WO1987007719A1

WO1987007719A1 - Apparatus for the detection of substances which can be excited for photon emission

Info

Publication number: WO1987007719A1
Application number: PCT/EP1987/000286
Authority: WO
Inventors: Wilhelm Ansorge; Christian Schwager; Josef Stegemann
Original assignee: Europäisches Laboratorium Für Molekularbiologie (E
Priority date: 1986-06-03
Filing date: 1987-06-03
Publication date: 1987-12-17
Also published as: DE3618605A1

Abstract

An apparatus for the detection of substances which migrate in a retarding medium, especially gel, and which can be excited for photon emission by energy irradiation by means of an excitation beam comprises a carrier for the retarding medium, in which the substances move along at least one path, a beam source for generating the excitation beam which crosses the path and a detector arrangement for the emission photons. In order to make the arrangement as sensitive as possible it is provided that at least in the area of the excitation beam the cross section, perpendicular to the direction of migration, of the retarding medium has sustantially the form of an elongate rectangle, the length of the short side of which is of the magnitude of the cross section dimensions, the excitation beam entering the cross section on one of the two short rectangle sides through an inlet part, crossing the cross section in the direction parallel to the two long rectangle sides and leaving on the other short rectangle side through an outlet part. A detector arrangement detects the emission photons passing through one of the long rectangle sides.

Description

Apparatus for the detection of substances which can be exci¬ ted for photon emission

The invention relates to an apparatus for the detection of substances which migrate in a retarding medium, especially gel, and which can be excited for photon emission by energy irradiation by means of an excitation beam, comprising a carrier for the retarding medium, through which the substan¬ ces move along at least one path, a beam source for genera¬ ting the excitation beam which crosses the path, and a de¬ tector arrangement for the emission photons.

In a large number of research processes the sample, the com¬ position of which is to be determined, is caused to move along a path (by means of, for example, electric or magnetic forces, capillary forces, applied gas or liquid pressure or the like) , a spacial distribution of the sample being ob¬ tained during this movement along a path because of the dif¬ ferent migration rates of the different substances forming the sample. Similar substances have similar migration rates and therefore form migrating bands along the path, which can be detected. If stained substances of sufficiently high concentration are used in the bands, these can be visually detected or, optionally, automatically with the aid of opti¬ cal scanning devices (scanners, photodiodes or the like). If the concentration of the substances inside the bands is low. -2- detection is often no longer possible. In such cases the substances can be radioactively labelled and an instantaneous photograph taken of the band structure of the path formed af¬ ter a certain running time by applying a photograph film to the carrier. This process, used especially in gel-electropho- resis, has a number of disadvantages, especially the large expenditure necessary (handling of radioactive substances with limited life (half life) ) and the time-consuming eva¬ luation of the photographs which can be carried out only by trained personnel and which is not substantially improved even with optical scanning of the photograph and subsequent computer evaluation (Nucleic Acids Research journal 14, 417 to 424, 1986) because of the often poor resolution of neigh¬ bouring bands. Another example of an area of application of the apparatus of the above-mentioned type is to be found in chromatograph , in which the different migration rates of the substances to be investigated are similarly used for their analysis. However, the invention is to be used preferably, but not exclusively, in the field of electrophoresis, espe- σially thin-film electrophoresis.

An apparatus of the above-mentioned type is known from the journal BIO/TECHNOLOGY 3, Hay 19S5,pages 395,396 and GB-A-2155176, in which in gel-electrophoresis for sequencing DNA the primer of the DNA fragments obtained according to the dideoxy method is labelled, with differently coloured fluorescent dye for each of the four batches corresponding to the four dideoxy nucleo- tides (c.f. also the journal Nachrichten Che . Tech. Lab. 34, 5, pages 430, 431, 1986). One of these dyes is rhodamine iso- thiocyanate (emission wavelength 580 mm-) . The DNA fragments can then be separated in a single track of a polyacrylamide gel and measured photometrically with a laser. The gel is lo¬ cated in a thin tube. The laser is arranged at the end of the gel towards which the bands run, and lights up each band as soon as it runs past it. The emission photons emitted as a result by the fluorescent substances with the corresponding wavelengths are recorded by the detector arrangement and re- registered with allocation to one of the four wavelengths. In principle, the DNA sequence can be read off directly from the sequence of the emission maxima allocated to the four deoxy- nucleotides. In practice, however, there are serious measu¬ ring problems, especially problems of resolution, as the con¬ centration of the fluorescent substances in the bands is ex¬ tremely small. Further drawbacks are variations in the electrophoretic mobility due to different dyes, spectral over¬ lap of the dyes, additional software needed to correct these problems, a need for moving parts in filtering or scanning and slow run to compensate for low counting time per track and due to time for scanning and time for change of excitation and emission filters.

The problem on which the invention is based is that of rai- ' sing the measuring sensitivity in a device of the above-men¬ tioned type.

To solve this problem the apparatus according to the inven- tion is characterized in that at least in the area of the ex¬ citation beam the cross section, perpendicular to the direc¬ tion of migration, of the retarding medium has substantially the shape of an elongate rectangle, the length of the short sides of which is preferably of the magnitude of the beam cross section dimensions, that the excitation beam enters the cross section on one of the two short rectangle sides through an inlet part, crosses the cross section in the direction pa¬ rallel to the .two long rectangle sides and leaves on the other short rectangle side through an outlet part, and that the detector arrangement detects the emission photons passing through one of the long rectangle sides. It was recognized that in the known arrangement with gel electrophoresis inside a small thin glass tube, the laser beam, on passing through the tube wall twice, o ^'entry and exit from the glass tube, generates a relatively high back¬ ground radiation (especially light diffusion) , which practi¬ cally cannot be prevented from reaching the detector arrangement fo¬ cussed on the inner chamber of the tube (by means of a dia¬ phragm or the like) . It was also recognized that the laser beam only detects a small partial volume of the gel. If the laser beam were widened to correspond approximately to the inner diameter of the tube, the laser light intensity scattered by

the glass into the detector arrangement would be raised un¬ reasonably. In the invention, on the other hand, practically the whole cross section is covered for detection -by the laser beam, which runs between the two long rectangle sides of the cross section and is at only a slight distance from these sides. The path or paths to be monitored inside the cross section can run with such a distance from the inlet and out¬ let parts that all the scattered radiation created in the area of the inlet and outlet parts can be reliably prevented from reaching the detector arrangement.

Reference should be made to the fact that the excitation beam is particularly preferably a laser beam, because of the high intensities which can be achieved, although an exci- tation beam emitted by an incoherent beam source is also pos¬ sible. In accordance with the multiplicity of possible exci¬ tation mechanisms (chemiluminiscence; thermoluminiscence or the like) , other types of excitation beams are also possible, for example X-rays; the wavelength of the emission photons also does not have to lie within the visible range.

In order for the excitation beam to enter- the cross section and to exit therefrom with as little loss as possible, it is suggested that at least one of the inlet and outlet parts has parallel end faces perpendicular to the axis of the exci¬ tation beam. The coupling-in and coupling-out of the exci¬ tation from the inlet or outlet parts respectively are im¬ proved still further by the measures that the end faces are optically polished.

It is further suggested that in the retarding medium the po¬ larisation direction of the excitation beam runs substantially parallel to the direction of the detector arrangement. This brings the advantage of minimum scattered radiation from the excitation beam in the retarding medium, especially gel, as then the intensity of the Raman scattering and the Ra leigh scattering in the direction towards the detectors is minimal. To obtain this polarisation direction even with conventional lasers with vertical polarisation, it is proposed that the beam source comprises a 90° polariser.

To enable exact adjustment of the beam path of the excita¬ tion beam through the inside of the housing with structurally simple means, it is suggested to provide a small plate, which can be rotated around an axis of rotation substantially pa¬ rallel to the direction of migration and has plane-parallel side faces, in the excitation beam between the beam source and the retarding medium.

The arrangement of at least two parallel paths, which are each detected separately by the detector arrangement is es- pecially preferred. With a single excitation beam several paths can, in this way, be monitored in on-line operation. In DNA sequencing, in the processes both according to Maxam and Gilbert and according to Sanger, four batches, corresponding to the four nucleotide bases G, A, T, C, of the DNA to be in- vestigated are prepared and subjected to electrophoresis with one path for each batch. With the arrangement according to the invention these four paths can be monitored simultaneous¬ ly by a single laser beam. As one detector arrangement is al¬ located to each path, the same dye, especially fluorescent dye, can be used for all the batches. It is especially prefe¬ rable thatthis dye be a fluoro dye,especially fluorescein and tetrame- thylrhc3amine,attached to the primer. This is characterized by high absorption and therewith greatefficiency as well as by small band width (52 nm line width at half maximum intensity) with an absorption wavelength of 560 nm and an emission wave¬ length of 575 nm. By means of this on-line measuring of the four separate paths with optimum fluorescent dye in each path and separate registration of the emission of each path there results an exceptionally good separation of the individual emission peaks of directly successive bands both inside a single path as from path to path. To further suppress the signal background by preventing stray ra¬ diation from reaching the detector arrangement comprises a collec¬ ting and imaging optical system having an aperture in the image plane allocated to each path for collimating stray ra- diation. However, it is also possible to use a detector ar¬ rangement without limiting aperture and/or without a collec¬ ting device. In this case it is preferred to arrange on one of said two long rectangle sides in front of each path a re¬ ceiving end of a light guide means. To enhance the signal-to- noise ratio filters, especially Rayleigh- or Raman-filters, sensitive for the wavelength of the photons to be detected can be used.

The arrangement according to the invention can be used with particular advantage for DNA sequencing with division into four paths, as described above. Because of the substantial improvement of the signal-to-noise ratio aimed at according to the invention, the apparatus according to the invention can, however, be used successfully for the known four-colour method described above with a single path. In this connection it is also possible to analyse several samples at the same time in adjacent paths each with four-colour mixture by means of the apparatus according to the invention. The respective detector arrangement of each path then comprises several sen- sors corresponding to the number of different emission photon wavelengths. By appropriate beam filtering the photons of the allocated wavelengths can be supplied to the sensors. However, it is also possible to split the beam (by prisms, groove gra¬ tings, Fresnel zone plates) into partial beams of different wavelengths travelling in different direction. The sensors can then be position-sensitive detectors, preferably in the form of a charge-coupled-device line, a photodiode line or a photo ultiplier with subdivided position areas.

To be able to effect a rapid exchange of the housing contai¬ ning the retarding medium for the substances to be investiga¬ ted, especially a gel layer, for quick preparation of the next measurement without time-consuming adjustment operations, a holder for the carrier is connected rigidly with a holder for the beam source. The carrier for the housing-can be con¬ nected with the holder for the beam source via an optical bench, an optical table or the like.

The housing preferred for gel electrophoresis is the housing serving as a gel-carrier according to the features of claim 15. The gel layer can be produced according to DE-OS 30 24 288; the further of the two parallel plates from the detector arrangement can be applied to a thermostatic plate according to German Patent 30 46 729 or can even form a wall of this thermostatic plate. The construction of the carrier as the housing enclosing the gel is particularly preferable but the invention is also applicable to non-masked gel layers on a carrier. Such gel layers generally have no completely con¬ stant thickness along the respective path; as what matters in the on-line process according to the invention is only the relative sequence to the bands, however, this uneven thick- ness can be accepted.

The apparatus according to the invention is preferably con¬ structed for gel electrophoresis and fluorescent excitation by laser beam.

The apparatus according to the invention is particularly suitable for DNA sequencing, although other uses are possible. DNA sequencing can be carried out according to the invention in on-line operation with automatic evaluation, the high sen- sitivity being worth particular emphasis. An "instantaneous photograph" of the bands on the carrier can be made if neces¬ sary, either the excitation beam being displaced in the mi¬ gration direction in relation to the carrier or vice versa for the corresponding scanning of the band pattern.

Preferred exemplary embodiments of the invention are des¬ cribed below with the aid of drawings, in which: Fig. 1 is a schematic view of the whole system;

Fig. 2 is a partially sectional detailed view of -the arrange¬ ment in Fig. 1 in the area of the gel carrier;

Fig. 3 is a perspective, partially broken-away, simplified representation of a holder for the gel carrier and

Fig. 4 shows a partial perspective view of another embodi- ment of the invention with spectral detection distri¬ bution.

The general construction of a first exemplary embodiment of the apparatus according to the invention can be seen in Fig. 1. The reference numeral 10 is given to a laser (Argon laser, Spectra-Physics, Model 166, wavelength 514.5 nm) , which emits an excitation beam 12 with vertical polarisation, indicated by a directional arrow A'. A 90° polarizer 14 in the beam path between the laser 10 and an electrophoresis gel carrier 16 rotates the polarisation direction to horizontal (arrow B¹). A focussing system 18 follows, which reduces the beam cross section from 2 mm diameter to 0.3 to 0.4 mm. The laser output lies between 1 and 800 mW. The parts 10, 14 and 18 form a beam source 20. They are mounted in the usual way on a car- rier, not shown in any more detail, especially on optical table or optical bench, on which the plate 16 is also rigidly mounted, especially with use of the holder 21 shown in Fig. 3, This consists of a base plate 24 connected rigidly with the carrier, especially fixed by means of the fastening holes 22 in a manner not shown, to which base _.plate a bearing body 26, substantially U-shaped in horizontal cross section, is rigid¬ ly attached. The respective plate 16 is applied to vertical end faces 28 of the side legs 30 of the ϋ-shape and rigidly attached to the side legs 30, for example by means of spring members, which, on the one hand, engage with the cover plate 32 of the carrier 16 facing away from the body 26 and, on the other hand, engage in fastening recesses 34 in the side legs 30. The lower ends of the two side legs 30 are each provided with a recess 36, into which a tray (not shown) can be inser¬ ted, the tray receiving the lower end of the plate 16 in the usual way and being provided with the lower electrode for ge- nerating the electrical field for the electrophoresis.

As Fig. 2 shows, a small glass plate 38 with a vertical axis of rotation 40 parallel to the plane-parallel plate sides 42 can also be provided in the beam path between the laser 10 and carrier 16, in order to enable parallel displacement of the excitation beam 12 in the horizontal direction correspon¬ ding to the angle of rotation of the plate 38. The beam 12 is doubly deflected in the known way on passing through the two side faces 42 each time by the same angle, but in opposite directions, so that the desired parallel displacement in the horizontal plane is obtained. The possibility of exact ad¬ justment of the excitation beam 12 is advantageous, because the beam cross section, with a diameter of, for example, from 0.3 to 0.4 mm, almost corresponds to the gel layer thickness of approximately 0.5 mm.

The gel layer thickness is determined by the correspondingly thick spacing strips 46 indicated in Figures 1 and 2 and pa¬ rallel to the migration direction W, which strips 46 are ar- ranged at the vertical longitudinal edges of two parallel plates 48 and 50 of uniform shape and which hold these at the distance from each other. At least one of the two plates 48, 50, namely the plate 50 facing a detector arrangement 52, consists of low fluorescent, colourless glass with highly ^* precise, plane-parallel side faces (e.g. B 270, DESAG company, FRG) . Gel production can be effected according to DE-OS 30 24 288. The plate 48 facing away from the detector arrangement 52 can be thermostated by means of a thermostatic plate, for example according to DE-OS 30 46 729, or can optionally it- self form a front plate of this thermostatic plate. A pos¬ sible gel is a polyacrylamide gel with a (horizontal) width of 100 mm and a length of 400 mm. Shorter gels (200 mm) are also possible. The irradiation point of the excitation beam 12 is located, for example, approximately 100 mm in front of the lower end of the plates 48, 50.

In order to enable the laser light to enter the gel cross section with as little loss as possible, an inlet part 56 is inserted into a corresponding gap in the edge strip on the left in figures 1 and 2, said inlet part 56 having a thick¬ ness corresponding to the thickness of the edge strip and a width in the vertical direction of approximately 10 mm. The length of the inlet part 56 (in the horizontal direction) amount^'s, for example, to 15 mm. As the right-hand end (in Fig. 2) of the inlet part 56 is aligned with the inner longi¬ tudinal edge 58 of the edge strip 46 and the width of the edge strip 46 amounts in the horizontal direction to only 10 mm, the inlet part 56 projects beyond the plate outline. The end faces 60 of the inlet part 56 which are crossed by the excitation beam 12 and are perpendicular thereto are polished in plane-parallel manner to optical quality. An outlet part 62 arranged at the same height as the inlet part 56 has the same structure and is located in an opening in the opposite edge strip 46. The outlet part 62 aids the low-loss and therewith low-reflection coupling-out of the excitation beam from the gel cross section. This arrangement ensures that the laser beam with high intensity is coupled without noticeable divergence into the gel cross section and leaves it again without any greater reflection or light scatter effects. In gel production, the absolutely smooth insides of the inlet and outlet parts 56 and 62 respectively also result (with parts 56 and 62 already inserted) in a correspondingly smooth polymerized gel surface with minimum scatter and reflection of the incident laser beam. Thus, an advantageously low mea¬ suring background is obtained.

The detector arrangement aids the simultaneous, separate de¬ tection of paths (altogether four in the exemplary embodi¬ ment) designated A, T, G, C, along which, during electropho- resis, four samples to be investigated migrate in the di¬ rection W forming bands, owing to the different migration rates of the components of the samples. The designating in the exemplary embodiment of the four paths with the letters A, T, G and C should indicate the bases at which in the res¬ pective sample batch the DNA fragments are each broken off (corresponding to the usual DNA sequencing methods according to Maxam-Gilbert or Sanger) . In order to be able simply to detect the bands designated 66 in Fig. 1 and formed by the DNA fragments of the same molecule lengths despite their ex¬ tremely low concentration and without using radioactive la¬ belling, the DNA fragments are labelled with a fluorophore. The primer left on the fragments which is particularly, al¬ though not exclusively, possible for this is one provided with a tetramethyl rhodamine. This dye is characterized by high absorption and thus a high degree of efficacy and by a relatively small line width (52 nm line width at half maximum intensity) . Its absorption wavelength is 560 nm, and its emission wavelength 575 nm.

The emission photons, induced by the excitation beam 12 of the laser 10, of the band 66 crossing straight through the laser beam are detected separately by a collecting and ima¬ ging optical system 70 for each path A, T, G or C (numerical aperture (f-number) 1.2;focal length 50 mm) and focussed onto a colli- mator arrangement 72 with four apertures. Each aperture is formed by a horizontal slit in a diaphragm corresponding to the respective band length and with a slit height in the ver¬ tical direction of 0.4 mm, corresponding to the diameter of the scanning beam 12. On these the photons coming from areas outside the respective band are blocked out as far as pos¬ sible.

The light coming through the respective apertures 74 is sup- plied to light guides 76, each of which opens into a filter arrangement 78. The filter arrangements serve to filter out photons with wavelengths deviating from the emission photons. Each filter arrangement comprises a non-fluorescent filter for the passage of larger wavelengths (Schott No. KV 550) and a band-pass interference filter (Schott FRG, Type AL 575) with a width of 18 nanometers at half maximum intensity. The- se filters keep Raman scatter and Rayleigh scatter photons away from the following sensors. The sensors are installed in a block designated 80 and are indicated symbolically by the reference numeral 82. The sensors preferred are photomulti- pliers (EMI London, Type 9954 A) with appropriate supply electronics. Alternatively, photodiodes or other usual light detectors can also be used. The measured signals supplied by the detector block 80 are conveyed to a data-collecting and storage unit 86 via a line designated 84, said unit 86 in turn being connected with a microcomputer 88 for evaluating the data. Fig. 1 shows a screen 90 connected with the micro¬ computer and a printer 92 controlled by the microcomputer.

The structure and operation of the arrangement according to Fig. 1 is substantially clear from the above. After the ad- justment of the laser 10 and the carrier 16, simplified by the holder 20 already explained with the aid of Fig. 3 and by the adjustment plate 38 according to Fig. 2, and after lining up of the detector arrangement 52, electrophoresis is started, for example at a voltage applied to the gel 54 of from 1000 to 1500 V, at 50°C and at a wattage of 20 W. In the example shown a band 66' of the path G is the first and only one al¬ ready to have passed the beam 12, thereby generating a corresponding peak in the measured curve allocated to this path G on the screen 90 and in the printer 92. The result of this is that the shortest DNA fragment ends in a guanine base at the end opposite the primer. The next band to cross the beam 12 will be a band designated 66" in the path T, generating a corresponding peak in the allocated measured curve T. It is possible to infer directly from this that a thymidijie base follows the guanine base.

In this way the whole base sequence of a DNA piece to be in- vestigated can in principle be detected directly in on-line operation.

The embodiment according to Fig. 4 relates to the four-colour method according to the Journal Bio/Technology vol. 3, May

1986, 395, 396, in which method the four batches A, T, G and C of the DNA fragments are characterized by four different fluorescent dyes, for example by appropriately different-co¬ loured labelling of the primer. Owing to this distinguishabi- lity between the batches, the four batches can be mixed and a sample obtained from this mixture investigated in a single path M by gel electrophoresis. This is indicated in Fig. 4, where the excitation beam is again given the reference nume¬ ral 12 and the gel layer carrier the reference numeral 16 '. In the detection arrangement 52* the collecting and imaging optical system is omitted for the sake of simplicity. On the other hand, a device 94 can be seen for splitting the inci¬ dent detection beam 96 into partial beams 98 of different wa¬ velengths corresponding to the different wavelengths of the primer fluorophore and travelling in different directions.

The device 94 is symbolized by a prism; other structures are possible, such as for example groove gratings- or thin film filters.

The partial beams 98 are supplied to individual detectors 82', a filter arrangement 78' being connected therebetween with adaptation to the respective partial beam wavelength. Elec¬ tric signal lines 84' lead off from the individual detectors 82' and open into a data-collecting and storage arrangement 86 corresponding to Fig. 1.

In DNA sequencing according to the last-described method, therefore, a single DNA fragment sample as a mixture of the four differently labelled batches is subjected to gel layer electrophoresis, the bands in path M of Fig. 4, for example, which are split owing to their different migration rates, then being detected by the detector arrangement 52' on cros- ω CO t to H-¹

Ol o o tn O cn t-¹

CΩ 3 P H ιp o rt cn Pi ιq Φ rt H H tr rt tr TJ a ιq S3 TJ ι > TJ ft ct TJ rt H tr rt Pi 3 ft CΩ

Φ Φ ti *: Φ Hi ti d P- Φ X tr P £ >< 3^* Φ tr d H P- P 4^ P O tr P P- 3 P tr φ Φ P- P-

H P Φ H P- li H H Ω φ P. KJ H o Φ Φ rt ti z ft ϋ φ ft O 3 d rt P 3 3

Φ en tr Ω tr Φ Φ P- H ιq_ ct H H ti 3 P tr ft tr tr 3 πa Pi CΩ φ tΩ ι U-l

Ω d TJ : H o d 3 Ω H rt P- •<: tr P- Φ Ω rt P- Hi P D P- Ω d rt ti H P d rt Φ rt P P 3 *_* Φ ϋ 3 en Φ rt P O & H a S tr ιP cn rt rt H P- rt

Φ P- o P KJ TJ φ 3 P- rt < P tr ιq P- J tr H H H Φ • cn tr 0 φ 3 tr i 3 < Φ H i rl- 0 Φ P- Φ P P- en 0 cn P- H, φ P P φ ϋ 3 rt φ ι P- CΩ li P- 3 ti o 3 < cn P Pi P cn 0 tr en P 3 cn P 3 >P» ι_ι. CΩ Φ O

TJ Pi P- _* 3 rt cn 3 rt P 3 P tr ιq 3 Φ TJ 3 ιp Φ Pi d tr 3 φ

O tr φ 3 U3 o Φ o TJ P- 3 H. Φ 3 J >< Pi H φ P H i Φ Λ l_l. — - P- CΩ P 00 ft TJ X

H P Pi ιp > 3 Hi P tr o rt Φ P- ti Φ Φ P 3 P- 3 d P to rt ft en to P Ω

P Ω _^ H rt tr tn li Φ 3 P H P 3 P- Pi rt cn CΩ cn ft o Φ φ Ω _^ Φ - O H P-

H W Φ TJ tr ^■ P- 3 P P • ιp P- TJ Φ 3 P- Φ cn d rt Hi 3 3 Φ P- Pi en Hi rt rt

P- ι z H Φ rt P- H 3 Φ P TJ *» Φ ti ti H *£> P- li^¬ Ω 3 α CΩ Φ P P- P en ti tr Φ d rt P- ιP H *«• J 0 tr H ti Φ 3 P- φ 00 3 z P- rt a rt Ω H ft P rt

P o P- X H H tr <! H Φ tr d H P- Pi Ω P- CΩ IP tr en 3 P- Φ - H tr H P- rt d Ω Ω P P φ P- P H Z Φ cn Φ Ω φ Z ft 3 i P- to ιq rt 3 Ω 0 Φ O

P- 3 tr P- H cn rt rt tr H P tn P- Φ P Φ P- rt Ω = 3- cn Pi rt o Ω tr 3 o i rt P- Φ φ * P- rt P- Φ P 3 Ω rt ii Pi Ω cn CΩ 3^* tr P- Φ Φ P- Φ *» P li Φ

3 P- P ft ti Hi o o Ω X 3 rt ti tr φ tr Ω Φ •_* P- 3 Λ Ω i ft φ P tr

P- 3 rt Hi tr 3 tr Ω Ω Pi P- P P- P- H P- en TJ d P P3 Φ cn 3 φ

Pi cn Ω P- H P- P- ft P- O d tr P Pi 3 φ P- 3 P- tr rt P Φ rt Ω i TJ CΩ P

P- ti o o P- Ω rt tr TJ rt φ 3 Φ 1 < cr Pi 3 o TJ tr rt 3 Φ P P φ 3 ti ti Φ 3 Hi ιq P- tr TJ φ P P r P Pi Pi cn P- rt Φ P- Ω z •i φ tr Ω i 3 3 rt Ω o φ Φ P tr φ φ li CΩ rt P- CΩ Φ P- 3 tr P. i Ω P- φ O P- P. O ft 00 — -

Ω Pi en tr TJ rt 3 ti o ιq CΩ P- 3 * ϋ P- 3 φ P P. < < TJ S 3 rt tr P- t rt d Φ φ P rt rt < Φ φ o Φ 3 ιq P- P- P rt Φ φ P- P •» I tr Φ > rt <i P

P- Ω cn P rt ti o P- H CΩ 3 Pi ft rt H 3 O 3 tr Φ 3 li P. rt P *_* tr φ Ω P- o Φ 3 tr Φ P i O tr rt Φ ft 3 O Pi rt _^ Φ tr P- Ω rt P- Φ Ω a

3 Pi P • CΩ ω 3 d φ TJ rt o Z Φ tr O 1 rj < • Pi Hι P rt P- O

Ω TJ Pi 3 cn' H tr Hi P- Ω Φ Hi H a Φ Pi O _^ S 3 § 3^* P- 3 ti rt o Ω Ω a Ω φ P P ti rt P t H P P- > Φ 3 3 Z Φ Φ 3 rt i tr

Hi o d o P Ω cn Ω P 3 O rt tr P. P- P d 3 P rt H z Φ tr P- Pi i Φ P- Φ

3 ti 3 rt ct tr 3 Φ d tr li rt TJ O Ω φ m Ω Φ ; tr J Ω φ rt P- H p- 3 3 rt CΩ P 3 P- l P- IP φ rt • H TJ ti 0 Φ Ω Ω P- O Φ tr P Φ <! CΩ p O tr P- Ω Φ tr < d , φ Φ P tr tr Φ H O 3 TJ Λ O rt rt Ω en Ω rt CΩ p- P- 3

Φ Pi ^ Ω φ Φ Ω <J X 3 Hi φ 0 P- TJ J ti d H P- tr tr rt en P rt φ TJ i ft ft 1

Φ tr H rt φ rt Pi rt H d Ω tr d 0 Φ Pi O Φ tr P P. tr H Φ d P- 0 H

Φ ti o P i ^►< d i P. tr d P ft TJ P o ft Ω 3 P- 3 p- P- ti ti φ ^ Ω P φ P-

X P Hi 3 Φ li P TJ φ o TJ ϋ rt H φ φ Ω 3 TJ en ω * P- rt H CΩ Z 3

Ω tr P- rt P- P 0 Φ H. TJ P O P- Φ K cn P- I tr ti φ P φ P- P Φ

P- H 3 Ω Φ 3 H J t li Z Φ P 3 Ω 0 _^ • en 3 Φ p- P φ P Pi P- en < J tr < rt P Ω rt H tr Pi TJ tr CΩ H Pi Φ 3 ιq . rt ςu en H 3 rt i rt Φ P Φ TJ

P _^ P H rt o P- P- Φ 0 Ω P en 3 PJ i O 3 H Pi O P ti H li rt en H Φ CΩ 3 3 H Φ rt Ω cn O P t d O rt d O tr tr rt rt Φ O

P- pr fi • i P cn P Pi Φ 3 d P 3 3 Φ ti rt rt V£> O Ω P 3^* rt Hi H P P- tr 3 Ω o Φ 3 P- li P- Ω tn 3 Hi Φ p- tr tr en Hi P φ P- CΩ P φ ιP Φ

3 H Φ 3 CΩ Pi 3 cn P- Ω Z Φ O rt H 3 3 Φ Φ z ft cn P ft tn φ H ft cn

TJ 3 o P- TJ H d P- P tr ϋ 3^* KJ Φ ι ft Φ Φ tn H tr ^{^} P- tr en tr φ Φ < 3 o H P < H Pi tr Ω φ φ rt o P- P- tr Pi Ω P ω Φ Φ ft tr 3 •

Φ Pi 3 P- d d Φ H P ft *< Ω σ rt tr Φ a 3 3 Φ O 3 O Pi >< Φ Pi P

Q) rt 3 H rt cn tr n 3^* O J en Φ o H <J rt rt 3 Φ φ • TJ P P- 3 ω

3 tr • IP rt 3 0 P- P ti P > P rt P. Φ (D O Pi O Pi P- X Φ 3 < Pi

. K P o Φ ^q ft 0 Pi ti 3 ii Ω CΩ 3 Φ rt 3 Ω tn P- J J 3 Hi ct Ω tr o Hi H P- ft H φ P O ft Φ rt Hi <; rt Pi P- P- O Pi rt en

£ rt ^* P Φ tr o P 3 P- P 3 Hi li 1 P- O P- tr Φ 3 P rt Hi P d tr TJ tr Φ ti o rt O 3 3 ft rt cn Lq Ω tr Pi Φ O O d Ω Φ ft Φ P 3 P φ H o φ rt d tr Pi 1 Φ tr tr Φ d Φ P 1 H 1 3 ϋ Φ Φ 1 ft Pi H P-

Pi cn tn φ P Φ Φ ti ct 1 H K! P _^ Ω P tr rt

1 1 O 1 1 1 cn Φ 1

counting statistics result, as the excitation beam continuous¬ ly crosses the path or paths to be investigated. In contrast to a possible arrangement in which the laser beam is directed more or less rectangularly onto the gel layer and the four tracks are scanned in turn, the invention provides the sub¬ stantial advantage that the laser beam detects all four tracks at the same time without interruption. The effects of irregularities or contamination of the glass covering the gel layer are eliminated with the arrangement according to the invention. No difficulties are caused by manufacturing the relatively small inlet part to optical quality. If the pre¬ ferred embodiment is deviated from and a pulsed laser is used instead of a continuously operating laser, the possibility arises of detecting the fluorescence after completion of the laser pulse, with the advantage of a further reduced back¬ ground.

With the arrangement described above automatic DNA sequencing can be carried out, beginning with base 1 , without further measures, compared with base 40 to 50 in conventional ra- dioautogram technology. The automatic data analysis possible according to the invention makes possible- a quantification of the band distances and the relative band intensities.

On the subject of Fig. 4 it should be mentioned that the in¬ dividual detectors 82' can be formed from individual photo- diodes. However, other detector forms are also possible, such as, for example, a charge-coupled-device line or a photodiode line. The gel can also be oriented differently from the ar- rangement shown, as the substance transport is effected on the basis of the electrical field applied. This is true also when an arrangement (not shown) is used for gas-chromatogra- phy, in which the driving force for the substance particles to be investigated stems from gas pressure (or liquid pres- sure in the case of liquid chromatograph ) . Detection of the substance particles by an excitation beam passing through the elongate cross section of the retarding medium in the longi- tudinal direction is common to the arrangements according to the invention.

As indicated in Fig. 1 by dash-dot-lines 76' each of said light guides 76 may be prolonged to end at the cover plate 32 in front of the respective path A, T, G or C for direct de¬ tection of photons emitted in these paths without use of the collecting and imaging optical system 70, 72. To this aim the respective light guide end is connected to the cover plate 32 in a suitable way (optical coupling) . A limiting aperture may be provided or not depending on circumstances.

Claims

n

H

P

3 en

detects the emission photons passing through one of the long rectangle sides.

2. An apparatus according to claim 1 , c h a r a c t e r i z e d in that at least one of the inlet (56) and outlet (62) parts has parallel end faces (60) per¬ pendicular to the axis of the excitation beam.

3. An apparatus according to claim 2, c h a r a c t e r i z e d in that the end faces (60) are op¬ tically polished.

4. An apparatus according to claim 1 , c h a r a c t e r i z e d in that in the retarding medium the polarisation direction (B') of the excitation beam (12) runs substantially parallel to the direction of the detector arrangement (52) .

5. An apparatus according to claim 4, c h a r a c t e r i z e d in that the beam source (20) com¬ prises a 90° polariser (14) .

6. An apparatus according to claim 5, c h a r a c t e r i z e d by a small plate (38) , which can be rotated around an axis of rotation (40) substantially pa¬ rallel to the direction of migration (W). and has plane-pa¬ rallel side faces (42), in the excitation beam (12) between the beam source (10) and the retarding medium.

7. An apparatus according to claim 1, c h a r a c t e r i z e d by at least two parallel paths (A; T; G; C) , which are each detected separately by the detector arrangement (52) .

8. An apparatus according to claim 1, c h a r a c t e r i z e d in that the detector arrangement (52) comprises a collecting and imaging optical system (70) having an aperture (74) in the image plane allocated to each path (A;T,G C) to prevent stray radiation from reaching the detector arranσeπient.

9. An apparatus according to claim 1, c h a r a c t e r i z e d in that the detector arrangement (52) comprises at least one filter arrangement (78) for fil¬ tering out photons with wavelengths deviating from the emis¬ sion photons .

10. An apparatus according to claim 9, c h a r a c t e r i z e d in that the filter arrangement (78) comprises filters for Rayleigh or Raman scattered light.

11. An apparatus according to claim 1 , c h a r a c t e r i z e d in that the detector arrangement (52') comprises at least two sensors (82') for different emission photon wavelengths .

12. An apparatus according to claim 11, c h a r a c t e r i z e d in that the detector arrangement (52') comprises a device for splitting the incident beam (96) into partial beams (98) of different wavelengths, as well as sensors (82') allocated to each of the partial beams.

13. An apparatus according to claim 12, c h a r a c t e r i z e d in that the sensors (82') comprise position- sensitive detectors, preferably in the form of a charge-coupled -device line, a photodiode line or a photomul- tiplier with subdivided position areas.

14. An apparatus according to claim ^"1, c h a r a c t e r i z e d in that a holder (21) for the carrier (16) is connected rigidly with a holder for the beam source (20) .

15. An apparatus according to claim 1, c h a r a c t e r i z e d in that the carrier (16) is formed of a housing enclosing the retarding medium (54) , which housing comprises two parallel plates (32,48), which define the long rectangle sides of the cross section, and spacing strips (46) parallel to the direction of migration, which define the short rectangle sides of the cross section, and that the inlet part (56) and the outlet part (62) are each inserted into gaps formed between spacing strip sections .

16. An apparatus according to claim 15, c h a r a c t e r i z e d in that the inner chamber of the housing is provided with a gel layer (54) for gel electropho¬ resis.

17. An apparatus according to claim 16, c h a r a c t e r i z e d in that the excitation beam (12) is a light beam.

18. An apparatus according to claim 17, c h a r a c t e r i z e d in that the excitation beam is a laser beam.

19. An apparatus according to claim 1 , c h a r a c t e r i z e d in that on one side of said two rectangle sides in front of each path a receiving end of a light guide means is arranged.

20. An apparatus according to claim 1, c h a r a c t e r i z e d in that the excitation beam can be displaced in relation to the carrier in the direction of migration.

21. The use of the apparatus according to any one of the preceding claims for DNA sequencing.