WO1995028636A1 - Method and apparatus for electrophoretic analysis - Google Patents
Method and apparatus for electrophoretic analysis Download PDFInfo
- Publication number
- WO1995028636A1 WO1995028636A1 PCT/SE1995/000396 SE9500396W WO9528636A1 WO 1995028636 A1 WO1995028636 A1 WO 1995028636A1 SE 9500396 W SE9500396 W SE 9500396W WO 9528636 A1 WO9528636 A1 WO 9528636A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lane
- different
- fluorophore
- electrophoresis
- labelled
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- 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
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
- G01N2021/6419—Excitation at two or more wavelengths
Definitions
- the present invention relates to electrophoretic analysis, and more particularly to a method and an apparatus for electrophoretic separation of fluorescence- labelled mixtures of substances, particularly nucleic acid fragments obtained in sequencing reactions .
- the DNA strand to be analysed is labelled at one end with a detectable tag.
- the sample is divided into four parts, each of these parts being treated with a respective reagent capable of cleaving specifically at one of the four bases.
- the reaction conditions are adapted to obtain approximately one or a few cleavages per molecule.
- Each reaction mixture will then contain a mixture of a number of fragments of different lengths, among them end-labelled fragments of lengths corresponding to all possible cleavage sites, i.e. the base that is specifically cleaved by the respective reagent.
- a detectable ladder of labelled bands representing the relative positions of one of the four bases is obtained. From these fragment ladders the DNA sequence in question may then directly be read.
- the DNA fragment to be analysed is instead used as a template for DNA synthesis in four different reaction mixtures by means of a starter sequence, or so-called primer, hybridised to the 5 ' -end of the strand, and DNA polymerase in the presence of the four deoxynucleoside triphosphates.
- Each reaction mixture contains a low concentration of a chain terminator in the form of a respective one of the four dideoxynucleoside triphoshate analogues which upon incorporation in the growing chain prevents continued growth.
- Either the primer, one or more of the deoxynucleoside triphosphates or the terminators are labelled with a detectable tag.
- each reaction mixture is then obtained a population of partially synthesized labelled DNA molecules having a common 5 ' -end but varying in length to a base-specific 3' -end. Electrophoretic separation of the four different reaction mixtures side by side on a gel as in the Maxam-Gilbert method gives four fragment ladders from which the desired DNA sequence thus may be read.
- a commercial automated development of this type of apparatus for DNA sequence analysis based on the chain termination method and using labelling with one and the same fluorescent tag, or fluorophore is marketed by Pharmacia Biotech AB, Uppsala, Sweden, under the trade name A.L.F. DNA SequencerTM (where A.L.F. stands for "Automated Laser Fluorescent”) .
- That apparatus has 40 electrophoresis lanes where the excitation is effected by laser light and the light emitted from the fluorophore-containing DNA fragment bands is detected by an equal number of separate fixed detectors (photodiodes) , one for each lane. After the samples have been loaded onto the gel, the detection signals are collected automatically and sent to a computer for storing and processing. Since each sample requires four lanes, i.e. one lane for each of the four different base- related terminators, 10 different DNA samples may thus be analysed at the same time.
- the object of the present invention is to increase the capacity of DNA sequencing apparatus of this type by making it possible to analyse two or more samples simultaneously in one and the same electrophoretic gel lane. According to the invention this is achieved by labelling these samples with different fluorophores excited at different wavelengths by means of separate excitation sources . By having the excitation/detection of the respective fluorophores take place at different times, the different sample-related signals may be distinguished from one another.
- the inventive concept is, however, not restricted to DNA sequencing but may equally well be applied to any electrophoretic separation, not only of nucleic acids but also of other types of substances, such as e.g. proteins.
- One aspect of the invention therefore relates to a method of electrophoretically analysing a mixture of fluorophore-labelled substances by detecting the substances in an electrophoresis lane as they are separated and pass photometric detector means fixed relative to the electrophoresis lane, which method is characterized by simultaneously analysing in the same electrophoresis lane two or optionally more different substance mixtures which are labelled by different fluorophores capable of being excited at different wavelengths or wavelength bands by alternately illuminating the detection area with light of these different wavelengths, and relating a detected fluorescent light emission to the respective fluorophore on the basis of the time for the excitation thereof.
- Another aspect of the invention relates to a system for electrophoretic analysis of fluorophore-labelled substance mixtures, comprising an electrophoresis zone having at least one lane, and photometric detector means fixed relative to this lane for detecting separated fluorophore-labelled substances as they pass the detector means, which system is characterized by further comprising means for alternately emitting to said lane light of different wavelengths or wavelength bands corresponding to the excitation wavelengths of two or more different fluorophores, and synchronisation means for relating a detected fluorescent light emission to a respective excitation wavelength.
- the method and system of the invention are adapted to nucleic acid sequencing. While the "samples" labelled with different fluorophores in the present case are primarily meant to consist of fragment populations obtained by one and the same base-specific sequencing reaction of the above-mentioned type on several different DNA or RNA fragments to be analysed, the differently labelled samples in question may, of course, also be two or more different fragment populations obtained by base-specific sequencing reactions on one and the same DNA or RNA fragment to be analysed. Also so-called fragment analysis may advantageously be performed with the method and system according to the invention.
- Fig. 1 is a schematic illustration of a prior art system for nucleic acid sequencing
- Fig. 2 is a schematic illustration of an embodiment of a system according to the invention
- Fig. 3 is a schematic diagram which shows the filter characteristic of a filter for filtering out fluorescent light from the detector in the system of Fig. 2.
- the prior art analytical system shown in Fig. 1 is of the type mentioned above with A.L.F. DNA SequencerTM (Pharmacia Biotech AB, Uppsala, Sweden) as a commercial example and intended for a fluorescent label. It operates with a fixed laser beam and a number of fixed detectors arranged across the separation gel.
- the illustrated system has an electrophoresis unit 1 having a gel 2 with a number of lanes, here as example four lanes, each represented by a detection zone 3 monitored by a respective photodetector 4 (only one shown) in the form of a photodiode.
- a laser 5 introduces a light beam 6 of wavelength ⁇ -]_ into the gel plane 2 for excitation of the fluorescent label of the DNA fragments that pass the detection zones 3. Emitted fluorescence is detected perpendicularly to the gel plane by the photodiodes 4.
- An optical filter 7 prevents the excitation light from reaching the detectors 4 while simultaneously allowing emitted fluorescence to pass through.
- the detectors 4 are meant to be connected to a computer-based data collecting system so that the DNA sequence determined in each case may be presented in chart form.
- Fig. 2 the system of Fig. 1 has been modified according to the invention for the use of two different fluorophores, the capacity of the system thereby being doubled.
- this modified system has in addition to the laser 5 an extra laser 8 which emits light at another wavelength ⁇ 2 adapted to excite the additional fluorophore.
- a rotating chopper disc 9, or “chopper” is disposed in the light path of the beams from the two lasers 5, 8, so that the beams are chopped up by alternately being allowed to pass through the chopper disc.
- a dichroic mirror 10 arranged to reflect the beam from the laser 5 of wavelength ⁇ ] _ and transmit the beam from the laser 8 of wavelength ⁇ 2 is placed between the chopper disc 9 and the electrophoretic apparatus 1.
- the two laser beams are combined and aligned to the same physical path, i.e. the same optical axis, for a common incidence into the gel 2.
- the detector electronics is synchronized with the laser chopping through sync pulses from the chopper disc 9.
- the chopping or pulsing of the laser beams may, of course, be accomplished in other ways.
- the rotating disc and the dichroic mirrors may be combined to a single component that provides for both chopping and beam combination by replacing the rotating disc by a mechanical component of equivalent construction where mirrors have been substituted for the solid parts of the chopper disc.
- acousto- optical or electro-optical modulators may be used instead of the rotating disc. It is, of course, also possible to accomplish an analogous function with solid state components.
- the optical filter 7 in Fig. 1 is replaced by a filter 11 capable of blocking both laser wavelengths ⁇ ]_ and ⁇ 2 instead of only the wavelength ⁇ .
- a filter 11 capable of blocking both laser wavelengths ⁇ ]_ and ⁇ 2 instead of only the wavelength ⁇ .
- One embodiment of such a filter consists of a combination of a glass type absorption filter and an interference filter (thin film filter) , the absorption filter functioning as a substrate for the interference filter.
- An example of a filter characteristic obtained hereby (transmission versus wavelength) is shown in Fig. 3.
- such a filter with damping at laser wavelengths 488 n (argon laser) and 633 ran (He-Ne-laser or laser diode) has been obtained from Omega Optical Inc., U.S.A., which on behalf of applicant deposited an RB (rejection band) type interference filter on an OG 515 filter glass with high damping for 488 nm to also obtain a high damping for a band around 633 nm.
- RB rejection band
- fluorescein which exhibits a light absorption maximum at a wavelength of about 490 nm and a light emission maximum at a wavelength of about 530 nm
- Cy5 Biological Detection Systems, Inc., Pittsburgh, PA, U.S.A.
- Cy5 which is a carbocyanine-based dye containing a reactive succinimidyl ester and which exhibits a light absorption maximum at a wavelength of about 650 nm and a light emission maximum at about 670 nm.
- a doubled capacity is thus obtained in comparison with the prior art system according to Fig. 1.
- the capacity may, of course, be further increased if three, or optionally even more lasers/fluorophores are used.
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Electrochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP7526897A JPH09512102A (ja) | 1994-04-14 | 1995-04-12 | 電気泳動分析用の方法および装置 |
EP95916890A EP0755513A1 (en) | 1994-04-14 | 1995-04-12 | Method and apparatus for electrophoretic analysis |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE9401251A SE9401251D0 (sv) | 1994-04-14 | 1994-04-14 | Sätt och anordning för elektroforetisk analys |
SE9401251-5 | 1994-04-14 | ||
CA002187780A CA2187780A1 (en) | 1994-04-14 | 1996-10-11 | Method and apparatus for electrophoretic analysis |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1995028636A1 true WO1995028636A1 (en) | 1995-10-26 |
Family
ID=25678731
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/SE1995/000396 WO1995028636A1 (en) | 1994-04-14 | 1995-04-12 | Method and apparatus for electrophoretic analysis |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0755513A1 (sv) |
JP (1) | JPH09512102A (sv) |
CA (1) | CA2187780A1 (sv) |
SE (1) | SE9401251D0 (sv) |
WO (1) | WO1995028636A1 (sv) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2314622A (en) * | 1996-06-28 | 1998-01-07 | Hitachi Ltd | Electrophoresis separation and detection |
US11656220B2 (en) | 2016-09-08 | 2023-05-23 | Hemex Health, Inc. | Diagnostics systems and methods |
US11701039B2 (en) | 2016-09-08 | 2023-07-18 | Hemex Health, Inc. | Diagnostics systems and methods |
US11740203B2 (en) * | 2019-06-25 | 2023-08-29 | Hemex Health, Inc. | Diagnostics systems and methods |
-
1994
- 1994-04-14 SE SE9401251A patent/SE9401251D0/sv unknown
-
1995
- 1995-04-12 EP EP95916890A patent/EP0755513A1/en not_active Withdrawn
- 1995-04-12 WO PCT/SE1995/000396 patent/WO1995028636A1/en not_active Application Discontinuation
- 1995-04-12 JP JP7526897A patent/JPH09512102A/ja active Pending
-
1996
- 1996-10-11 CA CA002187780A patent/CA2187780A1/en not_active Abandoned
Non-Patent Citations (3)
Title |
---|
ORBIT SEARCH SERVICE, File WPAT, accession number 93-380040/48, TOYOBO KK; & JP,A,05 281 191 (29-10-93) (9348) 5p. * |
ORBIT SEARCH SERVICE, File WPAT, accession number 94-013193/02, TOYOBO KK; & JP,A,05 322 770 (07-12-93) (9402) 4p. * |
ORBIT SEARCH SERVICE, File WPAT, accession number 94-013194/02, TOYOBO KK; & JP,A,05 322 771 (07-12-93) (9402) 6p. * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2314622A (en) * | 1996-06-28 | 1998-01-07 | Hitachi Ltd | Electrophoresis separation and detection |
GB2314622B (en) * | 1996-06-28 | 2000-03-29 | Hitachi Ltd | Method and apparatus for electrophoresis separation and detection |
US6132578A (en) * | 1996-06-28 | 2000-10-17 | Hitachi, Ltd. | Method and apparatus for electrophoresis separation and detection |
US11656220B2 (en) | 2016-09-08 | 2023-05-23 | Hemex Health, Inc. | Diagnostics systems and methods |
US11701039B2 (en) | 2016-09-08 | 2023-07-18 | Hemex Health, Inc. | Diagnostics systems and methods |
US11740203B2 (en) * | 2019-06-25 | 2023-08-29 | Hemex Health, Inc. | Diagnostics systems and methods |
Also Published As
Publication number | Publication date |
---|---|
JPH09512102A (ja) | 1997-12-02 |
CA2187780A1 (en) | 1998-04-11 |
EP0755513A1 (en) | 1997-01-29 |
SE9401251D0 (sv) | 1994-04-14 |
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