1994-11-01 | 1998-05-07 | Bayer Healthcare Llc | Microgels for use in medical diagnosis and methods of making and using same |
WO1996018891A1 (en) * | 1994-12-15 | 1996-06-20 | University College London | Gel-matrix electrophoresis |
US6071396A (en) * | 1994-12-15 | 2000-06-06 | University College London | Gel-matrix electrophoresis |
WO1996042012A1 (en) * | 1995-06-08 | 1996-12-27 | Visible Genetics Inc. | Nanofabricated separation matrix for analysis of biopolymers and methods of making and using same |
WO1996042013A1 (en) * | 1995-06-08 | 1996-12-27 | Visible Genetics Inc. | Microelectrophoresis chip for moving and separating nucleic acids and other charged molecules |
US6261430B1 (en) | 1995-06-08 | 2001-07-17 | Visible Genetics Inc. | Micro-electrophoresis chip for moving and separating nucleic acids and other charged molecules |
US6176990B1 (en) | 1995-06-08 | 2001-01-23 | Visible Genetics Inc. | Micro-electrophoresis chip for moving and separating nucleic acids and other charged molecules |
US6110339A (en) * | 1995-06-08 | 2000-08-29 | Visible Genetics Inc. | Nanofabricated separation matrix for analysis of biopolymers and methods of making and using same |
AU698921B2 (en) * | 1995-06-08 | 1998-11-12 | Bayer Healthcare Llc | Nanofabricated separation matrix for analysis of biopolymers and methods of making and using same |
US5599434A (en) * | 1995-12-12 | 1997-02-04 | Visible Genetics Inc. | Electrophoresis gels and gel holders having adhesive affixed fiber spacers and method of making same |
US5618398A (en) * | 1995-12-12 | 1997-04-08 | Visible Genetics Inc. | Electrophoresis gels and gel holders having fiber spacers and method of making same |
AU722800B2 (en) * | 1996-03-29 | 2000-08-10 | Medical Research Council | Composite body and method of use |
WO1997037216A1 (en) * | 1996-03-29 | 1997-10-09 | Medical Research Council | Composite body and method of use |
US6027625A (en) * | 1996-05-17 | 2000-02-22 | Purdue Research Foundation | Miniaturized disposable gels for DNA analysis |
US5759375A (en) * | 1996-05-17 | 1998-06-02 | Purdue Research Foundation | Miniaturized disposable gels for DNA analysis |
WO1997043630A1 (en) * | 1996-05-17 | 1997-11-20 | Purdue Research Foundation | Miniaturized disposable gels for dna analysis |
Also Published As
Publication number | Publication date |
---|---|
EP0594673A1 (en) | 1994-05-04 |
JPH06508927A (en) | 1994-10-06 |
GB9115073D0 (en) | 1991-08-28 |
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