CA2187780A1 - Method and apparatus for electrophoretic analysis - Google Patents
Method and apparatus for electrophoretic analysisInfo
- Publication number
- CA2187780A1 CA2187780A1 CA002187780A CA2187780A CA2187780A1 CA 2187780 A1 CA2187780 A1 CA 2187780A1 CA 002187780 A CA002187780 A CA 002187780A CA 2187780 A CA2187780 A CA 2187780A CA 2187780 A1 CA2187780 A1 CA 2187780A1
- Authority
- CA
- Canada
- Prior art keywords
- lane
- fluorophore
- different
- electrophoresis
- labelled
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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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
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
In a method and system for electrophoretic analysis of fluorophore-labelled substance mixtures, an electrophoresis zone (I) having at least one lane (3), and photometric detector means (4) fixed relative to this lane for detecting separated fluorophore-labelled substances as they pass the detector means (4) are used. According to the invention, there are further used means (5, 8, 9, 10) for alternately emitting to the electrophoresis lane light of different wavelengths or wavelength bands (.lambda.1, .lambda.2) corresponding to the excitation wavelengths for two or more different fluorophores, and synchronisation means for relating a detected fluorescent light emission to a respective excitation wavelength.
Description
6 TIIE 15 ~ F.~ .3l;~ 08"3 RID~ T ~ M.~BEE
WO 95f286~6 2 1 8 7 7 ~3 0 PCTISE~1~10039G
-NETHOD ANl~ APPAR~T~S FO~ EI~ECTROPHO~:TIC ~LYSIS
The presen~ invention relates to electrophoretic analysis, and more part~cularly to a method and an apparatus for electrophoretic separation of fluorescence-labelled mixtures of substa~ces, particularly nucleic acid fragments obtained in sequencing reactions.
Since some time past there are two basic methods for DNA sequence determina~ion, ~iz. the chemic~l degrada~ion method (Maxam and Gilbert, Proc. nat. Acad. Sci. U.S.A., Vol. 74, p. 560-564 (1977)) and the chain termination method ~Sanger et al., Proc. nat. Acad. Sci. U.S.A., Vol.
74, p. 5~63-5467 ~1977)).
In the chemical degr~dation method, the D~A strand ~o be analy~ed is labelled at one end with a detectable ~ag.
The sample is divided into ~our parts, each of these parts being treated with ~ respective reagent capable of cle~ing specifically at one of the four b~ses. The reac~ion conditions are adapted to obtain approximately one or a few cleava~es per molecule. Each re~ction mixture will ~hen 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 reagen~ By separating ~he four reac~io~ mixtures according co frag~ent size in parallel in four lanes on an electrophoretic gel, a detectable ladder of labelled bands representing the relative positions o~ one o~ the four bases is ob~ained.
~rom these ~ra~ment ladders the DNA sequence in question 3 0 m~y then dir~ctly be read.
In the chain termination ~ethod, which is the one most used today, the DNA fragment to be analyse~ is instead used as a template for DNA synthesis in ~our different reaction mixtures by means of a star~er sequence, or so-called primer, hybridised to the 5~-en~ of the strand, and DNA
polymerase in the presence of the four deoxynucleoside triphosphates. Each reaction mixture con~ains a low concentration of ~ chain terminator in the form of a 11 19 ~)6 TIIE 15:111 F.~ 36" 11~').3 Rll)~ T ~ M.~BEE ~ 115 WO 9512863G 2 1 8 7 72~) 0 P~'/SEg5/0039~
-respective one of the four dideoxynu~leoside triphoshate analogues which upon incorporation in ~he growing chain preve~s continued growth. Ei~her the primer, one or more of the deoxynucleoside triphosphates or the te~minators are labelled with a detec~able tag. In each reaction mixture is then obtained ~ 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 reac~ion mixtures side by side on a gel as in the Maxa~-Gilbert method gives four fragment ladders from which the desired DNA sequence thus m~y be read.
While originally, radioactive phosphorus was used as l~bel and the fragment ladders obtained after the electrophoresis were imaged on autoradiograms, the use of fluorescent labels has m~de possible more or less automatic analysis with continuous detectio~ of the fragment bands that migrate i~ the different lanes using fluorescence detectors.
US-A-4,675,095 desc~ibes an advantageous electrophoretic apparatus for this purpose where the exciting light is introduced sideways between the two gl~ss plates tha~ hold the electrophoretic gel between them, a~d ~he emitted fluorescence is detected on one side of ~he gel ~5 p~rpendicularly to the excitation light path By such an arrangement a sing~e light source may be us~d for a n~mbe~
of electrophoresis l~nes, simultaneously as background light caused by fluorescence and light scattering in the glass pl~tes themselves are avoided.
A commercial automated development of this type of apparatus for DNA seq~ence analysls based on the chain termina~ion method and using labelling with one and the same fluorescent tag, or fluorophore, is marketed ~y Pharmacia Biotech AB, Uppsala, Sweden, under ~he trade name A.L.F. DNA Sequencer~ (where A.L.F. stands for ~Au~oma~ed Laser Fluorescent"), That apparatus h~s 40 electrophoresis lanes where the excitation is effec~ed by lase~ liyh~ and the light emitted from the fluorophore-containing DNA
11 1~ 9(3 T~rE lS:11 F.~ 3 36~ ".3 RIDCI[IT 6 ~ BEE [~J~
WO gsr2863~ 2 1 8 7 / ~3 0 PCI/SE;95~003~G
fragment bands is detected by an equal number of sep~rate fixed detec~ors (photodiodes), one for each lane. ~fter the samples have been lo~ded onto the gel, the detection signals ~re collected automatically and sent to a comp~ter S for storing and processing. Since each sample requires four lanes, i.e. one lane for each of the four different base--related termina~ors, 10 different D~A samples may thus be analysed at the same t ime.
The object of the present invention is to increase ~.h~
capacity of D~A sequencin~ apparatus of this type by making it possible to analyse two or more samples simul~aneously in one and the s~me electrophoretic gel lane. According to the invention this is achieved by labelling these samples with diff~rent fluoropho~es e~cited ~t different w~velengths by means of separate excitation so~rces. By having the excitation/dete~tion of the respective fluorophores t~ke place at differen~ times, the differen~
sample-related sign~ls ma~ be distinguished from one another.
The inventive concept is, however, not re~tricted to DNA sequencing bu~ may equally well be applied to a~y electrophoretic separ~tion, not only of nucleic acids but also of other types of subst~nces, such as e.g proteins.
One aspect of the invention therefore relates tO a ~ethod o~ electrophore~ically analysing a mixture of fluorophore-labelled substances by dete~tinq the su~stances in an electrophoresis lane as they are sep~rated and pass photometric detector means ~ixed relative to the electrophoresis lane, which method is charac~erize~ by ~0 simultaneously analysing in the same ele~trophoresis lane ~wo or optionally more di~ferent substance mix~ures which are labelled by different fluorophores capable of being excited at different wavelengths or wavelength bands h~
alternately illumina~in~ the ~etection area wi~h light of these different wavelengths, ~nd relatin~ a detected fluorescent light emission to the respective fluorophore on the basis of the time for the excitation thereof.
TUE 15 :11 F.~ .362 1l~g"3 RI[)~ T & M.~BEE
w09st~636 4 PCT/SE95/00396 ~ no~her aspect of the invention relates to ~ system for electrophoretic analysis of fluorophore-labelled substance mixtures, comprising an electrophoresis zone having at leas~ one lane, and photometric detector mea~s fixed relative to this lane for detec~ing separated fluorophore-labelled substances as they pass the detector means, which system is characterized hy further comprisinq means for al~ernately emitting to said lane light of different wavelengths or wavelength bands corresponding to the excitation wa~eleng~hs of two or more different fluorophores, and synchronisation means for rela~ing a de~ected fluorescent light emission to a respective excitation wavelength.
In a p~eferred embodimen~ the method and system of the invention are adapted to nucleic acid sequencing. While the "samples n labelled wi~h di~ferent fluorophores in ~he present case are prim~rily meant to consist of fragment populations obtained by one and the same base-specific sequencing reaction o~ the a~ove-mentioned type on several different DN~ or RNA fragments to be analysed, the differently labelled samples in question may, o~ course, also he two or more diffe~en~ fragment pop~lations obtained by base-specific sequencing reactions on one and the s~me DNA or RNA fragment to be analysed. Also so-called fragment analysis may ad~antageously be per~ormed with the method and system according to the invention.
In the following the invention will be descri~ed in more detail wit~ regard to a specific, non-limiting embodimen~, reference being made to the accompanying drawing~, wherein 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 t~e invention, and Fig. 3 is a schematic diagram which shows the filter characteristic of a filter for filtering ou~ fluorescent light from the de~ec~or in the system of Fig. 2.
6 TUE 1.5:12 F.~ 3~2 ~ 23 RI[)~:I['T & M.~-BEE
WO 9512863G PCI'/SE95/0039 S
The prior art analytical system shown in Fig. 1 is of the type mentioned above with A.L.F. DNA Sequencer~
(Pharmacia BioteCh AB, Uppsala, Sweden) as a commercial example and intended for a fluorescent label. It operates ~ith a fixed laser beam and a number of ~ixed detectors arranged across the separation gel. To ~his end the illustrated system has An electrophoresis ~nit 1 having a gel 2 with a num~er of lanes, here as example four lanes, each represented by a detection zone 3 monitored by a respective photodetec~or 4 (only one shown) in the form of a photodiode. A laser S introduces a light beam 6 of wavelength ~1 into the ~el plane 2 ~or excitation of ~he fluorescent label of the DNA fragments ~ha~ pass the detec~ion zones ~. 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 ~llowing emitted fluorescence to pass through. ~he detectors 4 ar-e meant to be co~nected to a computer-~ased da~a collecting system so tha~ the DNA sequence determined in each case may be presen~ed in char~ form.
In Fig. 2, the system of Fig. 1 has been modified according ~o the invention for the use of two different fluorophores, the capacity of the system therehy being doubled. In comparison with the system in Fig. 1 this modified system has in addition to the laser 5 an extra laser 8 which emits light a~ another waveleng~h ~2 adap~ed to excite the additional fluorophore.
Further, a rotating chopper di~ 9, or "chopper", is dis~osed in the light path of the beams ~rom 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 re~lect the beam from the laser 5 of wavelength ~1 and transmi~ the beam fro~ the laser 8 o~
~avelength ~2 is placed between the chopper disc 9 and the elPctrophoretic apparatus 1. Hereby the two laser beams are combined and aligned to the same ~hysical path, i.e. the same optical axis, fo~ a co~mon incidence into the gel ~
ll l9 i9~ T~IE 15 :12 F.~ .3~2 1.~2.3 RIr)t-l~TT ~ M~BEE @1~
WO 95/28~i36 2 1 8 7 7 8 0 PCl'lSE95tO039G
For the above-mentione~ automatic data collecting syste~ to know when the respective lasers 5 and 8 ~re active, the detector electronics is synchronized with the laser chopping ~hrou~h sync pulses from the chopper disc 9.
The chopping or pulsing of the laser beams may, of course, be accomplished in other ways. For example, the rotating disc and the dichroic mirrors may be combined to a single component that provides for both chopping and be~n combination by replacing the rotating disc ~y a mechanical component o~ equivalent construction where mirrors have been substituted fo~ che solid parts of the chopper dis~.
Further, i~ order to avoid movin~ mechanics, acous~o-optical or electro-optical modul~tors may ~e used instead of the-~otating disc. It is, of course, also possible to lS accomplish an analogous function wi~h solid state components.
In the modified sys~em according to ~ig. 2, the optical filter 7 in Fig. 1 is replaced by a filter 11 capable of blocking both laser wavelengths ~1 and ~2 instead of only the wavelen~th ~1 One embodiment o~ such a filter consists of a combination of a glass ~ype absorption filter and an interference filter ~thin film ~ilter~, the absorption filter functioning as a s~bs~rate for the interference fil~er An example of a filter characteristic 2S obtained hereby ~transmission versus wavelength) is shown in ~ig. 3. As a speci~ic example, such a filter with damping at las~r wavelengths 488 nm ~argon laser) and 633 nm (He-Ne-laser or l~ser diode) h~s been obtained from Omega Optical Inc., U.S.A., which on behalf of applicant deposited an ~B ~rejection band) type interference filter - on an OG 515 ~ilter glass with high damping for 488 nm to also ~btain a high damping for a band around 633 nm.
Exemplary of a suitable fluorophore combi~a~i~n for these laser wavelengths is fluo~escein, which exhibits.a ligh~
absorption maximum at a wavelength of about 490 nm and a light emission maxi~um at a wavelength of about 530 nm, and CyS (Biological Detection Systems, Ino., Pittsburgh, PA, U.S.A.), which is a carbocyanine-based dye containlng a 11 i19 Q~ T~rE 15: 13 F.~ .3~2 ~ "3 Rll)lll!T ~ 7~BEE G~
WO 95128636 7 PC:~T/sE95too396 reactive succinimidyl ester and which exhibits a light absorption maxi~um at a wavelength of about 650 ~m and a light emission maximum at about 670 n~
With ~he analytical system shown in Fig. 2, a doubled capacity is thus oh~ained in comparison with the prior art syste~ according to Fig. 1. The capacity may, o~ course, be fu~ther increased if three, or optionally eve~ more lasers~fluorophores are used.
~he invention is, of co~rse, not restricted to the embodimen~s specifically descri~ed above and shown in the drawings, but many variations and modi~ications may be made within the scope of the general inventive concept as defined in the following claims.
WO 95f286~6 2 1 8 7 7 ~3 0 PCTISE~1~10039G
-NETHOD ANl~ APPAR~T~S FO~ EI~ECTROPHO~:TIC ~LYSIS
The presen~ invention relates to electrophoretic analysis, and more part~cularly to a method and an apparatus for electrophoretic separation of fluorescence-labelled mixtures of substa~ces, particularly nucleic acid fragments obtained in sequencing reactions.
Since some time past there are two basic methods for DNA sequence determina~ion, ~iz. the chemic~l degrada~ion method (Maxam and Gilbert, Proc. nat. Acad. Sci. U.S.A., Vol. 74, p. 560-564 (1977)) and the chain termination method ~Sanger et al., Proc. nat. Acad. Sci. U.S.A., Vol.
74, p. 5~63-5467 ~1977)).
In the chemical degr~dation method, the D~A strand ~o be analy~ed is labelled at one end with a detectable ~ag.
The sample is divided into ~our parts, each of these parts being treated with ~ respective reagent capable of cle~ing specifically at one of the four b~ses. The reac~ion conditions are adapted to obtain approximately one or a few cleava~es per molecule. Each re~ction mixture will ~hen 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 reagen~ By separating ~he four reac~io~ mixtures according co frag~ent size in parallel in four lanes on an electrophoretic gel, a detectable ladder of labelled bands representing the relative positions o~ one o~ the four bases is ob~ained.
~rom these ~ra~ment ladders the DNA sequence in question 3 0 m~y then dir~ctly be read.
In the chain termination ~ethod, which is the one most used today, the DNA fragment to be analyse~ is instead used as a template for DNA synthesis in ~our different reaction mixtures by means of a star~er sequence, or so-called primer, hybridised to the 5~-en~ of the strand, and DNA
polymerase in the presence of the four deoxynucleoside triphosphates. Each reaction mixture con~ains a low concentration of ~ chain terminator in the form of a 11 19 ~)6 TIIE 15:111 F.~ 36" 11~').3 Rll)~ T ~ M.~BEE ~ 115 WO 9512863G 2 1 8 7 72~) 0 P~'/SEg5/0039~
-respective one of the four dideoxynu~leoside triphoshate analogues which upon incorporation in ~he growing chain preve~s continued growth. Ei~her the primer, one or more of the deoxynucleoside triphosphates or the te~minators are labelled with a detec~able tag. In each reaction mixture is then obtained ~ 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 reac~ion mixtures side by side on a gel as in the Maxa~-Gilbert method gives four fragment ladders from which the desired DNA sequence thus m~y be read.
While originally, radioactive phosphorus was used as l~bel and the fragment ladders obtained after the electrophoresis were imaged on autoradiograms, the use of fluorescent labels has m~de possible more or less automatic analysis with continuous detectio~ of the fragment bands that migrate i~ the different lanes using fluorescence detectors.
US-A-4,675,095 desc~ibes an advantageous electrophoretic apparatus for this purpose where the exciting light is introduced sideways between the two gl~ss plates tha~ hold the electrophoretic gel between them, a~d ~he emitted fluorescence is detected on one side of ~he gel ~5 p~rpendicularly to the excitation light path By such an arrangement a sing~e light source may be us~d for a n~mbe~
of electrophoresis l~nes, simultaneously as background light caused by fluorescence and light scattering in the glass pl~tes themselves are avoided.
A commercial automated development of this type of apparatus for DNA seq~ence analysls based on the chain termina~ion method and using labelling with one and the same fluorescent tag, or fluorophore, is marketed ~y Pharmacia Biotech AB, Uppsala, Sweden, under ~he trade name A.L.F. DNA Sequencer~ (where A.L.F. stands for ~Au~oma~ed Laser Fluorescent"), That apparatus h~s 40 electrophoresis lanes where the excitation is effec~ed by lase~ liyh~ and the light emitted from the fluorophore-containing DNA
11 1~ 9(3 T~rE lS:11 F.~ 3 36~ ".3 RIDCI[IT 6 ~ BEE [~J~
WO gsr2863~ 2 1 8 7 / ~3 0 PCI/SE;95~003~G
fragment bands is detected by an equal number of sep~rate fixed detec~ors (photodiodes), one for each lane. ~fter the samples have been lo~ded onto the gel, the detection signals ~re collected automatically and sent to a comp~ter S for storing and processing. Since each sample requires four lanes, i.e. one lane for each of the four different base--related termina~ors, 10 different D~A samples may thus be analysed at the same t ime.
The object of the present invention is to increase ~.h~
capacity of D~A sequencin~ apparatus of this type by making it possible to analyse two or more samples simul~aneously in one and the s~me electrophoretic gel lane. According to the invention this is achieved by labelling these samples with diff~rent fluoropho~es e~cited ~t different w~velengths by means of separate excitation so~rces. By having the excitation/dete~tion of the respective fluorophores t~ke place at differen~ times, the differen~
sample-related sign~ls ma~ be distinguished from one another.
The inventive concept is, however, not re~tricted to DNA sequencing bu~ may equally well be applied to a~y electrophoretic separ~tion, not only of nucleic acids but also of other types of subst~nces, such as e.g proteins.
One aspect of the invention therefore relates tO a ~ethod o~ electrophore~ically analysing a mixture of fluorophore-labelled substances by dete~tinq the su~stances in an electrophoresis lane as they are sep~rated and pass photometric detector means ~ixed relative to the electrophoresis lane, which method is charac~erize~ by ~0 simultaneously analysing in the same ele~trophoresis lane ~wo or optionally more di~ferent substance mix~ures which are labelled by different fluorophores capable of being excited at different wavelengths or wavelength bands h~
alternately illumina~in~ the ~etection area wi~h light of these different wavelengths, ~nd relatin~ a detected fluorescent light emission to the respective fluorophore on the basis of the time for the excitation thereof.
TUE 15 :11 F.~ .362 1l~g"3 RI[)~ T & M.~BEE
w09st~636 4 PCT/SE95/00396 ~ no~her aspect of the invention relates to ~ system for electrophoretic analysis of fluorophore-labelled substance mixtures, comprising an electrophoresis zone having at leas~ one lane, and photometric detector mea~s fixed relative to this lane for detec~ing separated fluorophore-labelled substances as they pass the detector means, which system is characterized hy further comprisinq means for al~ernately emitting to said lane light of different wavelengths or wavelength bands corresponding to the excitation wa~eleng~hs of two or more different fluorophores, and synchronisation means for rela~ing a de~ected fluorescent light emission to a respective excitation wavelength.
In a p~eferred embodimen~ the method and system of the invention are adapted to nucleic acid sequencing. While the "samples n labelled wi~h di~ferent fluorophores in ~he present case are prim~rily meant to consist of fragment populations obtained by one and the same base-specific sequencing reaction o~ the a~ove-mentioned type on several different DN~ or RNA fragments to be analysed, the differently labelled samples in question may, o~ course, also he two or more diffe~en~ fragment pop~lations obtained by base-specific sequencing reactions on one and the s~me DNA or RNA fragment to be analysed. Also so-called fragment analysis may ad~antageously be per~ormed with the method and system according to the invention.
In the following the invention will be descri~ed in more detail wit~ regard to a specific, non-limiting embodimen~, reference being made to the accompanying drawing~, wherein 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 t~e invention, and Fig. 3 is a schematic diagram which shows the filter characteristic of a filter for filtering ou~ fluorescent light from the de~ec~or in the system of Fig. 2.
6 TUE 1.5:12 F.~ 3~2 ~ 23 RI[)~:I['T & M.~-BEE
WO 9512863G PCI'/SE95/0039 S
The prior art analytical system shown in Fig. 1 is of the type mentioned above with A.L.F. DNA Sequencer~
(Pharmacia BioteCh AB, Uppsala, Sweden) as a commercial example and intended for a fluorescent label. It operates ~ith a fixed laser beam and a number of ~ixed detectors arranged across the separation gel. To ~his end the illustrated system has An electrophoresis ~nit 1 having a gel 2 with a num~er of lanes, here as example four lanes, each represented by a detection zone 3 monitored by a respective photodetec~or 4 (only one shown) in the form of a photodiode. A laser S introduces a light beam 6 of wavelength ~1 into the ~el plane 2 ~or excitation of ~he fluorescent label of the DNA fragments ~ha~ pass the detec~ion zones ~. 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 ~llowing emitted fluorescence to pass through. ~he detectors 4 ar-e meant to be co~nected to a computer-~ased da~a collecting system so tha~ the DNA sequence determined in each case may be presen~ed in char~ form.
In Fig. 2, the system of Fig. 1 has been modified according ~o the invention for the use of two different fluorophores, the capacity of the system therehy being doubled. In comparison with the system in Fig. 1 this modified system has in addition to the laser 5 an extra laser 8 which emits light a~ another waveleng~h ~2 adap~ed to excite the additional fluorophore.
Further, a rotating chopper di~ 9, or "chopper", is dis~osed in the light path of the beams ~rom 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 re~lect the beam from the laser 5 of wavelength ~1 and transmi~ the beam fro~ the laser 8 o~
~avelength ~2 is placed between the chopper disc 9 and the elPctrophoretic apparatus 1. Hereby the two laser beams are combined and aligned to the same ~hysical path, i.e. the same optical axis, fo~ a co~mon incidence into the gel ~
ll l9 i9~ T~IE 15 :12 F.~ .3~2 1.~2.3 RIr)t-l~TT ~ M~BEE @1~
WO 95/28~i36 2 1 8 7 7 8 0 PCl'lSE95tO039G
For the above-mentione~ automatic data collecting syste~ to know when the respective lasers 5 and 8 ~re active, the detector electronics is synchronized with the laser chopping ~hrou~h sync pulses from the chopper disc 9.
The chopping or pulsing of the laser beams may, of course, be accomplished in other ways. For example, the rotating disc and the dichroic mirrors may be combined to a single component that provides for both chopping and be~n combination by replacing the rotating disc ~y a mechanical component o~ equivalent construction where mirrors have been substituted fo~ che solid parts of the chopper dis~.
Further, i~ order to avoid movin~ mechanics, acous~o-optical or electro-optical modul~tors may ~e used instead of the-~otating disc. It is, of course, also possible to lS accomplish an analogous function wi~h solid state components.
In the modified sys~em according to ~ig. 2, the optical filter 7 in Fig. 1 is replaced by a filter 11 capable of blocking both laser wavelengths ~1 and ~2 instead of only the wavelen~th ~1 One embodiment o~ such a filter consists of a combination of a glass ~ype absorption filter and an interference filter ~thin film ~ilter~, the absorption filter functioning as a s~bs~rate for the interference fil~er An example of a filter characteristic 2S obtained hereby ~transmission versus wavelength) is shown in ~ig. 3. As a speci~ic example, such a filter with damping at las~r wavelengths 488 nm ~argon laser) and 633 nm (He-Ne-laser or l~ser diode) h~s been obtained from Omega Optical Inc., U.S.A., which on behalf of applicant deposited an ~B ~rejection band) type interference filter - on an OG 515 ~ilter glass with high damping for 488 nm to also ~btain a high damping for a band around 633 nm.
Exemplary of a suitable fluorophore combi~a~i~n for these laser wavelengths is fluo~escein, which exhibits.a ligh~
absorption maximum at a wavelength of about 490 nm and a light emission maxi~um at a wavelength of about 530 nm, and CyS (Biological Detection Systems, Ino., Pittsburgh, PA, U.S.A.), which is a carbocyanine-based dye containlng a 11 i19 Q~ T~rE 15: 13 F.~ .3~2 ~ "3 Rll)lll!T ~ 7~BEE G~
WO 95128636 7 PC:~T/sE95too396 reactive succinimidyl ester and which exhibits a light absorption maxi~um at a wavelength of about 650 ~m and a light emission maximum at about 670 n~
With ~he analytical system shown in Fig. 2, a doubled capacity is thus oh~ained in comparison with the prior art syste~ according to Fig. 1. The capacity may, o~ course, be fu~ther increased if three, or optionally eve~ more lasers~fluorophores are used.
~he invention is, of co~rse, not restricted to the embodimen~s specifically descri~ed above and shown in the drawings, but many variations and modi~ications may be made within the scope of the general inventive concept as defined in the following claims.
Claims (11)
1. 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, 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 said different wavelengths, and relating a detected fluorescent light emission to the respective fluorophore on the basis of the time for the excitation thereof.
2 The method according to claim 1, characterized in that said substance mixtures comprise fluorophore-labelled nucleic acid fragments.
3. The method according to claim 2, characterized in that said fluorophore-labelled nucleic acid fragments are DNA
fragments obtained in sequencing reactions, particularly according to the chain termination method.
fragments obtained in sequencing reactions, particularly according to the chain termination method.
4. The method according to claim 3, characterized in that at least two different reaction mixtures from different analytical samples, which reaction mixtures are specific for the same base, are run in each electrophoresis lane.
5. The method according to claim 3, characterized in that at least two different reaction mixtures from the same analytical sample, which reaction mixtures are specific for the same base, are run in each electrophoresis lane.
6. A system for electrophoretic analysis of fluorophore-labelled substance mixtures, comprising an electrophoresis zone (1) having at least one lane (3), and photometric detector means (4) fixed relative to said lane for detecting separated fluorophore-labelled substances as they pass the detector means (4), characterized in that the system further comprises means (5, 8, 9, 10) for alternately emitting to said lane light of different wavelengths or wavelength bands (.lambda.1, .lambda.2) 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.
7. The system according to claim 6, characterized in that said light of different wavelengths is directed into the electrophoresis zone along one and the same optical axis.
8. The system according to claim 7, characterized in that the system comprises at least two lasers (5, 8) and a chopping and beam combining device (9, 10) arranged between the lasers and the electrophoresis zone.
9. The system according to claim 8, characterized in that the chopping and beam combining device comprises a rotating chopper disc (9) and a dichroic mirror (10).
10. The system according to any one of claims 6 to 9, characterized in that the system comprises filter means (11) designed to selectively prevent excitation light of at least two different wavelengths from reaching the detector means (4).
11. The system according to claim 10, characterized in that said filter means (11) comprises a combination of absorption filter and interference filter.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE9401251A SE9401251D0 (en) | 1994-04-14 | 1994-04-14 | Methods and apparatus for electrophoretic analysis |
| PCT/SE1995/000396 WO1995028636A1 (en) | 1994-04-14 | 1995-04-12 | Method and apparatus for electrophoretic analysis |
| JP7526897A JPH09512102A (en) | 1994-04-14 | 1995-04-12 | Method and apparatus for electrophoretic analysis |
| EP95916890A EP0755513A1 (en) | 1994-04-14 | 1995-04-12 | Method and apparatus for electrophoretic analysis |
| CA002187780A CA2187780A1 (en) | 1994-04-14 | 1996-10-11 | Method and apparatus for electrophoretic analysis |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE9401251A SE9401251D0 (en) | 1994-04-14 | 1994-04-14 | Methods and apparatus for electrophoretic analysis |
| CA002187780A CA2187780A1 (en) | 1994-04-14 | 1996-10-11 | Method and apparatus for electrophoretic analysis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2187780A1 true CA2187780A1 (en) | 1998-04-11 |
Family
ID=25678731
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002187780A Abandoned CA2187780A1 (en) | 1994-04-14 | 1996-10-11 | Method and apparatus for electrophoretic analysis |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0755513A1 (en) |
| JP (1) | JPH09512102A (en) |
| CA (1) | CA2187780A1 (en) |
| SE (1) | SE9401251D0 (en) |
| WO (1) | WO1995028636A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3400650B2 (en) * | 1996-06-28 | 2003-04-28 | 株式会社日立製作所 | Electrophoretic separation detection method and apparatus |
| CA3036186C (en) | 2016-09-08 | 2021-03-30 | Hemex Health, Inc. | A point-of-care system for diagnosing disease, infection or condition of a patient |
| US10349589B2 (en) | 2016-09-08 | 2019-07-16 | Hemex Health, Inc. | Diagnostics systems and methods |
| WO2020264182A1 (en) * | 2019-06-25 | 2020-12-30 | Hemex Health, Inc. | Diagnostics systems and methods |
-
1994
- 1994-04-14 SE SE9401251A patent/SE9401251D0/en unknown
-
1995
- 1995-04-12 JP JP7526897A patent/JPH09512102A/en active Pending
- 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
-
1996
- 1996-10-11 CA CA002187780A patent/CA2187780A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| WO1995028636A1 (en) | 1995-10-26 |
| EP0755513A1 (en) | 1997-01-29 |
| SE9401251D0 (en) | 1994-04-14 |
| JPH09512102A (en) | 1997-12-02 |
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