DE19948391A1 - Electrophoresis device for analyzing labeled molecules, especially biological molecules - Google Patents

Abstract

The invention relates to an electrophoresis device (10) for analysing molecules labelled with fluorescent markers, in particular, biological molecules. According to one aspect of the invention, said device is suitable for resolving at least four different fluorescent markers independently of one another using a detector field assigned to each marker, according to the respective gel electrophoresis lane and the respective fluorescent marker. To this end, spectral filters are balanced with one another with regard to their spectral selectivity, the fluorescent markers are balanced with one another with regard to their spectral absorption and emission selectivity and the wavelengths of the laser beams (60, 62, 64, 66) which are used to excite the fluorescent markers are balanced with one another in a corresponding manner.

Description

The present invention relates to an electrophoresis device for Analyze fluorescent marker labeled molecules, in particular biological molecules (e.g. DNA, DNA fragments or proteins) that form a A plurality of substantially parallel to each other and along one Molecular direction of gel electrophoresis lanes, one Energy source for applying an electrical potential to one Start area and end area of the gel electrophoresis sheets and assigned a laser arrangement for excitation of the molecules Has fluorescent markers, the laser arrangement in Electrophoresis mode generates laser radiation, which as at least one in essentially transverse to the direction of molecular propagation Laser beam cuts at least several of the gel electrophoresis sheets, um in a detection area of the respective gel electrophoresis web to excite assigned fluorescent markers of the molecules present there, as well as a detection arrangement for the detection of the excited ones Fluorescence markers emitting emission radiation, the Detection arrangement at least one essentially parallel to has at least one laser beam extending detector field, which over a spectral filter and radiation guide arrangement to the detector field guided emission radiation detected. The detector field, which is along one preferably extends linear detection path and a spatially resolved Detection of the emission radiation along the course of the gel Defined electrophoresis path cutting laser beam section Detection path enables (and regarding a linear detection path is therefore also referred to below as a linear detector field) of a number of detection elements, such as photodiodes or CCD  Elements, or a matrix of several parallel rows of Detection elements (such as photodiodes or CCD elements) can be formed. However, there are basically no restrictions regarding the Functionality and of the spatially resolved detection Principle of operation of the detector field.

Such an electrophoresis device is in several Embodiments known from WO 96/23213 that a good Overview of the background to the analysis of biological molecules by means of gel electrophoresis and basic functions and design options for such electrophoresis devices and therefore their disclosure is fully incorporated by reference into Disclosure of the present description of the invention is included. In WO 96/23213 discloses several electrophoresis devices which have two or three laser beam sources, apparently by two or three To be able to analyze fluorescent dyes (fluorescent markers). With two Embodiments are two substantially transverse to that Direction of molecular expansion and essentially parallel to each other extending, offset against each other in the direction of molecular expansion Laser beams from two separate laser light sources into the electrophoresis Gel coupled, which each have a separate linear detector field and a separate spectral filter is assigned. As linear detector fields are alternatively linear CCD element fields and linear photodiode fields intended. For collecting and focusing the emission light, the WO 96/23213 the use of gradient index lens fields (specifically so-called SELFOC gradient index microlens arrays).

The simultaneous determination of two nucleic acid sequences under Use of two differently labeled primer molecules for one Sequencing reaction in a single reaction vessel was carried out by Wiemann et al. (Anal. Biochem. 224 (1995), 117-121). Further Methods in which sequencing reactions using two  different fluorescent labels in one Reaction vessel connected to a common separation of these two different fluorescent labels are in DE 195 15 552 A1 and WO 96/34114. The evaluation of the Sequencing reactions used detection systems, the two different lasers included. WO 96/23213 obviously stops Detection system ready that an evaluation of such Sequencing reactions enabled.

Based on the technical background of the DNA sequence analysis a laser-assisted detection of fluorescent dye-labeled DNA Fragments can also be added to the following basic articles Reference: Nature, Vol. 321, June 12, 1986, pages 674-679, L. M. Smith et al. "Fluorescence detection in automated DNA sequence analysis" and Journal of Biochemical and Biophysical Methods, Vol. 13, 1986, pages 315-323, W. Ansorge et al. "A non-radioactive automated method for DNA sequence determination ". Further for understanding the technical Background writings and essays are in WO 96/23213 called.

Also worth mentioning is EP 0 275 440 B1, which deals with the Minimization of background fluorescence from the gel by appropriate Selection of the fluorescent dye used and the Fluorescence excitation uses laser wavelength.

In addition to a primer-specific fluorescent dye label, the Practice also base-specific fluorescent dye labeling used, for which preferably fluorescent dye-labeled stop Nucleotides (labeled chain termination molecules, in particular Dideoxyribonucleoside triphosphates (ddNTPs)) or labeled Deoxyribonucleoside triphosphates (dNTPs) can be used. Especially for the Sequence determination based on a base-specific  Fluorescent dye labeling are from Perkin Elmer Biosystems (formerly Applied Biosystems) offered layer gel sequencing devices that one clone assigned four different fluorescent dyes for the four different nucleotides using one common for all dyes Laser excite and the resulting fluorescent radiation over four each Detect spectral filter arrangements assigned to a dye. Because of strong spectral crosstalk, the four dyes can dye-dissolved cannot be detected independently of one another. A Determination of the sequence is only based on a complex software Correction of the raw measurement data not yet resolving the dyes possible, which is based on the fact that the four specific, differently marked Sequencing approaches of a clone separated together in a gel track and be analyzed. A sample-specific detection in which for example, assigned to four different clones and different marked approaches are separated and analyzed in a gel track not possible despite the software correction.

Another major disadvantage of base-specific labeling is that the coupled dyes due to different chemical parameters influence the mobility of the DNA fragments and so-called "mobility Shifts "that need to be corrected by software Correction of the mobility shifts as well as the correction of the above spectral crosstalk lead to errors and inaccuracies with the As a result of shorter reading lengths and a higher error rate. This means that with higher redundancy must be sequenced. That means it must be the same Sequence can be read multiple times.

Problematic when analyzing with different Fluorescent dyes labeled sequencing products, generally from with different fluorescent dyes labeled molecules, in one single lane or gel track is generally that fluorescent dyes over a wide wave range can be excited and the excitation energy  also return over a wide range. Furthermore the excitation and emission spectra are subject to external conditions, given by parameters of the chemical environment of the dye are, among other things by the pH value, the temperature and the Dielectric constant of the solution. One had to assume that a simultaneous quantitative recording of four or more of each other independent dye spectra - such as those used for accurate Evaluation of sequencing reactions is required - not is feasible.

Surprisingly, it has now been found that a clever one Selection of the spectral filters used with regard to their spectral Selectivity, the fluorescent marker with regard to its spectral absorption and emission selectivity and the wavelengths of the laser beams are four or more fluorescent marker spectra (dye spectra) from each other can be differentiated recorded and evaluated, so that for example, a joint evaluation of four or more Sequencing reactions, each different Fluorescent dyes were used, is possible, specifically also a joint evaluation of four or more sample-specific (especially primer-specific) labeled sequencing reactions. This is of great practical relevance because by using four or more different marks in one Sequencing reaction the cost of performing the Sequencing reactions can be significantly reduced. Both the Chemicals and labor costs are reduced. This is especially true in large-scale sequencing projects important, such as the "Human Genome" project or others Genome projects.

Accordingly, the invention provides a first aspect Electrophoresis device for analyzing fluorescent markers  labeled molecules, especially biological molecules, ready includes: a plurality of substantially parallel and longitudinal a direction of molecular spreading gel electrophoresis tracks, an energy source for applying an electrical potential to one Start area and end area of the gel electrophoresis sheets, one Laser arrangement for exciting a plurality of different ones Fluorescent markers assigned to molecules, at least some of which mutually different absorption wavelength ranges or / and have different emission wavelength ranges, the laser arrangement generating laser radiation in electrophoresis mode, which are considered to be essentially transverse to the direction of molecular propagation and in essentially parallel and in Direction of molecular propagation of mutually offset laser beams cuts at least several of the gel electrophoresis sheets to in one Detection area assigned to the respective gel electrophoresis path To stimulate fluorescent markers of the molecules present there, one Detection arrangement for gel electrophoresis web-resolved and Fluorescence marker-resolved detection of the excited Fluorescence markers of outgoing emission radiation, the Detection arrangement a plurality of in the molecular propagation direction offset from each other and preferably essentially parallel to the laser beams extending detector fields, each are assigned to a specific one of the laser beams and are designed for this are along, preferably to, assigned to the respective laser beam this essentially parallel detection path at least a part that in the detection areas due to the excitation by the respective Laser beam emerging, via a respectively assigned spectral filter and Radiation guidance arrangement guided to the detector field To record emission radiation in a spatially resolved manner, the Spectral filter arrangement with regard to their spectral selectivity, the Fluorescence markers with regard to their spectral absorption and Emission selectivity, and the wavelengths of the laser beams so  are coordinated that using at least four Laser beams and at least four of at least four detector fields different fluorescent markers independently of one another by means of a assigned detector field according to the respective gel electrophoresis Web and the respective fluorescent marker are resolvably detectable.

In generalization of the proposed electrophoresis device underlying inventive concept is also an electrophoresis Device for analyzing fluorescence marker-labeled molecules, in particular biological molecules, which comprises: a A plurality of substantially parallel to each other and along one Molecular direction of gel electrophoresis lanes, one Energy source for applying an electrical potential to one Start area and end area of the gel electrophoresis sheets, one Laser arrangement for exciting a plurality of different ones Fluorescent markers assigned to molecules, at least some of which mutually different absorption wavelength ranges or / and have different emission wavelength ranges, the laser arrangement generating laser radiation in electrophoresis mode, which is considered to be substantially transverse to the direction of molecular propagation extending laser beams at least several of the gel electrophoresis Cuts paths in a detection area of the respective gel Fluorescence markers assigned to the electrophoresis path of those present there Excite molecules, with at least some of the laser beams spatially are offset from one another and / or offset from one another Time windows occur and / or at least one laser beam Allowing a phase-sensitive detection is modulated or / and wherein at least some of the laser beams are different from each other Have wavelengths, a detection arrangement for gel Electrophoresis web-resolved and fluorescent marker-resolved Detection of fluorescent markers originating from the excited Emission radiation, the detection arrangement being at least one  preferably extending substantially parallel to the laser beams Has detector field, which is designed for this purpose, along one of the laser beams assigned, preferably essentially parallel to this Detection path at least a part of that in the detection areas resulting from the excitation by the respective laser beam an associated spectral filter and radiation guide arrangement for Detector field guided emission radiation to record, where the spectral filter arrangement with regard to its spectral selectivity, the Fluorescence markers with regard to their spectral absorption and Emission selectivity, and the wavelengths of the laser beams so are coordinated that using at least four Laser beams are independent of at least four different fluorescent markers from each other according to the respective gel electrophoresis path and the respective fluorescent markers can be detected when resolved.

Looking at the emission and absorption spectra of the usual fluorescent dyes used in gel electrophoresis, so appears at first glance, it may be completely impossible to be four or more Fluorescence markers resolved after the respective fluorescence markers, that is distinguishing the fluorescent markers from one another, for example with a spectral "residual crosstalk" less than 10%. One easily becomes a combination of two fluorescent labels find different fluorescent markers whose absorption spectra differ do not overlap and their emission spectra do not overlap, so that obviously a detection after the frequency marker-resolved is possible is. However, it was found that there are also fluorescent markers overlapping absorption spectra and fluorescent markers overlapping emission spectra resolved fluorescence markers detected can be. It was recognized that the laser radiation selectively excitable fluorescent markers with overlapping Emission spectra are distinguishable from each other on the basis of a spatially and / or temporally separated laser beam excitation or / and  Basis of different wavelengths of the laser beams or / and Basis for phase-sensitive detection when excited with modulated laser radiation. It was also recognized that the Laser radiation with selectively or non-selectively excitable fluorescent markers at least partially non-overlapping emission spectra are distinguishable on the basis of spectral / selective detection of the Emission radiation and possibly based on spatially separated Laser beam excitation. It has been shown that based on this teaching spectral "residual crosstalk" between the fluorescent dyes in the Detection less than 10%, namely even better than 0.1%, is achievable.

The electrophoresis device according to the invention is preferred for this used, sequencing reactions based on a sample-specific (Clone-specific, especially primer-specific) fluorescent dye Evaluate the marking. For this purpose, the four belonging to a clone specific sequencing approaches in four different gel Electrophoresis sheets (gel traces) separated and analyzed.

By means of the electrophoresis device according to the invention, however, is also a Evaluation of sequencing reactions based on a base-specific fluorescent dye labeling possible, in which the to four specific, differently marked ones belong to a clone Sequencing approaches together in a gel electrophoresis lane (Gel trace) can be separated and analyzed. In the case of in Staggered molecular propagation direction, different ones used for base-specific labeling in relation to a clone Laser beams assigned to dyes can then be given different separation distances for sequence determination, for example software-compensated. So it can be a correction of the Runtime take place in order to compare the sequence determination To enable analysis of the mobility of the sequencing fragments. In the As a rule, it will suffice to correct the maturities according to a linear one  Approach (three-sentence with respect to the separation distances under the assumption constant fragment propagation speed along the gel Electrophoresis sheets). Furthermore, the usually necessarily occurring "mobility shifts" in a manner known per se and Be compensated as far as possible.

The spectral filter and associated with a fluorescent dye Radiation guidance arrangement can be associated with the Detector field as a detection unit assigned to this fluorescent dye or detector unit, if desired actually as "Detection unit that can be handled as a unit". For the Detection units preferably applies that for a detection unit Intensity ratio of from to the respective detection unit associated fluorescent dye to the light from a other fluorescent dye-derived light is 1: 0.2 or greater. The detection units preferably point to those to be detected Fluorescence radiation an acceptance angle of about ± 20 ° based on an optical axis of the detection unit.

It is proposed that the wavelengths of the laser beams or / and Spectral filter of the spectral filter arrangements on at least four different fluorescent markers are matched, their Absorption maxima under electrophoresis operating conditions at least each 30 nm, preferably at least 50 nm from each other are removed or / and their emission maxima under electrophoresis Operating conditions at least 30 nm each, preferably each are at least 50 nm apart.

It is extremely useful if at least one spectral filter is one has spectral characteristics such that due to the excitation of the fluorescence radiation of the respectively associated laser beam each assigned fluorescent marker for a transmission  Wavelength range around an emission maximum of the fluorescence spectrum this fluorescence marker or for a fluorescence maximum at this adjacent pass wavelength range to the assigned detector field to be led. Further training is proposed that in relation to at least one of the other fluorescent markers at least one of the following conditions a) and b) is fulfilled, according to the To enable fluorescence markers to be resolved: a) the laser beam Wavelength lies on a spectral side of an absorption spectrum of the other fluorescent markers beyond one of an absorption maximum to the laser beam wavelength down to at least one Residual absorption amplitude, which is at most a low residual absorption corresponds to falling edge of the absorption spectrum, b) the transmission Wavelength range lies on a spectral side of a Emission spectrum of the other fluorescent marker beyond one of an emission maximum down to the transmission wavelength range at least one residual emission amplitude, that of an at most low one Residual emission corresponds to falling edge of the emission spectrum.

The flank falling to the laser beam wavelength can be one to the longer one Wavelength falling edge, and that to the transmission The falling edge of the wavelength range can be too short Wavelengths falling edge.

Alternatively, it is possible that the one falling to the laser beam wavelength Flank is a flank falling to shorter wavelengths and that to the Passing wavelength range falling edge too long Is the falling edge.

It is generally proposed that the flank be at the laser beam wavelength or falls more steeply towards the pass wavelength range than one on the other side of the maximum of the spectrum, with the distance flank of the spectrum falling from the maximum.  

At least one spectral filter and radiation guiding arrangement can be used as Radiation guidance arrangement a lens arrangement, preferably a Gradient index lens field (possibly SELFOC lens field (SLA)). The Gradient index field should be trained, alone or in combination associated with other optical components fluorescent light And can map the detector field along the relevant detection path insofar as they are referred to as a "linear gradient index field". On an object side assigned to the gel electrophoresis tracks Radiation guidance arrangement can at least one object-side Spectral filter can be arranged. Furthermore, one of the detector fields associated image side of the radiation guide arrangement at least one spectral filter on the image side. The radiation guide arrangement can also have other optical means, for example an aperture arrangement include to increase the optical or spectral selectivity.

At least one spectral filter is preferably used as an interference filter formed or includes an interference filter. This is specifically proposed that at least one spectral filter arrangement as Combination of an absorption filter arrangement and one Interference filter arrangement is formed. For example, a Spectral filter include an absorption filter, possibly colored glass filter, which as Carrier substrate for thin-film films of an associated interference filter serves.

The use of an interference filter may come as a surprise as the spectral Selectivity of such a filter from the angle of incidence of the emission radiation depends in the filter. Especially if you have one Radiation guidance arrangement with high numerical aperture, for example a corresponding gradient index lens field is used, around the fluorescent photons emitted by the fluorescent markers to collect high efficiency appears to use a Interference filter with regard to the desired spectral selectivity of the  Filters extremely problematic. However, it was recognized that the Interference filter, the one as a transmission wavelength range Wavelength range defined, that of an overlay of Passband of the interference filter for different angles of light incidence in the interference filters corresponds to this pass wavelength range not necessarily alone, but with an appropriate arrangement in relation to the radiation guiding arrangement covers the transmission wavelength range defined together with the radiation guide arrangement. So that can Pass-through wavelength range can be defined as a wavelength range that all pass bands of the interference filter for all according to one Acceptance angle characteristic of the radiation guiding arrangement occurring light incidence angle includes. For example, Wavelength range a wavelength range can be defined, the one Passband of the interference filter for a light incidence angle of α = 0 ° and a pass band of the interference filter for one maximum acceptance angle of the radiation guide arrangement corresponding light incidence angle. If you take one into account such, the acceptance angle characteristic of the Radiation guidance arrangement taking into account Wavelength range in the design of the electrophoresis device, so opens up a variety of constructive options for the design and Specification of the spectral filter in relation to the emission spectra of the using fluorescent dyes, and it can be made using Interference filters Spectral filter arrangements with a comparatively high create spectral selectivity to resolve the fluorescent markers To enable detection.

Based on the principles explained, the inventive one Electrophoresis equipment can be designed from the following To detect fluorescent markers at least seven markers: Indodicarbocyanin CY 2, Alexa 488, Fluoreszein-Iso-Thio-Cyanat (FITC), 5- Carboxy-rhodamine 6G, Alexa 532, tetramethyl-rhodamine-iso-thio-cyanate  (TRITC), indodicarbocyanine CY 3, sulforhodamine 101 (TexasRed ™), Indodicarbocyanin CY 5, Indodicarbocyanin CY 5.5, IRD 700, Indodicarbocyanin CY 7, IRD 40 and IRD 800.

Specifically, at least the markers indodicarbocyanine CY 2, fluorescein Iso-thio-cyanate (FITC), 5-carboxy-rhodamine 6 G, sulforhodamine 101 (TexasRed ™), Indodicarbocyanin CY 5, Indodicarbocyanin CY 5.5 and IRD 800 be detectable.

The electrophoresis device can be specially designed by the following fluorescent markers at least three, preferably at least four, most preferably at least five markers at the same time Fluorescent markers resolved and gel electrophoresis web-resolved too detect: Indodicarbocyanin CY 2, Alexa 488, Fluoreszein-Iso-Thio- Cyanate (FITC), 5-carboxy-rhodamine 6G, Alexa 532, tetramethyl-rhodamine- Iso-thio-cyanate (TRITC), indodicarbocyanine CY 3, sulforhodamine 101 (TexasRed ™), Indodicarbocyanin CY 5, Indodicarbocyanin CY 5.5, IRD 700, Indodicarbocyanin CY 7, IRD 40 and IRD 800.

Specifically, it is suggested that at least the five markers Indodicarbocyanine CY 2, 5-carboxy-rhodamine 6G, sulforhodamine 101 (TexasRed ™), Indodicarbocyanin CY 5.5 and IRD 800 or at least the four markers fluorescein iso-thio-cyanate (FITC), sulforhodamine 101 (TexasRed ™), Indodicarbocyanin CY 5.5 and IRD 800 or at least the four markers indodicarbocyanine CY 2, 5-carboxy-rhodamine 6G, Indodicarbocyanin CY 5 and IRD 800 or at least the three markers Fluorescein iso-thio-cyanate (FITC), indodicarbocyanine CY 5 and IRD 800 fluorescent marker-resolved and gel electrophoresis-lane-resolved simultaneously are detectable.

When designing the spectral filter and when selecting the There are basically many possibilities of excitation wavelengths  the available laser systems and the available dependent spectral filters. Basic boundary conditions are only the selectivity of the excitation, for a particular one Excitation wavelength achievable excitation efficiency, the selectivity of detection and at a given pass band with respect to the spectral sensitivity of a detector used can be achieved Detection efficiency. In practice, however, others will Boundary conditions play an important role, namely the cost of Laser systems in terms of purchase and maintenance and the cost of Acquisition of spectral filters and detectors. The following are some Examples of applicable laser systems and spectral filters in relation to given various of the fluorescent markers mentioned, which by no means are restrictive, but should only give a clue. That's the way it is obvious that with lasers tunable in wavelength, such as laser diodes, also from the following information deviating excitation wavelengths within the regularly quite wide range Excitation spectra come into question. The above Excitation wavelengths are therefore only possible for one coming wavelength range of, for example, about ± 10 to 20 nm on both spectral sides of the wavelength mentioned. Corresponding applies to the passband ranges specified below Spectral filter arrangements. It is obvious that you definitely Spectral filter arrangements with spectrally narrow pass bands in the Area of the passband mentioned or / and Radiation guidance arrangements with a smaller acceptance angle range can use if you have a lower detection efficiency (higher photon losses). But you can also Spectral filter arrangements with larger pass bands or spectrally offset passband or / and Radiation guidance arrangement with larger acceptance angle ranges in Use dependence on the emission spectra of the fluorescent markers, as long as the detection selectivity of the electrophoresis device as a whole  remains preserved. Accordingly, the following give spectral limits of the pass band only a clue for the design of the spectral filter arrangements, and the above Passbands can vary by, for example, approximately ± 10 to 20 nm spectrally offset or towards smaller or / and longer wavelengths be expanded. The data given below for eligible Excitation wavelengths, pass bands and maximum acceptance angles in any case define preferred embodiments of a electrophoresis device according to the invention, with the Electrophoresis operation excellent analysis results have been achieved.

For the detection of the fluorescence marker indodicarbocyanine CY 2 proposed a frequency-doubled (possibly diode-pumped) Neodymium: YAG laser with an emission wavelength of approximately 473 nm to use. A spectral filter arrangement can be used for this fluorescence marker assigned with a pass range from about 494 nm to about 424 nm his. Play different light angles of incidence α in the Spectral filter arrangement a role, so the spectral filter arrangement passband areas of different light incidence angles α about 494 nm to about 524 nm for α = 0 ° and about 492 nm to about 514 nm for a maximum acceptance angle of Radiation guidance arrangement corresponding light incidence angle (for example of about 20 °). The spectral filter arrangement can an absorption short pass filter, an absorption pass filter and one Interference bandpass filters include.

For the detection of the fluorescence marker fluorescein iso-thio-cyanate (FITC) can be an argon ion laser with an emission wavelength of about 488 nm or a frequency-doubled (possibly diode-pumped) neodymium: YAG- Lasers with an emission wavelength of approximately 473 nm can be used. A spectral filter arrangement with a Pass range from about 505 nm to about 555 nm, possibly with  different passages associated with different angles of incidence α from about 505 nm to about 555 nm for α = 0 ° and about 500 nm to about 555 nm for a maximum acceptance angle of Radiation guidance arrangement corresponding light incidence angle (for example of about 20 °). The spectral filter arrangement can be an absorption short pass filter, an absorption long pass filter and an interference bandpass filter.

For the detection of the fluorescent marker 5-carboxy-rhodamine 6G proposed a frequency-doubled (possibly diode-pumped) Neodymium: YAG laser with an emission wavelength of approximately 532 nm to use. The one assigned to this fluorescent marker Spectral filter arrangement can have a pass band from about 555 nm to about 578 nm, possibly associated with different light incidence angles α Pass ranges from about 555 nm to about 578 nm for α = 0 ° and about 535 nm to about 569 nm for a maximum acceptance angle of Radiation guidance arrangement corresponding light incidence angle (for example of about 20 °). The spectral filter arrangement can an absorption short pass filter and an interference band pass filter.

For the detection of the fluorescent marker Sulforhodamin 101 (TexasRed ™) can be a helium-neon laser with an emission wavelength of about 594 nm are used. This fluorescent marker can be a Spectral filter arrangement with a pass range from approximately 613 nm to about 651 nm, possibly associated with different light incidence angles α Pass ranges from about 613 nm to 651 nm for α = 0 ° and about 613 nm to about 636 nm for a maximum acceptance angle of Radiation guidance arrangement corresponding light incidence angle (for example of about 20 °). The spectral filter arrangement can have an absorption long pass filter and an interference band pass filter include.  

A can be used for the detection of the fluorescence marker indodicarbocyanine CY 5 Helium-neon laser with an emission wavelength of approximately 633 nm or a semiconductor laser with an emission wavelength of about 640 nm be used. This fluorescent marker can be a Spectral filter arrangement with a pass range from about 655 nm to about 690 nm, possibly with different light incidence angles α assigned pass ranges from about 655 nm to about 690 nm for α = 0 ° and about 650 nm to about 683 nm for a maximum Appropriate angle of acceptance of the radiation guide arrangement Light incidence angle (for example of about 20 °) can be assigned. The Spectral filter arrangement can be an absorption long-pass filter and a Interference bandpass filters include.

For the detection of the fluorescent marker indodicarbocyanin CY 5.5 or / and the fluorescence marker IRD 700 can be a semiconductor laser with a Emission wavelength of about 660 nm or about 670 nm or about 675 nm are used. The spectral filter arrangement in question can be one Pass range from about 655 nm to about 690 nm, possibly different Passage ranges of approximately 695 nm to assigned to light incidence angles α about 745 nm for α = 0 ° and about 695 nm to about 730 nm for one a maximum acceptance angle of the radiation guiding arrangement corresponding light incidence angle (for example of about 20 ° C) exhibit. The spectral filter arrangement can be an absorption long-pass filter and an interference bandpass filter.

A semiconductor laser can be used to detect the fluorescent marker IRD 800 with an emission wavelength of about 775 nm or of about 780 nm be used. This fluorescent marker can be a Spectral filter arrangement with a pass range from about 795 nm to about 790 nm, possibly with different light incidence angles α assigned pass ranges from about 795 nm to about 835 nm for α = 0 ° and about 790 nm to about 830 nm for a maximum  Appropriate angle of acceptance of the radiation guide arrangement Light incidence angle (for example of about 20 °) can be assigned. The Spectral filter arrangement can be an absorption long-pass filter and a Interference bandpass filters include.

Preferred in the electrophoresis device according to the invention Measures taken to reduce the resolution of the Detection with respect to the gel electrophoresis pathway and / or with respect to the fluorescent markers and / or the signal-to-noise ratio interfering radiation, possibly fluorescent light background radiation or / and scattered laser radiation. For example, at least one the laser beams are linearly polarized with a polarization vector which according to an input-side light guiding direction of the spectral filter and Radiation guidance arrangement is aligned, at least to this at least is approximately parallel. This will make it in the direction of the detectors radiated scattered light is minimized, since elastic (Rayleigh scattering the scattered light preferably into one of the incident polarization vector Light vertical plane is emitted. Such The direction of polarization can be determined by arranging the laser accordingly or / and by using appropriate optical components, for example polarizers or / and λ / 2 platelets can be achieved.

With regard to gel electrophoresis sheets, it is preferred that at least 20, preferably at least 40 and particularly preferably at least 80 (e.g. 120 or even 192 or 384 lanes) evaluated in parallel can be. The gel electrophoresis sheets can be in one preferably plate-shaped gel matrix runs between glass plates made of floated borosilicate glass. So-called floated glass avoids multiple reflections due to its high planarity laser beams running between the glass plates, so that an increase of the scattered light and fluorescent light background due to a multiple Penetration of the laser radiation into the glass plates is avoided.  

Borosilicate glass is available in qualities with low intrinsic fluorescence than floated borosilicate glass, for example, under the trade name Borofloat from the Schott company in Jena (Germany).

It is special for the coupling of the laser beams into the gel matrix expedient, at least one (preferably a common one) Laser beam propagation direction essentially parallel coupling plate use the polished entry and / or exit surfaces for the each laser beam and one adapted to the gel matrix Has refractive index.

To prevent premature degradation of the fluorescent dyes and thus under Circumstances to avoid reduced signal levels can be spatial offset against each other, having different wavelengths Laser beams are offset against each other in such a way that the Laser radiation wavelength in the molecular propagation direction of laser beam decreases to laser beam. Pass through the fluorescent markers accordingly first the laser beams with longer wavelength before they reach the laser beams with a shorter wavelength. A degradation of longer wavelength to be excited by laser radiation Fluorescent dyes from laser radiation of shorter wavelength before Detection of the fluorescent marker in question becomes accordingly avoided.

The electrophoresis device described can be used to analyze Nucleic acids are used, for example in such a way that with at least four different fluorescent markers from Nucleic acid sequencing reactions together in one lane after their Size separated and analyzed independently. The Separation can be done by electrophoresis in a denaturing gel respectively. A parallel separation is preferably carried out in several Lanes, each with at least four differently marked products  contain. In this way, using conventional gels, an evaluation of z. B. 1,000-1,200 bases per Allow sequencing reaction and simultaneous separation of four sequencing reactions in one lane using one Plate gels with 192 tracks, more than 100,000 bases 99999 00070 552 001000280000000200012000285919988800040 0002019948391 00004 99880, e.g. B. 155,000 bases, can be determined on a single gel. In the case of gels with larger Number of tracks, as mentioned above, can be correspondingly more bases determine, So based on a doubling of the track density (384 tracks on a single gel) more than 200,000 bases, e.g. B. 300,000 to 384,000 bases can be determined on a single gel.

The gel used for analysis and used as a sequencing device The electrophoresis device is preferably largely automated (preferably especially with regard to sequence determination) and points preferably a separate excitation laser for each fluorescent dye and a separate detection unit. To increase the (spectral) If desired, selectivity is available in addition to the spectral filters mentioned other optical means, for example diaphragms, are also used.

The fluorescent markers can be selected, for example, in such a way that the absorption maxima under the measuring conditions at least 30 each nm, preferably at least 50 nm from each other. Furthermore, the fluorescent markers can be selected so that the Emission maxima under the measuring conditions at least 30 nm each, are preferably at least 50 nm apart.

The above-mentioned fluorescent dyes can be used as fluorescent dyes are used, for example the dye combination: fluorescein, TexasRed ™, CY 5.5 and IRD 800, or for example the Dye combination: 5-carboxy-rhodamine 6G, TexasRed ™, CY 5.5, IRD 800 and possibly CY 2, or for example the dye combination: 5-  Carboxy-Rhodamine 6G, TexasRed ™, CY 5.5, CY 7 and possibly CY 2 or / and IRD 800.

The sequencing products separated together in one lane can be made from a sequencing reaction performed in a single vessel come. At least four can be used for the sequencing reactions differently labeled primers can be used. The Sequencing reaction can be carried out as a quadruplex reaction, as a tandem duplex Reaction, as a sequence gap filling reaction or as a competing Tandem duplex reaction can be carried out.

With regard to such a use of the invention The invention further relates to an electrophoresis device for a reagent kit Sequencing of nucleic acids that are at least four different Contains fluorescent dye labels, with each label for the Determination of a different sequence is provided. The kit can contain at least four different primers, each one wear different markings.

Would you like in terms of high resolution and high Reading lengths the length of the available gel electrophoresis Make optimal use of webs to maximize the separation distance and fluorescent markers are spatially offset from one another Interrogate laser beams, so it is desirable to know the distance of the To keep laser beams in the molecular propagation direction relatively low, so that for the fluorescent markers assigned to the laser beams Separation distances of the same order of magnitude are available. For this It is proposed according to a second aspect of the invention that the Spectral filter and radiation guide arrangements that of the excited Fluorescence markers emitted emission radiation Receiving surfaces of the associated laser beam Detector field deflects, at least to the direction of molecular propagation  are arranged approximately orthogonally. This makes it compact Formation of the detection unit possible in the direction of molecular propagation, so that, accordingly, the laser beams are comparatively small Cut the gel electrophoresis sheets at a distance from each other.

This inventive concept is also independent of the maximization of Separation distance in the case of simultaneous separation of several Fluorescent dyes of interest, so according to the second aspect generally an electrophoresis device for analyzing Fluorescence marker-labeled molecules, especially biological ones Molecules is provided, which comprises: a plurality of im essentially parallel to each other and along one Molecular direction of gel electrophoresis lanes, one Energy source for applying an electrical potential to one Start area and end area of the gel electrophoresis sheets, one Laser arrangement for excitation of the molecules assigned Fluorescence markers, the laser arrangement in electrophoresis mode Laser radiation is generated which is essentially transverse to at least one at least in the direction of molecular propagation cuts several of the gel electrophoresis webs around in one Detection area assigned to the respective gel electrophoresis path To stimulate fluorescent markers of the molecules present there, one Detection arrangement for detecting the excited ones Fluorescence markers of outgoing emission radiation, the Detection arrangement at least one preferably essentially parallel to the at least one laser beam extending detector field has, which is designed along one of the laser beam assigned, preferably essentially parallel to this Detection path at least a part of that in the detection areas resulting from the laser beam excitation, via an assigned Spectral filter and radiation guide arrangement guided to the detector field Detect emission radiation spatially resolved, the spectral filter and  Radiation guidance arrangement of the excited fluorescent marker outgoing emission radiation on receiving surfaces or Deflects receiving surface sections of the detector field, which for Molecular direction of propagation at least approximately orthogonal are arranged.

It is particularly expedient to place the (respective) detector field on one Arrange carrier board, at least to the direction of molecular propagation is arranged approximately orthogonally. As already indicated, can several of a plurality of Laser beams preferably assigned different wavelengths Detector fields each with at least the direction of molecular propagation approximately orthogonally arranged receiving surfaces or Receiving surface sections may be provided, each preferably are arranged on their own carrier board. The distance of the Carrier board is primarily due to the thickness of any on the carrier board arranged components, possibly the detector fields, or a dimension of the radiation guide arrangement, but not by the size of the individual detector fields, so that the carrier boards at a short distance can be arranged from each other.

The spectral filter and radiation guide arrangement can have at least one assigned to a respective detector field and essentially parallel deflecting prism arrangement extending to this detector field, possibly an (in Longitudinal direction) continuous deflecting prism. The Deflection prism arrangement can be on an output side Lens arrangement, possibly a gradient index lens field, the spectral filter and radiation guiding arrangement can be arranged.

To adapt an image-side optical path between a Lens arrangement, possibly a / the gradient index lens field, the Spectral filter and radiation guide arrangement on the one hand and the  Receiving surfaces on the other hand to an image-side focus length of the Lens arrangement, the deflection prism arrangement from one high-refractive optical material, possibly flint glass SF4.

The deflecting prism arrangement is preferably part of an integral one optical module, which one / the lens arrangement and at least one Spectral filter includes. For example, the deflection prism arrangement, the lens arrangement designed as a gradient index lens field, the at least one spectral filter and, if desired, a carrier rail for Production of the integral optical module can be glued together. It can be on an optical input side of the Gradient index lens field or / and on an optical output side of the Gradient index field (possibly between the gradient index lens field and the Deflection prism arrangement) at least one spectral filter can be glued on.

At least one spectral filter of the optical module can be used as an interference filter be formed or include an interference filter. Preferably at least one spectral filter as a combination of an absorption filter and an interference filter. It is proposed that at least one spectral filter of the optical module, an absorption filter, optionally colored glass filter, which is used as a carrier substrate for thin-film films assigned interference filter is used.

The optical module can be used to adjust an optical path in one Module carrier in one of those that intersect the gel electrophoresis webs Laser beam sections and the direction of molecular propagation in the substantially orthogonal direction can be arranged displaceably.

A preferred embodiment is characterized in that a A plurality of detector fields, a plurality of each one of the Optical modules assigned to detector fields and the detector fields assigned detection electronics in a detector module with a  common housing are integrated, the detector module as one Unit is manageable and preferably by relocating to one Gel electrophoresis and laser cutting sections Molecular direction of propagation essentially orthogonal one common adjustment of assigned optical path lengths for the object enables all optical modules of the detector module.

With regard to the basic structure of an electrophoresis device, for example, the electrophoresis devices according to the invention In the first or second aspect of the invention, there are basically many Possibilities. With a view to a compact and simple construction of the Electrophoresis device or / and with a view to a high directional Stability of the laser beams (in this regard, for example, DNA Sequencing high demands will be made after a third aspect proposed that the laser arrangement at least one Has laser with an optical laser resonator, based on a the beam path of the laser beam emerging from the resonator characteristic resonator axis at least approximately parallel to the Detector fields is arranged, the one from the laser resonator emerging laser beam deflected by means of a beam guidance arrangement so that a crosswise to the molecular propagation direction Laser beam section cuts the gel electrophoresis sheets. Alternatively or in addition it is proposed that the laser arrangement at least has two lasers with a respective laser resonator, each based on the beam path from the respective resonator emerging laser beam characterizing resonator axis under a Angles to the detector fields are arranged, preferably at least are arranged approximately orthogonally to the detector fields, wherein the laser beam emerging from the respective laser resonator by means of a Beam guidance arrangement is deflected so that a cross to the Molecular propagation direction laser beam section the gel Cuts electrophoresis webs and covered the laser resonators  on the resonator axes at least approximately according to the Offset of those cutting the gel electrophoresis sheets Laser beam sections against each other in the direction of molecular propagation are offset as well as different side distance from the gel Have electrophoresis webs.

Since the specified structure of the electrophoresis device is not only for the Electrophoresis device after the first and / or after the second Aspect can be advantageous, the invention according to the third aspect generally an electrophoresis device for analyzing Fluorescence marker-labeled molecules, especially biological ones Molecules ready comprising: a plurality of substantially parallel to each other and along a direction of molecular propagation gel Electrophoresis webs, an energy source for applying an electrical Potential at a start area and an end area of the gel Electrophoresis webs, a laser arrangement for exciting a plurality of different fluorescent markers assigned to the molecules, where the laser arrangement in the electrophoresis mode generates laser radiation which as essentially across the direction of molecular propagation and in essentially parallel and in Direction of molecular propagation of mutually offset laser beams different wavelength at least several of the gel electrophoresis Cuts paths in a detection area of the respective gel Fluorescence markers assigned to the electrophoresis path of those present there To stimulate molecules, a sensing arrangement for gel electrophoresis Lane-resolved and fluorescent marker-resolved detection of the excited fluorescent markers emitting emission radiation, the Detection arrangement a plurality of in the molecular propagation direction offset from each other and preferably essentially parallel to the laser beams extending detector fields, each are assigned to a specific one of the laser beams and are designed for this are along, preferably to, assigned to the respective laser beam  this essentially parallel detection path at least a part that in the detection areas due to the excitation by the respective Laser beam emerging, via a respectively assigned spectral filter and Radiation guidance arrangement guided to the detector field Detect emission radiation spatially resolved, the laser arrangement has at least one laser with an optical laser resonator, the based on the beam path of the emerging from the resonator Laser beam characteristic resonator axis at least approximately is arranged parallel to the detector fields, the one from the Laser resonator emerging laser beam by means of a Beam guidance arrangement is deflected so that a cross to the Molecular propagation direction laser beam section the gel Cuts electrophoresis webs.

Several lasers can be connected with respect to a respective resonator axis the detector fields at least approximately parallel resonators be provided, the laser resonators based on the Resonator axes at least approximately according to the offset of the the laser beam sections intersecting the gel electrophoresis tracks Molecular direction of propagation are offset from each other.

Furthermore, according to the third aspect, the invention provides an electrophoresis Device for analyzing fluorescence marker-labeled molecules, especially biological molecules, comprising: a plurality of essentially parallel to one another and along one Molecular direction of gel electrophoresis lanes, one Energy source for applying an electrical potential to one Start area and end area of the gel electrophoresis sheets, one Laser arrangement for exciting a plurality of different ones Fluorescence markers assigned to molecules, the laser arrangement in the Electrophoresis produces laser radiation that is considered essentially transverse to the direction of molecular propagation and substantially parallel to each other  extending and mutually offset in the direction of molecular propagation Laser beams of different wavelengths at least several of the gel Electrophoresis slices to cut in a detection area fluorescent markers assigned to the respective gel electrophoresis lane there to stimulate existing molecules, a detection arrangement for gel Electrophoresis web-resolved and fluorescent marker-resolved Detection of fluorescent markers originating from the excited Emission radiation, wherein the detection arrangement a plurality of in Molecular propagation direction offset and against each other preferably extending substantially parallel to the laser beams Has detector fields, each a specific one of the laser beams are assigned and are designed for this, along one to the respective Laser beam assigned, preferably to this essentially parallel detection path at least part of the in the Detection areas based on the excitation by the respective Laser beam emerging, via a respectively assigned spectral filter and Radiation guidance arrangement guided to the detector field Detect emission radiation spatially resolved, the laser arrangement has at least two lasers with a respective laser resonator, the each based on the beam path of the respective Exiting laser beam characterizing resonator axis below are arranged at an angle to the detector fields, preferably arranged at least approximately orthogonally to the detector fields are, the laser beam emerging from the respective laser resonator is deflected by means of a beam guidance arrangement, so that a transverse to in the direction of molecular propagation, the laser beam section Cuts electrophoresis webs and covered the laser resonators on the resonator axes at least approximately according to the Offset of those cutting the gel electrophoresis sheets Laser beam sections against each other in the direction of molecular propagation are offset as well as different side distance from the gel Have electrophoresis webs. For this purpose, it is further proposed that  the resonator axes at least approximately in a respective Molecular direction of propagation lie orthogonal plane.

A particularly preferred embodiment is characterized by a Stepped optical bench arranged to the side of the gel electrophoresis sheets with arranged at an angle to the direction of molecular propagation, preferably at least approximately orthogonal to Molecular propagation stages that increase with increasing Side distance from the gel electrophoresis lanes opposite to Molecular propagation direction at least approximately according to the Offset of those cutting the gel electrophoresis sheets Laser beam sections rise and assigned to a respective one of the lasers are for holding a respective assigned to the laser Beam guiding arrangement and / or with its resonator axis extending at least approximately along the step Laser resonators.

With regard to the configuration of the beam guidance arrangement, there are various Variants conceivable. For example, it is proposed that the respective Beam guiding arrangement an optical fiber, preferably Single-mode fiber, or / and at least one deflecting mirror or / and at least one optical element for focusing the laser beam or / and setting a laser beam waist in relation to the gel Electrophoresis sheets, possibly an optical lens, an optical lens or an optical telescope, and / or an optical element for Conditioning, possibly spatial filtering, of the laser beam. In In this context, it is preferred that at least one deflecting mirror or / and the optical element for adjusting the beam path of the each laser beam is adjustable, preferably adjustable by motor.

For this purpose, it is further developed that a deflecting mirror from which the laser beam section intersecting the gel electrophoresis sheets  goes out, at least approximately in one to the Molecular propagation direction and the laser beam section orthogonal Direction, preferably at least approximately along the respective Stage of the optical bench, can be moved to adjust the gel Electrophoresis-cutting laser beam section or / and that at least one optical element at least approximately in the Molecular propagation direction is displaceable for adjusting the gel Electrophoresis path cutting laser beam section.

The use of an optical fiber, preferably single-mode fiber, in one Beam guiding arrangement also has independent of the rest of the training an electrophoresis device meaning, since an optical Fiber easily conditioning the laser radiation or / and guiding the laser radiation to the gel electrophoresis tracks can be achieved. The conditioning of the laser radiation is also independent of the optical arrangement used for this with regard to a high Triggering capability and minimizing the signal-to-noise Ratio of an / the electrophoresis device of interest. The According to a fourth aspect, the invention therefore provides an electrophoresis Device for analyzing fluorescence marker-labeled molecules, especially biological molecules, comprising: a plurality of essentially parallel to one another and along one Molecular direction of gel electrophoresis lanes, one Energy source for applying an electrical potential to one Start area and end area of the gel electrophoresis sheets, one Laser arrangement for exciting a plurality of the molecules assigned fluorescent markers, the laser arrangement in Electrophoresis mode generates laser radiation, which as at least one in essentially transverse to the direction of molecular propagation Laser beam cuts at least several of the gel electrophoresis sheets, um in a detection area of the respective gel electrophoresis web to excite assigned fluorescent markers of the molecules present there,  a detection arrangement for gel electrophoresis web-resolved and Fluorescence marker-resolved detection of the excited Fluorescence markers of outgoing emission radiation, the Detection arrangement at least one preferably essentially Has a detector field extending parallel to the laser beams, therefor is formed, preferably along one associated with the laser beam this essentially parallel detection path at least a part which in the detection areas due to the excitation by the associated laser beam arising via an assigned Spectral filter and radiation guide arrangement guided to the detector field Detect emission radiation spatially resolved, the laser arrangement has at least one laser, the laser beam into an optical fiber, preferably single-mode fiber, is coupled in to the laser beam to guide the gel electrophoresis tracks and / or the laser beam condition, and / or wherein the laser arrangement at least one laser has, the laser beam through at least one optical element for Conditioning, possibly spatial filtering, of the laser beam.

For this purpose, it is further developed that the laser beam, which may have a non-Gaussian profile beforehand, is conditioned in such a way that it is at least approximately a Gaussian profile and / or a reduced divergence or / and a reduced beam waist diameter or / and a reduced one , with the product of a divergence angle describing the divergence and the beam waist diameter increasing collimation factor, possibly so-called M ² factor.

By using such conditioned or from the outset such The quality of the laser beam is the selectivity of the Electrophoresis device maximized, as this is essentially due to the Width of the exciting laser beam determined in the direction of molecular propagation is. The area "illuminated" in the gel provides a kind of finish line for the migrating molecules, possibly DNA fragments. For a good one  Resolution, possibly temporal or vertical resolution, must be the illuminated one Area as narrow as possible. Furthermore, the individual cut gel electrophoresis webs in their illuminated areas Difference in width in the direction of molecular propagation as little as possible in order to achieve comparable results for all railways. This is specifically also suggested that at least one of the laser beams be guided in this way and possibly focusing that a laser beam area is minimal Laser beam diameter, possibly one / the beam waist, along the assigned Detector field, approximately in the area of a middle one of the laser beam cut gel electrophoresis sheets is arranged.

That with an electrophoresis device, possibly the electrophoresis Device according to one of the aforementioned aspects of the invention Resolving power and generally the quality of the measurement results depends to a large extent by adjusting the various components and in particular the course of the laser beams (generally: at least one Laser beam) in relation to assigned gel electrophoresis tracks. According to a fifth aspect of the invention, an adjustment method is used for this for adjusting the course of at least one laser beam with respect to associated gel electrophoresis webs of an electrophoresis device, the at least one excitation laser for generating the laser beam and at least one detector field for picking up excited Has fluorescent markers of outgoing emission radiation, provided, according to the invention the adjustment based on by means of a detector field assigned to the (respective) laser beam Detection signals recorded, one by the laser beam induced background radiation, possibly background fluorescence or / and Flare, represent.

For this purpose, it is further developed that in the course of the adjustment at least one optical element associated with the laser beam, possibly one Lens, a lens, a telescope or a deflecting mirror, for taking  a background radiation profile is adjusted, and the optical element is then set in such a way that a background radiation at least approximately according to a maximum and / or one Center of the profile is detected.

It is particularly preferred that the gel electrophoresis lanes be used first intersecting laser beam path and then one to the gel electrophoresis Paths at least approximately orthogonal laser beam path or / and one that is at least approximately parallel to the assigned detector field Laser beam path is set.

A laser beam path cutting the gel electrophoresis tracks can expediently particularly favorable on the basis of at least a detector field section at one end, preferably at one Laser beam coupling point into the gel electrophoresis lanes having the gel matrix next end, the detector field recorded Detection signals are set and a to the gel electrophoresis Paths at least approximately orthogonal laser beam path or / and a laser beam path at least approximately parallel to the detector field can be particularly useful on the basis of at least one Detector field section recorded at the other end of the detector field Detection signals can be set. This applies provided that that with regard to the parallelism of the laser beam to one of the gel Electrophoresis paths defined level, possibly the gel matrix, no or only there is still a low degree of freedom. Is there a one in this regard? Degree of freedom requiring adjustment, so this parallelism should be first can be set, for example, using appropriate constructive arrangements of the gel electrophoresis device.

The adjustment can be carried out with appropriately equipped gel electrophoresis Device motorized and preferably automatic or automated respectively.  

So-called thermostatic plates are often used in gel electrophoresis used, which serve the gel temperature in the direction perpendicular to Wall direction (direction of molecular propagation) of the molecules if possible to keep constant, so for an even temperature distribution across To worry about molecular propagation direction. At least one like that Thermostat plate, as for example in DE 30 46 729 C2 or in the article "Improvements of DNA Sequencing Gels" by H. Garoff and W. Ansorge, Analytical Biochemistry 115, 450-457 (1991). can also be used in the electrophoresis devices according to the named Aspects of the invention can be used advantageously.

It has now been found that it can be advantageous to use the Thermostatic plate in the gel (in the gel matrix) a temperature gradient in the direction of molecular propagation and / or in a thickness of the gel matrix assigned to the thermostatic plate essentially orthogonal Set the direction, in the case of a thermostatic plate, which is used for Thermostatization of a heat exchange medium (in general Water) is flowed through (such as that from DE 30 46 729 C2 or the above-mentioned thermostat plate), preferably in Condition of continuous flow through the thermostat plate with the Heat exchange medium.

In general, the invention provides a sixth aspect Electrophoresis device for analyzing labeled molecules, especially biological molecules, comprising: a plurality of essentially parallel to one another and along one Molecular direction of gel electrophoresis pathways run in a gel matrix, possibly a plate gel, at least one with are in heat exchange connection on a gel matrix surface Thermostatic plate, an energy source for applying an electrical Potential at a start area and an end area of the gel Electrophoresis sheets, the thermostatic plate being suitable for  a uniform temperature distribution in the gel matrix in the gel Electrophoresis webs cutting transverse direction to Establish and / or maintain molecular propagation direction wherein the at least one thermostatic plate is also suitable for a temperature gradient in the direction of molecular propagation in the gel matrix or / and in a thickness associated with the gel matrix, for Thermostat plate to adjust in the substantially orthogonal direction, in the case of which a heat exchanger means flows through Thermostat plate this is preferably suitable for relevant temperature gradient in the state of continuous flow the thermostat plate with the heat exchanger medium. Alternatively or additionally, the at least one thermostatic plate be able to use a temperature profile with a local Temperature rise or a local temperature drop in set at least one transverse region of the gel matrix, in the case of a thermostat plate through which heat exchange medium flows preferably in the state of continuous flow.

With regard to the functional principle of the thermostatic plate, there are none Limitations; this is particularly not due to the use of the plate flowing heat exchanger means limited. It is coming for example, direct thermostatting of the plate electrical means (e.g. heating elements, Peltier elements and the like) in Consideration.

It is proposed that two thermostatic plates are provided between which the gel matrix is arranged. Preferably is a Temperature profile with decreasing in the direction of molecular propagation Temperature adjustable, preferably such that the temperature along the Gel electrophoresis tracks by several degrees (for example 2-10 °), decreases. As the viscosity of the gel matrix decreases by about 2.4% per degree and a higher viscosity the mobility of the molecular bands in the gel  accordingly decreased, the decrease in temperature in Direction of molecular propagation means that the leading edge of each Bound from an area of higher mobility to an area of less Mobility moves. This has the consequence that the under the influence of electric field through the gel moving bands towards the Detection area become sharper or at least less strong diverge, usually during electrophoresis band diffusion occurring at least to a certain extent is counteracted. This "focusing effect" leads to a higher one Dissolution and in the case of analysis of products by Sequencing reactions to longer sequence reading lengths.

Under certain circumstances, however, it can also be useful to have a temperature profile with increasing temperature in the direction of molecular propagation, for which the thermostat plate is preferably suitable. A special one High flexibility is given when the plate is an optional one Different temperature profiles can be set, for example one with increasing in the direction of molecular propagation and one with in Molecular direction of spread of decreasing temperature.

Alternatively or additionally, it can be based on the thickness of the gel matrix assigned direction a temperature profile with one in a middle Temperature maximum in the area of the gel matrix. This leads to increasing mobility towards the middle area a concentration of the molecules in the central region of the gel matrix, what also counteracts a divergence of the gang and a kind Focusing with higher resolution and longer sequence reading lengths results. One explanation for this could be that in the middle area of the gel opposite edge areas of the gel adjacent to the gel boundaries There are more defined migration conditions for the molecules. This concentration in the middle range is with fluorescence marker labeled molecules also with a view to high excitation and  Detection yield by, for example, a Gaussian beam profile having laser beam advantageous. Basically, that's the Thermostat plate invention proposal but of the type of Labeling of the molecules and the type of detection of the labeled Molecules independent. To set the temperature profile with an in can lie in a central region of the gel matrix it may be particularly convenient to use the gel matrix between two Thermostat plates to be arranged.

The thermostatic plate can be suitable for a local Excess temperature in a starting area Adjust the transverse area of the gel matrix. Is a laser arrangement for Excitation of a plurality of the molecules assigned Fluorescence markers are provided which are used in electrophoresis Laser radiation is generated which is essentially transverse to at least one at least in the direction of molecular propagation cuts several of the gel electrophoresis webs around in one Detection area assigned to the respective gel electrophoresis path To excite fluorescent markers of the molecules present there, the Detectability of the fluorescence marker-labeled molecules be improved that in a respective detection area enclosing transverse area set a local temperature increase becomes. The thermostatic plate is preferably suitable for this. The Excessive temperature in the named cross region leads to a Broadening of the fluorescence marker bands and an enlargement the band gaps, which makes the gangs better through the regular "scan" the spatially limited laser beam. The attainable Sequence reading length is not significantly worsened if at least over a larger part of the gel electrophoresis routes to a lower temperature than in the respective detection range Cross area prevails.  

When operating an electrophoresis device with one of one Heat exchanger means flowable thermostat plate surprisingly found that a significant improvement in Measurement results in terms of reading length and reliability achieved could be that the flow of the heat exchanger (Heat exchange medium, possibly water) by the through the Heat exchanger means preheated thermostat plate after reaching a predetermined gel temperature and / or a predetermined Gel temperature distribution was interrupted. Accordingly, the Invention according to a seventh aspect, a method ready for operation an electrophoresis device for analyzing labeled molecules, especially biological molecules, comprising: a plurality of im essentially parallel to each other and along one Molecular direction of gel electrophoresis pathways run in a gel matrix, possibly a plate gel, at least one with a planar gel matrix surface in heat exchange connection, Thermostat plate through which a heat exchanger medium flows, which is suitable for an even temperature distribution in the Gel matrix in the transverse direction cutting the gel electrophoresis sheets To establish and / or maintain molecular propagation direction Energy source for applying an electrical potential to one Start area and end area of the gel electrophoresis sheets. The According to the invention, the method has the step of interrupting the Flow of the heat exchanger through the through the Heat exchanger means preheated thermostat plate after reaching a predetermined / predeterminable gel temperature or / and one predetermined / predeterminable gel temperature distribution.

According to the invention, the thermostatic plate can be used in a known manner used for even temperature distribution in Set transverse direction to the direction of molecular propagation and maintain. After preheating the thermostat plate (also under  known as the thermostatic plate or thermal plate) however the heat exchanger medium supply is interrupted, for example a water cycle from a thermostatted water bath interrupted by the plate. This has positive effects electrophoresis analysis on the one hand by preventing vibrations, which are induced by the circulating heat exchanger means and especially affect the glass surfaces of the thermostatic plates. A Avoiding these small vibrations is particularly important in that these vibrations in the case of laser-assisted fluorescence marker detection on the surface running through the gel layer Thermostatic plate grazing or even in the adjacent glass plate the penetrating laser beam are transmitted. There for example longer DNA fragments in the gel only very narrow bands in the direction of molecular propagation (about 0.1 mm and less), such vibrations of the laser beam around the bands can dissolve them for longer DNA fragments and thus reduce the sequence reading length limit.

On the other hand, a positive effect results from the fact that after Interruption of the heat exchanger medium flow the even Temperature to some extent first by that in the neighboring gel layer developing during electrophoresis Joule heat is maintained. Because of a Heat exchange especially by convection (convection of the Heat exchange medium in the thermostat plate or / and the surrounding air in a the thermostat plate with the gel housing of the electrophoresis device) will look after set a temperature profile in the gel for some time, which the mentioned can have positive effects, such as focusing the molecular bands. So can occur with a vertical arrangement of the gel matrix and the Thermostatic plate with vertical direction of molecular expansion from above down a higher temperature in an upper area of the gel matrix  and a lower temperature in a lower region of the gel matrix adjust, the thermostat plate still making sure that in Transverse direction to the direction of molecular propagation (and to the electrophoresis field) uniform temperature conditions are maintained. Because of due to the interruption of the heat exchanger medium circuit adjusting temperature distribution in the vertical direction will, as has been done described about influencing the viscosity of the water buffer in the Gel and thus the mobility of the bands in the gel as described Focusing effect on the molecular bands achieved.

In the aforementioned method for operating an electrophoresis device it may also be appropriate that two thermostatic plates are provided, between which the gel matrix is arranged. Depending on The design and construction of the electrophoresis device will change Interrupting the flow in the gel matrix a temperature gradient in Molecular direction of propagation (preferably according to a temperature profile with decreasing temperature in the direction of molecular propagation) or / and in a thickness of the gel matrix assigned to the thermostat plate in substantially orthogonal direction (preferably according to one of the Thickness of temperature profile assigned to the direction of the gel matrix with one lying in a middle area of the gel matrix Temperature maximum). It is also possible that at Corresponding arrangement of the thermostat plate, a temperature profile with increasing temperature in the direction of molecular propagation what can also be useful. Furthermore, under certain circumstances Temperature profile with a local temperature increase or a local one Lowering of temperature in at least one transverse region of the gel matrix to adjust. To achieve such a temperature profile, for example - the heat radiation from the gel matrix Shielding means, such as at least one shielding plate, targeted be influenced, for example by a temperature increase in one a transverse area of the gel matrix corresponding to an initial area or / and -  in the case of laser-assisted fluorescence marker detection with regard to the Detectability - in one of the detection areas of gel electrophoresis Obtain transverse area including webs. In the latter case, it offers itself, a / the detector unit receiving the fluorescent radiation train and arrange that for the appropriate shielding the gel matrix and thus - as a result of less heat radiation from the Gel matrix in the mentioned cross-section - for setting the Temperature rise in the transverse area ensures.

The operating method according to the invention can be advantageous in the Electrophoresis devices or methods according to the first to fifth Aspect of the invention can be applied.

The invention and its various aspects are described below several embodiments with reference to the accompanying figures and Text or tables included in the figures explained in more detail.

Fig. 1 shows schematically the structure of an embodiment of an electrophoresis device according to the invention.

FIG. 2 shows a detector module of the device of FIG. 1 in a view corresponding to a top view of input sides of five optical and detector field modules of the detector module arranged one above the other.

FIG. 3 shows an optics and detector field module according to FIG. 2 in an exploded view with a linear detector field and a spectral filter and radiation guide arrangement formed by a gradient index lens field and a spectral filter, which arrangement can be designed as an integral optical module.

FIG. 4 shows a horizontal section through an electrophoresis gel plate arrangement of the device of FIG. 1 in accordance with an embodiment variant in the vertical region of a spectral filter and radiation guide arrangement designed as an integral optical module with a schematic indication of a linear detector field formed by a series of photodiodes with associated control and Evaluation electronics.

FIG. 5 schematically shows the beam path of the optical module of FIG. 4 through a so-called notched plate, a first optical spectral filter, the gradient index lens field, a second optical spectral filter, a deflection prism on the photodiode field.

FIG. 6 shows a preferred embodiment of the thermal plate (thermostatic plate) shown schematically in FIG. 4.

FIG. 7 shows a diagram indicating the spectral sensitivity of the linear detector field formed from silicon photodiodes as a function of the wavelength.

Fig. 8 normalized absorption and emission spectra showing the four fluorescent dyes in the electrophoresis using an inventive electrophoresis device in the event of a simultaneous analysis of labeled with four different fluorescent dyes molecules, for example products from nucleic acid sequencing reactions, preferably used (fluorescent label fluorochromes) FITC , TexasRed ™, CY 5.5 and IRD 800 with details of the excitation wavelength preferred for the excitation of the respective fluorescent dye and the preferred type of laser used.

Fig. 9 shows the chemical structure of the invention with an electrophoretic device preferably fluorescent dyes used.

FIG. 10 specifies a spectral filter and radiation guide arrangement which is preferably used for the detection of the fluorescent dye CY 2 with regard to a preferred spectral passband and a preferred construction of the spectral filter arrangement, with in a table ( FIG. 10a) a transmission wavelength of 50% corresponding edge wavelengths of the spectral filter arrangement and data for the transmission of the spectral filter arrangement and the absorption and fluorescence spectrum of the dye CY 2 and filter characteristics of the spectral filter arrangement for maximum and minimum light incidence angles are indicated in the diagram in FIG. 10b.

FIG. 11 specifies the spectral filter and radiation guide arrangement preferably used for the detection of the dye FITC in a representation corresponding to FIG. 10.

FIG. 12 specifies the spectral filter and radiation guide arrangement preferably used for the detection of the dye rhodamine 6G in a representation corresponding to FIG. 10.

FIG. 13 specifies the spectral filter and radiation guide arrangement preferably used for the detection of the TexasRed ™ dye in a representation corresponding to FIG. 10.

FIG. 14 specifies the spectral filter and radiation guide arrangement preferably used for the detection of the dye CY 5 in a representation corresponding to FIG. 10.

FIG. 15 specifies the spectral filter and radiation guide arrangement preferably used for the detection of the dye CY 5.5 in a representation corresponding to FIG. 10.

FIG. 16 specifies the spectral filter and radiation guide arrangement preferably used for the detection of the dye IRD 800 in a representation corresponding to FIG. 10.

FIG. 17 shows a diagram which summarizes some of the data from FIGS. 10 to 16 and the spectral position of the absorption and emission spectra corresponding to a relative amplitude of at least 0.2, the proposed spectral passband of the spectral filter arrangements respectively assigned to the respective fluorescent dye according to a transmission of at least 0.5 for angle of incidence α = 0 ° and, for example, suitable excitation wavelengths and laser types.

FIG. 18 shows a variant of the thermostatic plate of FIG. 6, with which a temperature gradient in the direction of propagation of the molecules can be set during electrophoresis.

FIG. 19 shows a cross-sectional view in FIG. 19a with an electrophoresis gel layer sandwiched between two thermostatic plates, and FIG. 19b schematically shows a temperature gradient in the gel that can be adjusted by the arrangement of FIG. 19a in a direction assigned to the thickness of the gel layer.

Reference is first made to Figs. 1-3. Fig. 1 is a gel electrophoresis device schematically shows 10 with a vertically arranged layer of gel or gel slab 12 which is arranged between a so-called Notched plate or notch plate 14 and a plate 16 or thermal Thermostatisierplatte. In the production of the gel layer 12 , a row of upwardly open recesses or pockets in the gel was formed on an upper edge of the gel layer 12 , each of which lies at the beginning of an associated gel electrophoresis path and receives the samples to be analyzed. On a gel width of 305 mm, for example, 192 gel pockets 18 and thus 192 gel electrophoresis sheets lying next to one another can be provided, corresponding, for example, to a sheet width of 1 mm and a sheet space of 0.6 mm, so that, for example, a sequencing analysis Have 48 clones (4 lanes per clone) analyzed per selectively detectable fluorescent marker. With regard to the manner in which the electrophoresis gel of the device according to the invention is loaded with samples to be analyzed, there are basically no restrictions; reference can be made, for example, to the techniques and methods known from WO 96/27787 and WO 98/30911.

During the electrophoresis operation, an electrical potential is applied between an upper buffer tank and a lower buffer tank 24 by means of a voltage source 20 in accordance with the charge of the molecules to be analyzed, the upper buffer tank 22 being at a more negative potential than the lower buffer tank in the case of the analysis of sequencing products , so that the negatively charged DNA fragments move along the electric field lines from top to bottom through the gel, usually a poly-acrylamide gel, from top to bottom (molecular propagation direction M). In the course of their movement along the respective gel electrophoresis path, the molecules, possibly DNA segments, are separated according to their molecular weight or their size or length. The migration rate of the molecules decreases disproportionately with increasing molecular size or molecular weight, so that molecules of smaller molecular weight (shorter DNA fragments) move faster than molecules of larger molecular weight (longer DNA fragments) and first reach a detection area located in a lower area of the gel . Here, the molecules marked with fluorescent dyes are optically detected via fluorescence radiation emanating from the respective fluorescent marker, which is induced by optical excitation of the respective fluorescent marker by laser radiation. Due to the dependence of the molecular propagation speed (migration speed) on the molecular weight (the molecule size, possibly DNA fragment length), the molecules of smaller molecular weight (the smaller molecules or the shorter DNA fragments) first pass through the laser beam used for excitation, so that spatial separation the molecules in the vertical direction (their vertical separation distance) is converted into temporal information, namely in the order in which the molecules pass through the laser beam. In the case of the analysis of DNA sequencing products, this temporal information can be converted into sequence information, for example by means of a computer unit 26 .

The thermostat plate 16 is assigned a thermostattable heat exchange medium reservoir, preferably a thermostattable water bath 28 , a heat exchanger circuit, possibly a water circuit, being guided through the thermostat plate 16 via a pump 30 in order to set and / or set uniform temperature conditions, especially in the horizontal direction, over all gel electrophoresis tracks to maintain. Apart from the mentioned number of electrophoresis lanes and the lane widths and interstices, the gel electrophoresis device 10 , as far as described so far, corresponds to conventional electrophoresis devices. For the basic structure of such electrophoresis devices and the background of the analysis of sequencing products using such a device, reference is expressly made to WO 96/23213. The thermostat plate 16 can be designed in accordance with the thermostat plates known from DE 30 46 729 C2. Reference is expressly made to DE 30 46 729 C2, and the disclosure of this document is incorporated by reference into the disclosure of the present description.

Further details of an electrophoresis device according to the invention in accordance with the exemplary embodiment shown in FIG. 1 are now explained below, which lead to significant advantages over the prior art.

The electrophoresis device 10 of FIG. 1 is designed to use laser radiation from five different lasers for the detection of the fluorescent markers of the marked molecules migrating along the direction M, of which only four lasers are shown in FIG. 1, namely one at 488 nm emitting argon ion laser 50 , a helium-neon laser 52 emitting at 594 nm, a semiconductor laser emitting in the region of 675 nm and tunable, for example, by means of a Peltier element to the emission wavelength of 675 nm (also referred to as laser diode) 54 and an im Range of 775 nm emitting semiconductor lasers 56 , for example tunable to a wavelength of 775 nm by means of a Peltier element. Of the lasers, only the actual laser or laser head having the laser resonator is shown, but not associated control devices and the like. The argon-ion laser 50 and the helium-neon laser 52 are arranged at least approximately horizontally in a respective horizontal plane with a resonator axis defining the laser beam propagation direction, and at least approximately parallel to the gel layer 12 . The semiconductor lasers 54 and 56 are likewise arranged with their corresponding resonator axis at least approximately horizontally in a respective horizontal plane, and at least approximately orthogonally to the gel layer 12 . The horizontal planes assigned to the four lasers are arranged in a vertical staggered manner, preferably with a vertical distance of approximately 10 mm corresponding to a vertical distance of the laser beams 60 , 62 , 64 and 66 emitted by the lasers. The laser beams are deflected twice by deflecting mirrors 70 and 72 in the case of beams 60 and 62 from argon ion laser 50 and helium-neon laser 52 and in the case of laser beams 64 and 66 by semiconductor lasers 54 and 56 once each by at least approximately 90 ° in order to couple the laser beams laterally into the gel layer 12 in a vertical staggered manner with the aforementioned vertical spacing of, for example, approximately 10 mm. To focus the laser beams, an optical lens 74 , for example a plano-convex lens, is arranged in the beam path of the laser beams, by means of which the waist of the laser beam profile, ideally a beam profile corresponding at least approximately to a Gaussian beam profile, approximately in the center of the gel in relation is placed on the width b. For fine adjustment of the course of the laser beams in the vertical direction, the lenses 74 are preferably adjustable in height, preferably adjustable in height by motor. The deflecting mirrors 72 which deflect the laser beams in the direction of the gel are preferably adjustable in a direction at least approximately orthogonal to the gel layer 12 , preferably by means of a motor, in order to offset the laser beams parallel in a respective horizontal plane in the transverse direction to the gel layer and thus as precisely as possible in the middle the gel layer (based on the thickness of the gel) into the gel. The laser beams are preferably adjusted by means of the adjustable optical elements on the basis of detection signals from the respective detector field, which are based, for example, on scattered light or background fluorescent light from the gel layer and / or adjacent glass plates. A background radiation profile is preferably recorded and then a laser beam profile corresponding to a maximum and / or a center of the background radiation profile is set, advantageously in each case with respect to at least one detector field section at opposite ends of the detector field. The coupling into the gel is preferably carried out via a common coupling plate which laterally delimits the gel.

It should also be added to the semiconductor lasers that their comparatively strongly divergent laser beams are bundled by means of a respective telescope, not shown in FIG. 1, in order to bring the beam diameter to less than 1 mm. The laser beams can additionally pass through a spatial filter, for example a pinhole, in order to achieve a Gaussian beam that is as ideal as possible. In the case of the argon-ion laser and the helium-neon laser, the laser beam profile generally corresponds quite well to a Gaussian beam profile, so that beam conditioning of this type is generally unnecessary.

The optical components mentioned (deflection mirrors 70 and 72 , lenses 74 etc.) and the semiconductor lasers 54 and 56 are mounted on a step-shaped optical bench 80 , the steps 82 of which rise laterally in accordance with the offset between the laser beams, as can be seen in FIG. 1. By means of the stepped optical bench, a compact arrangement with high stability can be achieved in a particularly simple manner while enabling the laser beam paths mentioned. The top stage of the stepped optical bench 82 is unused according to FIG. 1, here a further semiconductor laser with corresponding optical components or a further arrangement of deflection mirrors 70 and 72 for a further laser arranged parallel to the lasers 50 and 52 could be mounted around to couple five laser beams vertically graded into the gel layer.

The laser beams with the four different wavelengths serve to excite assigned fluorescent markers, for example the fluorescent markers FITC, TexasRed ™, CY 5.5 and IRD 800, which have only slightly overlapping excitation and emission spectra and thus enable selective excitation and detection (see Fig . 8). A detection arrangement for detecting the emission radiation emitted by the excited fluorescence markers is assigned to the fluorescent dyes used and thus to the laser beams. The detector arrangements are integrated in a detector module 90 which, in the view in FIG. 1, is arranged behind the notch plate 14 and is explained in more detail with reference to FIGS . 2 and 4.

FIG. 2 shows the detector module 90 in a top view of input sides of five optics and detector field modules 94 , 96 , 98 and 100 , of which the modules 92 to 98 are assigned to the laser beams 60 , 62 , 64 and 66 , with FIG. 2, the ideal course of the laser beams with respect to the input sides of the optics and detector field modules is indicated in dashed lines, namely parallel to a longitudinal axis of the modules in the middle of the respective input side.

A possible construction of the optics and detector field modules is shown in FIG. 3. The optics and detector field module shown there is formed by a linear detector field 102 , which has, for example, a series of photodiodes 104 , and a spectral filter and radiation guide arrangement, which in the example of FIG. 3 has a spectral filter 106 and a gradient index lens field, for example comprises a so-called SELFOC lens array (SLA) 108 which, for example, has six rows of gradient index lenses vertically one above the other.

The gradient index lens field 108 is preferably a lens field with a numerical aperture of approximately 0.6, corresponding to a maximum angle of incidence of α = ± 22.5 °, that is to say an opening angle of an incoming beam of rays of approximately 45 °, the individual lenses being cylindrical Elements with a diameter of 1 mm and a length of 10 mm are formed in the 6 lines mentioned, each with 300 lenses. A lens array contains approximately 1,800 such lenses and there is a total light entry area of 305 mm × 6 mm. Such lens fields are available as custom-made products from the Nippon Sheet Glass Company, Japan.

The linear detector field 102 preferably comprises highly sensitive photodiodes with a dimension of the light-sensitive surfaces of approximately 3 mm, with at least two photodiodes (pixels) per electrophoresis path being provided with a view to differentiating the gel electrophoresis paths, so that the linear detector field preferably has at least 384 photodiodes juxtaposed. The linear detector array 104 is preferably formed from six modules lined up in a linear manner, which can be implemented in hybrid construction and can each carry 64 photodiodes on a common chip with a width of 2 inches (5.08 cm), preferably together with associated integrating operational amplifiers. Good results were achieved with a light-sensitive total area of 30.5 cm × 3 mm per detector line, divided into the 384 photodiodes mentioned, each 0.8 mm wide. To minimize the light-insensitive area, the light-insensitive zones between the individual photodiodes should be as narrow as possible. In the preferred embodiment, the light-insensitive zone between the photodiodes is only 0.02 mm each, so that the entire light-insensitive area 383 × 0.2 mm is equal to 7.66 mm, corresponding to a very small proportion of only 2.5% of the total Detector area. Such photodiode modules are available as custom-made products from Hamamatsu Photonics, Japan. The spectral sensitivity of the silicon photodiodes is shown in FIG. 7 and certain limits can be set by changing the doping.

The gradient index lenses of the gradient index lens field 108 each generate mutually overlapping individual images on the image side, that is to say on the photodiodes, with a spatially continuous 1-to-1 imaging occurring. Because of this and because of the high numerical aperture on the one hand and the dimensions of the photodiodes in the direction of molecular propagation M (3 mm in the example described here) on the other hand, there are no high requirements with regard to the orientation of the gradient index lens field 108 with respect to the detector field 108 .

Instead of a linear detector field formed from photodiodes, it is also possible a CCD field or another spatially resolving detector field is used be that along a detection path, which is determined by the course of the Laser beams are defined in the gel, a spatially resolved detection of the Fluorescent light enables. The detector fields used can quite a matrix of detection pixels with several to each other have parallel rows of pixels. The one in relation to the detector fields The term "linear" used is not restrictive and refers to this on the possibility of spatially resolved fluorescence light detection along a linear detection section according to the embodiment.

Absorption filters (for example colored glass filters) and interference filters and, above all, combinations of at least one absorption filter and at least one interference filter come into consideration as spectral filters. The passband of the spectral filter of the various optics and detector field modules is to be selected in accordance with the excitation wavelengths and the emission spectra of the fluorescent dyes used, suitable spectral filter specifications being given below as examples for a number of usable fluorescent markers. Remarkably, fluorescent markers with overlapping emission spectra can also be distinguished from one another, provided that these can be selectively excited and spatially separated laser beam excitation takes place, for example by means of mutually offset laser beams as in the exemplary embodiment in FIGS. 1 and 2. Alternatively, temporally separated laser beam excitation and excitation with differently modulated laser beams can also be considered to enable phase-sensitive detection. Furthermore, it is also possible to differentiate selectively or non-selectively excitable fluorescent markers with overlapping emission spectra, provided that the emission spectra only overlap in regions, i.e. do not have overlapping spectral ranges and the emission radiation is detected in a spectrally selective manner, possibly in connection with spatially separated laser beam excitation or / and a temporally separated laser beam excitation or / and an excitation with modulated laser radiation to enable phase-sensitive detection.

A particularly preferred embodiment of the spectral filter and radiation guide arrangements of the detector module, each as an integral optical module, is shown in FIGS. 4 and 5 in spatial and functional relation to a linear detector field that is separate from the integral optical module. To explain the embodiment variant of FIGS. 4 and 5, the same reference numerals are used as for the embodiment of FIGS. 1 to 3 for analog or identical components followed by the letter "a", only the differences from the previously described embodiment being described and otherwise, reference is expressly made to the preceding description.

FIG. 4 schematically shows the arrangement of the optical and electronic elements of a detector unit assigned to a laser beam with the detector field (the detector line) in a sectional illustration with a direction of view corresponding to the direction of molecular propagation M (in the arrangement corresponding to FIG. 1, therefore, from above) and FIG. 5 shows the elements of the detector unit in a sectional plane orthogonal to the gel layer, the notch plate and the thermostat plate, the photodetectors of the detector line being only indicated schematically in FIG. 4.

According to the variant of FIGS. 4 and 5, an integral optical module for the respective laser is provided for guiding the fluorescence marker emission radiation resulting from the excitation by an excitation laser, which consists of a gradient index lens field 108 a, an absorption filter 106 a on the image side of the gradient index lens field 108 a, an optical interference filter 107 a on the object side of the gradient index lens field 108 a and a mirrored deflection prism made of highly refractive flint glass SF 4 110 a. The spectral filters 106 a, 107 a and the deflecting prism 110 a are glued directly onto the gradient index lens field 108 a and onto a support rail made of aluminum in order to obtain an optical unit with a defined optical path length. The beam path for the emission radiation from the excited fluorescent markers can be seen in FIG. 5. The optical path length Z (λ) is approximately 20 mm, the optical path length required for the imaging being achieved on the basis of a relatively short geometric path length due to the highly refractive deflection prism. In order to match the optical path length to the different fluorescence wavelengths, deflection prisms with different refractive indexes can be used for the individual optical modules assigned to the detector rows. To a certain extent, however, different path lengths caused by chromatic aberration can also be compensated for by the fact that the optical units 112 a can be adjusted by shifting on the optical axis. To form the respective optical unit 105 a from the filters 106 a, 107 a, the gradient index lens field 108 a and the deflecting prism 110 a, it must also be added that the bonding can be carried out, for example, by means of a silicone-based optical adhesive, which preferably has a low viscosity and in terms of its refractive index is preferably adapted to the refractive indices of the filter glasses and the gradient index lens field.

The linear detector field 102 a assigned to the respective fluorescent dye, like the detector field 102, consists of six modules lined up linearly, which are inserted on a common carrier board 114 a, on which clock generators and multiplexers 116 a are also accommodated. The individual modules are designed in hybrid construction and each carry 64 photodiodes on a common chip with a width of two inches (= 5.08 cm), together with the associated integrating operational amplifiers. The entire light-sensitive area of a detector line is thus 30.5 cm wide and 3 mm high, divided into 385 photodiodes 0.8 mm wide. The light-insensitive zone between the individual photodiodes is 0.02 mm wide. The entire light-insensitive area is accordingly 383 × 0.02 mm = 7.66 mm wide, corresponding to a very small proportion of only 2.5% of the total detector area.

The gradient index lens field 108 a can correspond to the gradient index lens field 108 and, for example, have a high numerical aperture NA of approximately 0.6 and six rows of lens elements one above the other in order to ensure a large vertical detection range for fluorescence radiation (object height) of 3 mm with a full numerical aperture. Due to the overlapping upright image of the individual lenses in the field, a horizontal alignment of the lens elements with the photodiodes is not absolutely necessary.

The carrier board 114 a with the detector fields 102 a arranged thereon and the optical modules 112 a are received and held in a common detector module according to FIG. 2 with a common, shielding housing. Referring to Fig. 1, the detector module 90 may therefore five carrier boards 114 a with a respective detector array 102 a and have five associated integral optical modules 112 a. Due to the bent beam path achieved by the deflection prisms 110 a, the minimum vertical distance between the individual detection arrangements is determined by the structural height of the gradient index lens fields 108 a, which is approximately 10 mm. When using approximately 60 cm long glass plates to limit the gel layer, separation distances (running distance of the molecules or fragments to be analyzed) from 46 cm to the top detector or from 50 cm to the bottom detector can be achieved.

The linear detector fields can be formed by silicon photodiodes, which have a wide spectral sensitivity range (see FIG. 7). However, other materials, for example gallium arsenide phosphite, can also be used. The latter semiconductor material has the great advantage that the spectral sensitivity can be shifted into a wide range by varying the proportions of arsenide and phosphite in order to provide a special adaptation to the emission radiation to be detected in each case. Furthermore, this material is relatively insensitive to thermal background radiation compared to silicon. One could also think of further increasing the pixel density of the detector fields in order to achieve a higher track density, for example a doubling or tripling of the track density.

The electrophoresis device in the form shown in FIG. 1 can be used, for example, to analyze molecules which are marked by four different fluorescent markers, for example FITC, TexasRed ™, CY 5.5 and IRD 800 (cf. FIG. 8). In order to avoid premature degradation of the fluorescent markers by laser radiation which has a shorter wavelength than the laser radiation used for excitation, the lasers and the laser beams are staggered vertically in such a way that the excitation wavelengths increase along the direction of molecular propagation M, that is to say the molecules marked with fluorescent markers first the long-wave laser radiation of 775 nm, then the shorter-wave laser radiation of 675 nm, then the laser radiation of 594 nm and finally the laser radiation of the shortest wavelength of 488 nm. A corresponding arrangement is preferably also provided when other excitation wavelengths and other laser sources are used.

Basically, there are fluorescent dyes that can be used no restrictions. An overview of DNA sequencing usable fluorescent dyes with the maxima of each Excitation and emission spectrum and suitable excitation light sources is given in Table 1.  

Table 1

In connection with an electrophoresis device according to the invention according to FIG. 1, the dyes CY 2, FITC, Rhodamine 6G, TexasRed ™, CY 5, CY 5.5 and IRD 800 have proven particularly useful. For many applications it will be sufficient if the electrophoresis device is designed with regard to the design of its detectors in such a way that only four of the laser-dye combinations can be used simultaneously. For some of the dyes listed in Table 1, the chemical structure is shown in Fig. 9.

It should be noted that the excitation lasers shown in Fig. 1 serve only as an example. Table 2 gives examples of excitation lasers that can be used for DNA sequencing with their emission wavelength λ _em and fluorochromes that can be excited thereby. The right column of the table shows the product of the extinction coefficient ε (λ _em ) and the quantum yield q (λ _em ) of the respective dye at the corresponding excitation wavelength λ _em , which is a measure of the fluorescence yield that can be achieved. Since the excitation takes place selectively at the respective absorption maximum, only low laser output powers are required.

Table 2

An overview of in an electrophoresis device according to the invention for example usable laser types with wavelengths, Output powers and the respective absorption maximum selectively stimulable fluorescent dyes are given in Table 3. By selective excitation of the fluorochromes close to each Absorption maximums are only low laser output powers between 3 to 5 mW required. Higher laser powers can even be harmful due to effects such as fading of the dyes (photo-bleaching) Radiation induced chemical reactions of the dyes with the gel matrix and change in their refractive index as well as an increase in the Background can occur due to scattered radiation. Moreover, the The yield of fluorescence is not proportional to the performance of the excitation Light source as a saturation by emptying the energetic Basic state of the fluorochromes can occur. Laser types whose Output power is not or only poorly controllable, can therefore weakened, for example, by optical neutral filters in the beam path become.  

Table 3

With reference to FIGS. 10 to 16, the spectral properties of the detector units used for the detection of the respective emission radiation according to the exemplary embodiment described here should now be used for the dyes CY 2, FITC, Rhodamine 6 G, Texas Red, CY 5, CY 5.5 and IRD 800 which are preferably used , specifically the spectral filter arrangements used in connection with the properties of the respective gradient index lens field. The use of the optical radiation filter arrangement makes it possible to separate the fluorescent light from the excitation light of the lasers and partly from the background fluorescence as well as from Rhaleigh and Raman scattered light, so that the comparatively significantly weaker fluorescent light can be detected at all. Selective excitation and detection of the individual fluorescent markers (fluorochromes) also ensure that several differently colored molecules, possibly DNA fragments, can be analyzed simultaneously but independently of one another in the same electrophoresis gel. The optical radiation filters can be band-pass filters in which areas with low transmission (cut-off areas) are connected to a region of high transmission (pass band) towards shorter and longer wavelengths and / or edge filters in which a region of high transmission is followed by a region of low transmission connects, or vice versa, where long-pass filters in which the pass band is at longer wavelengths than the stop band, and / or short-pass filters in which the pass band is shorter than the stop band can be used as edge filters. Interference filters, which can be combined with absorption short-pass and / or absorption long-pass filters, are preferably used as bandpass filters. Depending on the gradient index lens field used, the possible total thickness of the filter arrangement can be set to a certain value, for example approx. 2 mm, for optimal imaging properties of the optical system, but in principle compensation is also possible by using materials with a correspondingly adapted refractive index. A particularly simple optical structure, which can also be easily adjusted, is achieved if the spectral filters have a predetermined total thickness for all detectors. The filter characteristic of the respective absorption filter can then be adapted primarily via the melt used for the production of the absorption filter. In this context, it is appropriate that a respective absorption filter is used as the substrate for the production of the interference filter. Short-pass and long-pass filters from Schott (Mainz, Germany) can be used as absorption filters.

The central data of the spectral filter arrangement used to detect the fluorescent light of the fluorescent marker CY 2 according to a preferred exemplary embodiment are given in the table in FIG. 10a. A combination of absorption short-pass filter, absorption long-pass filter and interference band-pass filter is used as the filter with edge wavelengths λ _C1 , λ _C2 of 494 nm and 524 nm for a light incidence angle α = 0 ° and λ _C1 = 492 nm and λ _C2 = 514 nm for a light incidence angle α = 20 ° corresponding to a maximum acceptance angle of the gradient index lens field. Here, an edge wavelength is denoted in each case by a wavelength giving a transmission of 0.5 (50%). At the excitation wavelength of 473 nm which is preferably used, the transmission of the spectral filter arrangement is less than 10 ^-4 in the light incidence angle range 0 ° to 20 °. For the emission spectrum of the dye, a transmission greater than 0.8 is obtained at a light incidence angle of 0 ° for the wavelength range 495 to 515 nm. With a light incidence angle of 20 °, a transmission greater than 0.8 is obtained for the wavelength range 490 to 505 nm. For wavelengths greater than the emission spectrum of the dye, a transmission less than 10 ^{-3 is obtained} for the light incidence angle range 0 ° to 20 °. Fig. 10b shows a graph indicating the transmission of the spectral filter for α = 0 ° and α = 20 ° as a function of wavelength. In addition, the absorption spectrum A and the emission spectrum E of the dye CY 2 as well as the excitation wavelength of 473 nm, which is preferably used, are shown. Furthermore, the excitation wavelength of 532 nm, which is preferably used for the excitation of the dye rhodamine 6G (cf. FIG. 12), is shown.

Fig. 11 shows corresponding data for the dye FITC associated detector. The spectral filter arrangement in turn preferably comprises an absorption short-pass filter, an absorption long-pass filter and an interference band-pass filter. In addition to excitation by means of an argon ion laser with a wavelength of 488 nm, the use of a frequency-doubled neodymium: YAG laser with a wavelength of 473 nm can also be considered, for example.

The data from a detector associated with the Rhodamine 6G dye are shown in FIG . The spectral filter arrangement preferably consists of an interference bandpass filter and an absorption shortpass filter, the transmission of which is additionally indicated in FIG. 12b (curve K).

The TexasRed ™ fluorescent marker can be detected, for example, by means of a detector whose spectral specifications are given in FIG. 13. The spectral filter arrangement is preferably formed by an interference bandpass filter and an absorption longpass filter. The transmission of the long-pass filter is also shown in Fig. 13b (curve L).

A spectral filter arrangement formed by an absorption long-pass filter and an interference band-pass filter can be used, for example, for the detection of the fluorescent light emitted by the dye CY 5, the spectral data of which are given in FIG. 14. In addition to excitation by means of a helium-neon laser with the emission wavelength of λ = 632.8 nm, excitation by means of a semiconductor laser, for example with a wavelength of 640 nm, can also be considered.

The dye CY 5.5 can be detected, for example, by means of a spectral filter arrangement which is formed by an absorption long-pass filter and an interference band-pass filter. The spectral data of a suitable detector are given in FIG. 15. The dye CY 5.5 can be excited, for example, by means of a laser diode that emits at 675 nm. A semiconductor laser diode emitting at 660 nm can also be used. The detector specified in FIG. 15 can also be used for the detection of the dye IRD 700.

For the detection of the dye IRD 800, which is chemically identical to the fluorochrome IRD 41, a detector can be used, the spectral specifications of which are given in FIG. 16. The spectral filter arrangement can be formed by an absorption long-pass filter and an interference band-pass filter. The dye can be excited with a laser diode that emits at 775 nm, for example. With regard to the emission spectrum (E) in FIG. 16b and the corresponding spectrum in FIG. 8, it should be noted that the decrease in emission shown towards longer wavelengths should be somewhat exaggerated.

The excitation and absorption spectra of the dyes mentioned, the pass bands of the assigned spectral filter arrangements to be understood as examples and the possible excitation wavelengths to be understood as examples are summarized in the diagram in FIG. 17, with the absorption spectra (A) and the emission spectra ( E) the range of a relative signal strength ≧ 0.2 standardized to 1 and the passband for the light incidence angle α = 0 ° are given for the spectral filters. As can be seen, there are spectrally wide overlaps between the absorption spectra of the dyes and between the emission spectra of the dyes. Despite these overlaps, the use of four independent detection systems enables four samples labeled with different fluorescent dyes to be detected simultaneously and independently of one another, for example samples labeled with the dyes fluorescein, TexasRed, CY 5.5 and IRD 800 (four different dyes). Furthermore, these five dyes can be detected simultaneously and independently of one another by means of the detector units specified for the five dyes CY 2, Rhodamine 6G, TexasRed, CY 5.5 and IRD 800. On the basis of the teaching of the present invention, the person skilled in the art will easily be able to set up an electrophoresis device with five or more independent detection systems which enables simultaneous detection independently of one another for another dye combination of five dyes or for more than five assigned different fluorescent dyes .

The crosstalk of the four detectors for the dyes fluorescein, TexasRed, CY 5.5 and IRD 800 were quantified as follows: Measurement of the thermal background in complete darkness, so switched off lasers, were assigned to the four detectors Laser beams coupled into an electrophoresis gel, on which no samples were applied. The lasers were used one after the other time interval of about 15 minutes switched on. The measured values for the Background radiation was averaged over 40 photodiodes. In the Table 4 below is the rise in the output voltage of the detector in digital units of the A / D converter used, absolute and percentage (based on the purely thermal radiation background). The increase caused by the laser assigned to the respective detector is highlighted in bold in the table.

Table 4

Further experiments showed that the increase in Radiation background (maximum 34% for the detector for CY 5.5) in time is constant and does not change in the course of electrophoresis operation. This increase can therefore be calculated as a constant background from Useful electrophoresis signal are subtracted, so that the influence of the laser radiation increases the background radiation to the signal Noise ratio of the respective detector and thus on the Separation capacity of the system can be largely neglected.

For possible irradiation of the fluorescent emission light from the dyes in the detectors not assigned to them was investigated for the four dyes each performed a sequencing run with samples using only this one of the four dyes were marked. There have been marked Primer used. During the sequencing run, the Signals from the respective non-corresponding detectors are recorded. The strongest signals came at the beginning of each sequencing run Pass through the primer by the laser beams. In all cases only this passing through the primers was detected in the other detectors be, but not going through the actual Sequencing products. So there was no detectable interference in the area of the actual sequence signals. The spectral selectivity the detector system was sufficient with different dyes labeled DNA samples simultaneously, but independently of each other analyze.

Table 5 and Table 6 show results on the resolving power of the Detection system with regard to the distances of the electrophoresis lanes (Traces) and the separation distance.  

Table 5

Table 6

Table 5 shows that the achievable resolving power is largely is independent of the distance between the tracks on the electrophoresis gel, as long as the Nyquist sampling theorem is adhered to, at least two Pixels (formed by photodiodes in the present embodiment) per Track demands. Table 6 makes it clear that doubling the Separation distance from 21 cm to 42 cm by using 60 cm long ones thermostated glass plates brings a significant advantage, namely one Increase the average number of bases read by 40% so that were read with 60 cm long gels in a run over 1200 bases. To  However, it must be taken into account that the beam diameter in the electrophoresis Gel is not constant in the direction of beam spread. Ideally, the Beam waist of the laser beam placed in the middle of the gel, so here the highest resolution is expected. Towards the edges Resolution then according to the beam diameter and Divergence angle of the laser beam. On average, reading lengths were reached from over 800 bases.

The following are examples of the analysis of nucleic acids below Given the use of an electrophoresis device according to the invention:

example 1 1.1 Material and methods Primer design and synthesis

Either standard primers (UPO, UP-40, RPO, T7, T7term) or the specific primers 1 to 8 given below used. Suitable primers were used as matrices Vectors with inserts to be sequenced used. As markings the fluorescent dyes were fluorescein isothiocyanate (FITC), Texas Red (TR), CY 5.5 and IRD 800 used.

The sequences and labels of the primers used are in Table 7 summarized.  

Table 7

Sequencing protocols

All of the sequencing reactions were considered thermal cycling reactions (Murray et al., Nucleic Acids Res. 17 (1989), 8889) using a thermal cycler (MJ Research, PTC-200) with the following profile carried out: 25 or 40 cycles at 97 ° C for 15 sec, 55 ° C for 30 sec, 68 ° C for 60 sec or 30 sec.

The protocols for the reactions (number of primers - number of matrices - number Reaction tubes) were as follows:

Tandem duplex response (4-2-1)

3 µl DNA template 1 (1 µg / µl) were mixed with 2 µl DNA template 2 (0.5 µg / µl) and 3 µl each of the primers FITC-T7term (4 µM), TR-T7, CY 5.5-RPO and IRD 800-UPO (2 µM each), 2.1 µl 10 × cycling buffer, 3 µl AmpliTaqFS  (Applied Biosystems) and 1.9 µl water mixed. The mixture was in four aliquots (5 µl each) divided. 3 µl of each Termination mixes (A, C, G, T) were in each aliquot given. Then thermal cycling was started. After 25 cycles 1 µl stop solution was added to each reaction and the volume during denaturation at 95 ° C for about 10 minutes by about half decreased. Then the batch was put on hold.

Quadruplex reaction (4-4-1)

2 µl DNA template 1 (0.5 µg / µl) were mixed with 3 µl DNA template 2 (0.3 µg / µl), 5 µl DNA template 3 (0.2 µg / µl), 3 µl DNA template 4 (0.3 µg / µl) and each 1 µl of primers 1 to 4 (5 µM each), 2.4 µl 10 × cycling buffer, 3 µl AmpliTaqFS and 1.5 µl DMSO mixed. The mixture was divided into four Aliquots (5 µl each) and 3 µl of each Termination mixes (A, C, G, T) were in each aliquot pipetted. Then thermal cycling was started. After 25 cycles 1 µl stop solution was added to each reaction and the volume during denaturation at 95 ° C for about 10 minutes by about half decreased. Then the approaches were put on hold.

Sequence gap filling reaction (4-1-1)

1.5 µl DNA template 1 (0.7 µg / µl) were added with 2 µl each of primers 5 to 8 (2 µM each), 2.1 µl 10 × cycling buffer, 3 µl AmpliTaqFS, 1.5 µl DMSO and 4.9 ul water mixed. The mixture was divided into four aliquots (each 5 µl) divided and 3 µl of the respective termination mixture (A, C, G, T) were pipetted into the individual aliquots. Then thermal cycling started. After 25 cycles, 1 µl stop solution was added to each reaction given and the volume was at 95 ° C during denaturation reduced by about half for about 10 min. Then the approaches put on ice.

Competitive tandem duplex reaction (4-1-1)

4 µl DNA template 1 (1 µg / µl) were each with 2 µl primers (FITC-RP, TR- 40, RPO-CY 5.5, IRD 800-40, 2 µM each), 2.0 µl 10 × cycling buffer, 3 µl AmpliTaqFS and 3 µl water mixed. The mixture was divided into four Aliquots (5 µl each) and 3 µl of each Termination mixes (A, C, G, T) were each in the individual aliquots pipetted. Then thermal cycling was started. After 40 cycles (Extension at 68 ° C for 60 sec) 1 µl stop solution was added to each Reaction batch was given and the volume was during a Denaturation at 95 ° C for about 10 min reduced to about half. Then the approaches were put on hold.

Similar results were also obtained with the reagents from commercial cycle sequencing kits from Applied Biosystems (TaqFS Dye Primer Cycle Sequencing Kit).

1.2 Automated DNA sequencing device

Four lasers were used to excite the four fluorescent dyes different emission wavelengths used, which correspond to the selected absorption spectrum of the fluorescent dye had been. The laser types and powers are in the following table 8 shown.

Table 8

The lasers were placed in the gel via a single coupling plate on four different positions with a vertical distance of 10 each mm coupled between the beams. A fine adjustment of the Beam position was done individually for all four lasers by a motorized one optical stage.

According to the emission spectra of the four fluorescent dyes optical bandpass filter for the optimal transmission of each Emission light spectra and the maximum suppression of scattered Excitation laser light and infrared background due to glass and Gel fluorescence selected. The high selectivity of the transmission profile was created by a combination of absorption and multilayer Interference filtering reached (Schott, Mainz, Germany).

The light was collected and through an optical system, which a spatially continuous and upright image, high resolution and high monomeric aperture (i.e. minimal photon loss) to which Detectors focused. For collecting and imaging fluorescent light a gradient index lens array (Nippon Sheet Glass Company, Japan) used.

The fluorescent light was detected by means of four one above the other stacked detector arrays (Hamamatsu Photonics, Japan) on which by a respective gradient index lens array imaged the fluorescent light was (cf. the above statements on the embodiment of Electrophoresis device).

1.3 Gel electrophoresis

The products of the sequencing reactions were separated to 5.0% or 4.3% Hydrolink Long R 16894 00070 552 001000280000000200012000285911678300040 0002019948391 00004 16775anger denaturing gels (AT Biochem, Malvern, PA, USA) with 42% urea. Running and gel buffers were 1.2 ×  TBE. 100 ml of gel solution was added by adding 84.5 µl of N, N, N ', N'- Tetramethylethylenediamine (TEMED) and 650 µl 10% Ammonium peroxodisulfate (APS) solution polymerized in water. The gels were 0.30 mm thick. The temperature was 45 ° C. The separation took place over a distance of about 50 cm using 60 cm long and 34 cm wide glass plates. The electrophoresis was performed with a performance of 60 W and an initial voltage of about 1500 V.

The sequences of all four differently labeled reaction products with A single gel lane was detected online and in a computer recorded. The evaluation took place automatically after the end of the run using the software packages Lanetracker and Geneskipper (EMBL, Schwager et al.).

1.4 Gel loading

For handling the large number of samples and the simultaneous Loading the 120 traces of a gel were made of porous combs Filter material and a robot system (Beckman Biomek 2000) are used. This method is described in detail by Erfle et al. (Nucleic Acids Res. 25 (1997), 2229-2230) and includes loading the Wells of a polyacrylic (plexiglass) block with the sample suspension by a pipetting robot. By immersing the teeth of the comb in the Plexiglas block pockets absorbs the porous material Sample liquid by capillary action. The comb containing the samples is then placed between the glass plates just above the straight edge of the polymerized gel used. When applying the electric field the samples are pulled out of the comb and into the gel.

In each case 0.6 µl of the sequencing samples were applied to the Comb loaded. In addition, four standard Sequencing reactions (4-4-4) and two duplex  Sequencing reactions (4-2-2) in separate reaction tubes performed, premixed and loaded onto the comb or without Premix all four reactions separately on the same teeth on the comb loaded. The results were identical to those of the previous ones receive the protocols described.

1.5 results

The fluorescence quadruplex sequencing method allows the application different strategies. So four different matrices were in a reaction vessel using four differently labeled Primer (4-4-1), two different matrices with a tandem duplex Sequence reaction in two directions and four differently marked Primers in a vessel (4-2-1) and sequence gap filling reactions using four "walking primers" for simultaneous closure of four gaps on the same template in a vessel (4-1-1) successfully carried out.

With a resolution of 1000 bases per strand, the techniques described about 4000 bases per sequencing reaction be determined. This means that there are 120 sequencing reactions can be separated into a single gel, making 120 kb Information can be obtained per gel.

The excitation of fluorescent dyes and the collection of Emission light occurred continuously across the width of the gel and continuously over the entire duration. Any dye could be selective be excited and detected. The accuracy and readable Sequence length showed no difference compared to standard Sequencing reactions.  

That by means of the electrophoresis device according to the invention feasible fluorescence quadruplex sequencing is also available for inner markings or dye-marked terminators are suitable.

Example 2

With the protocol described in Example 1, 192 Sequencing reactions (i.e. 48 sequencing reactions on each independent cloning) on a single gel. Could 155 kb or more information can be obtained per gel.

Example 3

The dye FITC used in Example 1 was modified by 5-carboxy Rhodamine 6 G replaced. Instead of the argon laser, a neodymium-YAG laser was used with an emission wavelength of 532 nm and a power of 5 to 10 mW used. Again, there was a selective suggestion and selective Detection of four dyes possible on a gel sheet.

Example 4

In addition to the dye combination described in Example 3 the dye CY 2 used. A neodymium YAG laser with a wavelength of 473 nm and a power of 5 to 10 mW.

It has been found that selective excitation and selective detection even when using five fluorescent dyes at the same time a gel track is possible.

In the following the invention is based on further aspects of exemplary embodiments explained in more detail.  

To temper the gel, an electrophoresis device according to the invention can have a thermostattable plate of the type shown in FIG. 6 as an example. FIG. 6 corresponds to FIG. 1 of DE 30 46 729 C2 already referred to. The thermostattable plate 16 b consists of a lower glass plate 112 and an upper glass plate 214 , which are arranged in parallel one above the other and are held at a defined distance from each other by a multi-part spacer strip 216 . The two glass plates 212 and 214 preferably have the same outline, but can have different thicknesses, for example 3 mm for the lower glass plate 212 and 1.5 mm for the upper glass plate 214 which is in contact with the gel layer. The outer upper side of the upper glass plate 214 can be pretreated in order to be gel-repellent and thus to facilitate the detachment of the gel layer coming into contact with the upper side. The two glass plates, like the multi-part spacer strip 216, are made of mirror glass or floated glass in order to ensure high planicity.

The spacer strip 216 runs along the rectangular outline of the glass plates 212 and 214 . It consists of two six sections running from top to bottom in FIG. 1, of which the left one is designated 220 and the right one is designated 222 , and two cross sections 224 and 226 connecting the ends thereof.

The spacer strip 216 is self-contained and therefore includes an interior 228 between the glass plates 212 and 214 . To through the interior 218 heat exchanger means, for. B. via a separate thermostat heating water or from the heating bath and pump arrangement 28 , 30 ( FIG. 1) water, two connections are provided, an inlet connection 230 and an outlet connection 232 . The supply connections are integrated by means of the adhesive used for gluing tightly into the spacer strips 216th

A plurality of guide strips 240 are used in the interior 228 in order to achieve a meandering (zigzag-shaped or serpentine) flow path 238 for the heat exchange medium. Between the guide strips 240 , the flow direction C of the heat exchanger means preferably runs perpendicular to the molecular propagation direction M, which in the illustration in FIG. 6 runs from bottom to top in deviation from the illustration in FIG. 1. The thermostattable plate can be arranged for use in electrophoresis for vertical separation from top to bottom as desired. The conductive strips 240 can be made of glass or - alternatively - made of a good heat-conducting material, e.g. B. copper exist. The latter can improve the temperature constancy in the transverse direction to the molecular propagation direction M.

A modified embodiment of the thermostattable plate is shown in FIG. 18. The thermostattable plate 16 c shown here has transverse guide strips 240 'which divide the interior 228 ' into seven separate flow areas 239 '. In addition to the connections 230 'and 232 ', further connections 234 'are provided, so that each flow area 239 ' has an inlet connection and an outlet connection. Connections immediately following one another along the direction of molecular propagation M can be connected to one another in pairs in order to achieve a meandering passage of the heat exchanger means through the plate, as in the embodiment of FIG. 6. Alternatively, each flow area 239 'can be supplied with heat exchange medium with a differently set temperature in order to set a temperature gradient in the thermostating plate 16 c along the molecular propagation direction M. For example, a temperature profile T, indicated schematically on the right of the plate 16 c, can be set, according to which the temperature of the plate and thus the adjacent gel layer increases in the direction of molecular propagation M, while the temperature in the transverse direction is essentially constant. It can be a temperature profile that increases linearly or also parabolically. In the case of 60 cm long plates, a temperature rise over a few degrees, for example 2 to 10 °, along the plate in the direction of molecular propagation has proven to be advantageous in order to achieve a kind of focusing effect for the bands moving in the direction of molecular propagation M by influencing the viscosity of the gel. This focusing effect is based on the front flanks of the bands moving from a higher mobility area to a lower mobility area. This focusing compensates at least to a certain extent for the band diffusion that naturally occurs in electrophoresis, and higher resolutions and longer sequence reading lengths can be achieved.

The thermostattable plate enables a wide variety of settings Temperature profiles, for example a temperature profile with in Molecular direction of propagation of increasing temperature. Furthermore, it can be advantageous in an initial area of the gel electrophoresis lanes to provide a locally elevated temperature in order to make it more denaturing To create conditions for DNA.

The detection of the bands by means of at least one laser beam can are supported in that in the area cut by the laser beam of the gel a temperature increase, i.e. a locally higher temperature, is set. This leads to a targeted broadening of the bands and Increase in the bandgap, which means better detectability of the Gangs results.

It has been found that a focusing temperature gradient in the molecular propagation direction can also be achieved in other ways using a thermostatted plate as shown in Fig. 6, based on a special mode of operation for the thermostatted plate. According to this mode of operation, the thermostattable plate, which is arranged vertically in a housing, is first heated to a predetermined temperature, for example 45 ° C., by means of the heat exchanger medium circuit in order to create denaturing conditions in the gel and to heat the gel uniformly in order to meet the requirements for a uniform and parallel migration of the molecules to be analyzed in the gel. The heat exchanger medium circuit is then interrupted by the thermostattable plate, for example by decoupling the plate from the water bath. In this way, on the one hand it is achieved that vibrations of the thermostat plate induced by pressure fluctuations of the heat exchanger means are eliminated, which lead to non-planarities of the thermostat plate and thus via a modification of the laser beam guidance in the gel, in particular additional reflections of the respective laser beam on the glass of the thermostat plate, an increase in the background - Radiation background (additional scattered light and / or additional fluorescent light due to an inherent fluorescence of the glass) causes, whereby the signal-to-noise ratio is deteriorated and thus the sequence reading length is limited. Furthermore, due to heat exchange with the surroundings of the thermostattable plate in the surrounding housing of the electrophoresis device (not shown in FIG. 1), especially the ambient atmosphere, and possibly due to convection of the surrounding atmosphere and / or the heat exchange medium in the plate Temperature gradient in the molecular propagation direction M, whereby a higher temperature occurs at the upper end of the gel, i.e. at the beginning of the gel electrophoresis webs, and a lower temperature at the lower end of the gel, i.e. at the end of the gel electrophoresis webs. Despite the interruption of the heat exchange medium circuit, the general temperature level, apart from the temperature gradient, is maintained at least to some extent due to the Joule heat developed in the gel during electrophoresis, so that the denaturing conditions are maintained in the gel. In this way, even with the use of a thermostattable plate as shown in FIG. 6, a temperature profile which drops by a few degrees in the direction of molecular propagation can be achieved in order to achieve the focusing effect explained.

By arranging the thermostat plate accordingly and if necessary additional shielding measures can also be used by others Temperature profiles, possibly with a local temperature increase in at least one cross-gel area can be achieved.

A kind of focusing effect can also be obtained on the basis of a temperature profile with a temperature gradient in a direction corresponding to a thickness of the gel layer. On the basis of the Joule heat generated in the gel during electrophoresis and, for example, two thermostattable plates 16 d1 and 16 d2, between which the gel layer 12 d is arranged (cf. FIG. 19a), a temperature gradient in the direction mentioned can be set accordingly a temperature profile at which the temperature in the middle of the gel is at a maximum and decreases towards the thermostattable plates. A corresponding temperature profile is shown as an example in FIG. 19b. Since the migrating molecules concentrate in an area of higher mobility, i.e. lower viscosity, there is an accumulation of molecules of the same molecular weight (same fragment length) in a central area of the gel layer, which reduces disruptive effects at the interface between adjacent glass plates and the gel . This also has a focusing effect on the migrating bands. A temperature gradient can be provided both in the direction of molecular propagation M and in the direction associated with the gel thickness. Alternatively, a temperature gradient can only be provided in the direction of molecular propagation M or only in the direction associated with the gel thickness.

The invention relates to an electrophoresis device for analyzing Fluorescence marker-labeled molecules, especially biological ones Molecules which, according to one aspect of the invention, are suitable for at least four different fluorescent markers independently of one another by means of an assigned detector field according to the respective gel Electrophoresis web and the respective fluorescent marker resolved to detect. Here are spectral filters with regard to their spectral Selectivity, the fluorescent markers in terms of their spectral absorption and emission selectivity and the wavelengths used to excite the Fluorescence markers used laser beams in a corresponding manner coordinated.

Claims (98)

1. Electrophoresis device ( 10 ) for analyzing fluorescent marker-labeled molecules, in particular biological molecules, comprising:

• a plurality of gel electrophoresis tracks ( 12 ) which run essentially parallel to one another and along a molecular propagation direction (M),
• an energy source ( 20 ) for applying an electrical potential to a start region and an end region of the gel electrophoresis tracks ( 12 ),
• a laser arrangement ( 50 , 52 , 54 , 56 ) for exciting a plurality of different fluorescent markers assigned to the molecules, at least some of which have different absorption wavelength ranges (A) and / or different emission wavelength ranges (E),
  wherein the laser arrangement in the electrophoresis mode generates laser radiation which cuts at least several of the gel electrophoresis tracks ( 12 ) as laser beams ( 60 , 62 , 64 , 66 ) which are essentially transverse to the direction of molecular propagation and essentially parallel to one another and offset in the direction of molecular propagation, in order to excite fluorescent markers of the molecules present in a detection area of the respective gel electrophoresis path,
• a detection arrangement ( 90 ) for gel electrophoresis web-resolved and fluorescence marker-resolved detection of emission radiation emitted by the excited fluorescence markers,

wherein the detection arrangement comprises a plurality of detector fields ( 102 ; 102a ) which are offset from one another in the direction of molecular propagation and preferably extend essentially parallel to the laser beams, each of which is assigned to a specific one of the laser beams and is designed for this purpose, preferably along one associated with the respective laser beam to detect at least a part of the essentially parallel detection section of the emission radiation which is generated in the detection areas due to the excitation by the respective laser beam and which is guided to the detector field via an associated spectral filter and radiation guide arrangement ( 106 , 108 ; 112 a),
wherein the spectral filter arrangement ( 106 ; 106 a, 107 ) in terms of their spectral selectivity, the fluorescence markers in terms of their spectral absorption and emission selectivity, and the wavelengths of the laser beams are matched to one another such that at least four laser beams and at least four detector fields are used to match at least four different fluorescent markers can be detected independently of one another by means of an assigned detector field according to the respective gel electrophoresis path and the respective fluorescent marker. 2. Electrophoresis device ( 10 ) for analyzing fluorescence marker-labeled molecules, in particular biological molecules, comprising:

• a plurality of gel electrophoresis tracks ( 12 ) which run essentially parallel to one another and along a molecular propagation direction (M),
• an energy source ( 20 ) for applying an electrical potential to a start region and an end region of the gel electrophoresis tracks ( 12 ),
• a laser arrangement ( 50 , 52 , 54 , 56 ) for exciting a plurality of different fluorescent markers assigned to the molecules, at least some of which have different absorption wavelength ranges (A) and / or different emission wavelength ranges (E),
  wherein the laser arrangement in the electrophoresis mode generates laser radiation which, as laser beams ( 60 , 62 , 64 , 66 ) which run essentially transversely to the molecular propagation direction, cuts at least several of the gel electrophoresis tracks ( 12 ) in order to detect in a detection area of the respective gel electrophoresis Excite fluorescent markers of the molecules present there, which are assigned to the path, at least some of the laser beams ( 60 , 62 , 64 , 66 ) being spatially offset from one another and / or occurring in mutually offset time windows or / and wherein at least one laser beam is modulated to enable phase-sensitive detection or / and wherein at least some of the laser beams have different wavelengths,
• a detection arrangement ( 90 ) for gel electrophoresis web-resolved and fluorescence marker-resolved detection of emission radiation emanating from the excited fluorescence markers,
  wherein the detection arrangement has at least one detector field ( 102 ; 102 a) which preferably extends essentially parallel to the laser beams and which is designed to along at least a portion of the in the detection areas along a detection path assigned to the laser beam, preferably essentially parallel to this To detect excitation caused by the respective laser beam, using an assigned spectral filter and radiation guide arrangement ( 106 , 108 ; 112 a), to detect emission radiation guided to the detector field in a spatially resolved manner,
  wherein the spectral filter arrangement ( 106 ; 106 a, 107 ) in terms of their spectral selectivity, the fluorescent markers in terms of their spectral absorption and emission selectivity, and the wavelengths of the laser beams are matched to one another in such a way that at least four different fluorescent markers can be used independently of one another using at least four laser beams the respective gel electrophoresis path and the respective fluorescent marker can be detected in a resolved manner.

3. Device according to claim 1 or 2, characterized in that the laser radiation selectively excitable fluorescent markers with overlapping emission spectra (E) can be distinguished from one another on the basis of spatially and / or temporally separate laser beam excitation and / and on the basis of different wavelengths of the laser beams ( 60 , 62 , 64 , 66 ) or / and on the basis of a phase-sensitive detection when excited with modulated laser radiation or / and that by means of the laser radiation selectively or non-selectively excitable fluorescence markers with at least partially non-overlapping emission spectra (E) can be distinguished on the basis of spectrally selective detection of the emission radiation and possibly spatially separated laser beam excitation. 4. Device according to one of claims 1 to 3, characterized in that the wavelengths of the laser beams ( 60 , 62 , 64 , 66 ) and / or spectral filter ( 106 ; 106 a, 107 a) of the spectral filter arrangements are matched to at least four different fluorescent markers whose absorption maxima are at least 30 nm, preferably at least 50 nm apart from one another under electrophoresis operating conditions and / and whose emission maxima are at least 30 nm, preferably at least 50 nm apart from one another under electrophoresis operating conditions. 5. Device according to one of claims 1 to 4, characterized in that at least one spectral filter ( 106 ; 106 a, 107 a) has a spectral characteristic such that due to the excitation of the respectively associated laser beam emerging fluorescent radiation of the respectively assigned fluorescent marker for a passage -Wavelength range around an emission maximum of the fluorescence spectrum of this fluorescence marker or for a transmission wavelength range adjacent to this fluorescence maximum is guided to the assigned detector field ( 102 ; 102 a). 6. Device according to claim 5, characterized in that in relation to at least one of the other fluorescent markers at least one of the following conditions a) and b) is fulfilled in order to enable the detection resolved after the fluorescent markers:

• a) the laser beam wavelength lies on a spectral side of an absorption spectrum (A) of the other fluorescence marker beyond a slope of the absorption spectrum falling from an absorption maximum to the laser beam wavelength down to at least a residual absorption amplitude which corresponds to an at most minimal residual absorption,
• b) the transmission wavelength range lies on a spectral side of an emission spectrum (E) of the other fluorescent marker beyond a flank of the emission spectrum falling from an emission maximum to the transmission wavelength range down to at least one residual emission amplitude, which corresponds to a minimal residual emission.

7. Device according to claim 6, characterized in that the for Laser beam wavelength falling edge is too long Is the falling edge and the Wavelength range falling edge is too short Is the falling edge. 8. Device according to claim 6, characterized in that the for Laser beam wavelength falling edge is too short Is the falling edge and the Wavelength range falling edge is too long Is the falling edge. 9. Device according to one of claims 6 to 8, characterized characterized in that the flank to the laser beam wavelength or Pass wavelength range drops more steeply than one on the other side of the maximum of the spectrum (A or E), with the distance from the maximum falling edge of the spectrum. 10. Device according to one of claims 1 to 9, characterized in that at least one spectral filter and radiation guide arrangement as a radiation guide arrangement comprises a lens arrangement, preferably a gradient index lens field ( 108 ; 108 a). 11. Device according to one of claims 1 to 10, characterized by at least one object-side spectral filter ( 107 a) arranged on an object side of the radiation guiding arrangement assigned to the gel electrophoresis tracks and / or at least one image-side spectral filter arranged on an image side of the radiation guiding arrangement assigned to the detector field ( 106 ; 106 a). 12. Device according to one of claims 1 to 11, characterized in that at least one spectral filter arrangement ( 106 a, 107 a) is designed as an interference filter or comprises an interference filter ( 107 a). 13. Device according to one of claims 1 to 12, characterized in that at least one spectral filter arrangement ( 106 a, 107 a) is designed as a combination of an absorption filter arrangement ( 106 a) and an interference filter arrangement ( 107 a). 14. The device according to claim 13, characterized in that at least one spectral filter ( 107 a) comprises an absorption filter, possibly colored glass filter, which serves as a carrier substrate for thin-film films of an associated interference filter. 15. Device according to one of claims 12 to 14, characterized in that the interference filter ( 107 a), possibly in conjunction with the radiation guide arrangement ( 108 a), defines a wavelength range as a transmission wavelength range, which is a superposition of transmission ranges of a / the Interference filter for different light incidence angles in the interference filter corresponds. 16. The device according to claim 15, characterized in that a wavelength range is defined as the transmission wavelength range, which includes all transmission ranges of the interference filter ( 107 a) for all occurring according to an acceptance angle characteristic of the radiation guide arrangement ( 108 a) light incidence angle. 17. The device according to claim 15 or 16, characterized in that a wavelength range is defined as the transmission wavelength range, a transmission range of the interference filter ( 107 a) for a light angle of incidence of α = 0 ° and a transmission range of the interference filter for a maximum acceptance angle the radiation guide arrangement ( 108 a) corresponding light incidence. 18. Device according to one of claims 1 to 17, characterized characterized by the following fluorescent markers at least seven markers are detectable: indodicarbocyanine CY 2, Alexa 488, fluorescein iso-thio-cyanate (FITC), 5-carboxy Rhodamine 6G, Alexa 532, tetramethyl-rhodamine-iso-thio-cyanate (TRITC), indodicarbocyanine CY 3, sulforhodamine 101 (TexasRed ™), Indodicarbocyanin CY 5, Indodicarbocyanin CY 5.5, IRD 700, Indodicarbocyanin CY 7, IRD 40 and IRD 800. 19. The device according to claim 18, characterized in that at least the markers indodicarbocyanin CY 2, fluorescein-iso-thio- Cyanate (FITC), 5-carboxy-rhodamine 6G, sulforhodamine 101 (TexasRed ™), Indodicarbocyanin CY 5, Indodicarbocyanin CY 5.5 and IRD 800 are detectable. 20. Device according to one of claims 1 to 19, characterized in that at least three, preferably at least four, most preferably at least five markers of the following fluorescent markers are simultaneously resolved fluorescent markers and gel electrophoresis web-resolved are detectable:
Indodicarbocyanin CY 2, Alexa 488, Fluorescein-Iso-Thio-Cyanate (FITC), 5-Carboxy-Rhodamin 6G, Alexa 532, Tetramethyl-Rhodamine-Iso-Thio-Cyanate (TRITC), Indodicarbocyanin CY 3, Sulforhodamine 101 (TexasRed ™ ), Indodicarbocyanin CY 5, Indodicarbocyanin CY 5.5, IRD 700, Indodicarbocyanin CY 7, IRD 40 and IRD 800. 21. The device according to claim 20, characterized in that at least the five markers
Indodicarbocyanin CY 2, 5-carboxy-Rhodamine 6G, Sulforhodamine 101 (TexasRed ™), Indodicarbocyanin CY 5.5 and IRD 800
or at least the four markers
Fluorescein iso-thio-cyanate (FITC), Sulforhodamine 101 (TexasRed ™), Indodicarbocyanin CY 5.5 and IRD 800
or at least the four markers
Indodicarbocyanin CY 2, 5-carboxy-Rhodamine 6G, Indodicarbocyanin CY 5 and IRD 800
or at least the three markers
Fluorescein iso-thio-cyanate (FITC), indodicarbocyanine CY 5 and IRD 800
fluorescent marker-resolved and gel electrophoresis web resolved can be detected simultaneously. 22. Device according to one of claims 18 to 21, characterized characterized in that for the detection of the fluorescent marker Indodicarbocyanin CY 2 a frequency-doubled, possibly diode-pumped neodymium: YAG laser with an emission wavelength of approximately 473 nm is provided. 23. Device according to one of claims 18 to 22, characterized characterized in that the fluorescent marker indodicarbocyanine CY 2 shows a spectral filter arrangement with a pass band of approximately 494 nm to about 524 nm, possibly with different light  Passages of about 494 nm assigned to angles of incidence α to about 524 nm for α = 0 ° and about 492 nm to about 514 nm for a maximum acceptance angle of Radiation guidance arrangement corresponding light incidence angle, for example of about 20 °. 24. Device according to one of claims 18 to 23, characterized characterized in that the fluorescent marker Spectral filter arrangement assigned to indodicarbocyanine CY 2 Absorption short pass filter, an absorption long pass filter and one Interference bandpass filter includes. 25. Device according to one of claims 18 to 24, characterized characterized in that for the detection of the fluorescent marker Fluorescein Iso Thio Cyanate (FITC) is an argon ion laser with a Emission wavelength of about 488 nm or a Frequency-doubled, possibly diode-pumped neodymium: YAG laser with an emission wavelength of about 473 nm is provided. 26. Device according to one of claims 18 to 25, characterized characterized in that the fluorescence marker fluorescein iso-thio Cyanate (FITC) a spectral filter arrangement with a pass band from about 505 nm to about 555 nm, possibly with different light Passages of about 505 nm assigned to angles of incidence α up to about 555 nm for α = 0 ° and about 500 nm to about 545 nm for a maximum acceptance angle of Radiation guidance arrangement corresponding light incidence angle, for example of about 20 °. 27. Device according to one of claims 18 to 26, characterized characterized in that the fluorescein iso- Spectral filter arrangement associated with thio-cyanate (FITC)  Absorption short pass filter, an absorption long pass filter and one Interference bandpass filter includes. 28. Device according to one of claims 18 to 27, characterized characterized in that for the detection of the fluorescent marker 5- Carboxy-Rhodamine 6 G a frequency-doubled, possibly diode-pumped neodymium: YAG laser with an emission wavelength of about 532 nm is provided. 29. Device according to one of claims 18 to 28, characterized characterized in that the fluorescent marker 5-carboxy-rhodamine 6G shows a spectral filter arrangement with a pass band of approximately 555 nm to about 578 nm, possibly with different light Passages of about 555 nm assigned to angles of incidence α to about 578 nm for α = 0 ° and about 553 nm to about 569 nm for a maximum acceptance angle of Radiation guidance arrangement corresponding light incidence angle, for example of about 20 °. 30. Device according to one of claims 18 to 29, characterized characterized in that a / the 5-carboxy- Spectral filter arrangement assigned to Rhodamine 6G has an absorption Short pass filter and an interference band pass filter. 31. Device according to one of claims 18 to 30, characterized characterized in that for the detection of the fluorescent marker Sulforhodamine 101 (TexasRed ™) a helium-neon laser with a Emission wavelength of about 594 nm is provided. 32. Device according to one of claims 18 to 31, characterized characterized in that the fluorescent marker sulforhodamine 101 (TexasRed ™) a spectral filter arrangement with a pass band  from about 613 nm to about 651 nm, possibly with different light Passages of about 613 nm assigned to angles of incidence α to about 651 nm for α = 0 ° and about 613 nm to about 636 nm for a maximum acceptance angle of Radiation guidance arrangement corresponding light incidence angle, for example of about 20 °. 33. Device according to one of claims 18 to 32, characterized characterized in that the fluorescent marker sulforhodamine 101 (TexasRed ™) associated spectral filter arrangement one Absorption long pass filter and an interference band pass filter. 34. Device according to one of claims 18 to 33, characterized characterized in that for the detection of the fluorescent marker Indodicarbocyanin CY 5 a helium-neon laser with a Emission wavelength of about 633 nm or a semiconductor laser an emission wavelength of about 640 nm is provided. 35. Device according to one of claims 18 to 34, characterized characterized in that the fluorescent marker indodicarbocyanine CY 5 shows a spectral filter arrangement with a pass band of approximately 655 nm to about 690 nm, possibly with different light Passages of about 655 nm assigned to angles of incidence α up to about 690 nm for α = 0 ° and about 650 nm to about 683 nm for a maximum acceptance angle of Radiation guidance arrangement corresponding light incidence angle, for example of about 20 °. 36. Device according to one of claims 18 to 35, characterized characterized in that the fluorescent marker Spectral filter arrangement assigned to indodicarbocyanine CY 5 Absorption long pass filter and an interference band pass filter.   37. Device according to one of claims 18 to 36, characterized characterized in that for the detection of the fluorescent marker Indodicarbocyanin CY 5.5 or / and the fluorescent marker IRD 700 a semiconductor laser with an emission wavelength of about 660 nm or about 670 nm or about 675 nm is provided. 38. Device according to one of claims 18 to 37, characterized characterized in that the fluorescent marker indodicarbocyanine CY 5.5 or / and the fluorescent marker IRD 700 a Spectral filter arrangement with a pass band of approximately 655 nm up to about 690 nm, possibly with different light incidence angles α assigned pass ranges from about 695 nm to about 745 nm for α = 0 ° and about 695 nm to about 730 nm for one maximum acceptance angle of the radiation guide arrangement corresponding light incidence angle, for example of approximately 20 °, assigned. 39. Device according to one of claims 18 to 38, characterized characterized in that the fluorescent marker Indodicarbocyanin CY 5.5 or / and the fluorescent marker IRD 700 assigned spectral filter arrangement an absorption long-pass filter and an interference bandpass filter. 40. Device according to one of claims 18 to 39, characterized characterized in that for the detection of the fluorescent marker IRD 800 is a semiconductor laser with an emission wavelength of approximately 775 nm or from about 780 nm is provided. 41. Device according to one of claims 18 to 40, characterized characterized in that the fluorescent marker IRD 800 a Spectral filter arrangement with a pass band of approximately 795 nm up to about 790 nm, possibly with different light incidence angles α  assigned pass ranges from about 795 nm to about 835 nm for α = 0 ° and about 790 nm to about 830 nm for one maximum acceptance angle of the radiation guide arrangement corresponding light incidence angle, for example of approximately 20 °, assigned. 42. Device according to one of claims 18 to 41, characterized characterized in that the fluorescent marker IRD 800 assigned spectral filter arrangement an absorption long-pass filter and an interference bandpass filter. 43. Device according to one of claims 1 to 42, characterized through measures to reduce the resolving power detection with respect to the gel electrophoresis pathway and / or in Reference to the fluorescent markers and / or the signal-to- Noise ratio impairing interference radiation, if necessary Fluorescent light background radiation or / and scattered Laser radiation. 44. Device according to claim 43, characterized in that at least one of the laser beams is linearly polarized with one Polarization vector which is after an input side Direction of light of the spectral filter and Radiation guidance arrangement is aligned, possibly to this is at least approximately parallel. 45. Device according to claim 43 or 44, characterized in that the gel electrophoresis tracks run in a preferably plate-shaped gel matrix ( 12 ) which is arranged between glass plates ( 14 , 16 ) made of floated borosilicate glass. 46. Device according to one of claims 43 to 45, characterized in that the gel electrophoresis tracks run in a preferably plate-shaped gel matrix ( 12 ), the laser beams ( 60 , 62 , 64 , 66 ) via at least one to the laser beam propagation direction essentially parallel coupling plate in the gel matrix ( 12 ) are coupled into the gel matrix, the coupling plate having a polished entry surface which is essentially orthogonal to the respective laser beam or / and a polished exit surface or / and which is essentially orthogonal to the respective laser beam and / or one adapted to the gel matrix , the refractive index of the gel matrix may have at least approximately the corresponding refractive index. 47. Device according to claim 46, characterized in that a common coupling plate is provided for all laser beams ( 60 , 62 , 64 , 66 ). 48. Device according to one of claims 1 to 47, characterized in that the spatially offset, different wavelengths having different laser beams ( 60 , 62 , 64 , 66 ) are offset from one another such that the laser radiation wavelength in the molecular propagation direction (M) from laser beam to laser beam decreases. 49. Electrophoresis device ( 10 ) for analyzing fluorescence marker-labeled molecules, in particular biological molecules, comprising:

• a plurality of gel electrophoresis tracks ( 12 ) which run essentially parallel to one another and along a molecular propagation direction (M),
• an energy source ( 20 ) for applying an electrical potential to a start region and an end region of the gel electrophoresis tracks ( 12 ),
• a laser arrangement ( 50 , 52 , 54 , 56 ) for exciting fluorescent markers assigned to the molecules,
  the laser arrangement in the electrophoresis mode generating laser radiation which, as at least one laser beam ( 60 , 62 , 64 , 66 ) running essentially transversely to the molecular propagation direction, cuts at least several of the gel electrophoresis tracks in order to detect in a detection area of the respective gel electrophoresis track to excite assigned fluorescent markers of the molecules present there,
• a detection arrangement ( 90 ) for detecting emission radiation emanating from the excited fluorescent markers,
  wherein the detection arrangement has at least one detector field ( 102 ; 102 a) which preferably extends essentially parallel to the at least one laser beam and is designed for this purpose along at least part of the in the detection areas along a detection path assigned to the laser beam, preferably essentially parallel to it to detect emission radiation that is caused by the laser beam excitation and is spatially resolved via an assigned spectral filter and radiation guide arrangement ( 106 , 108 ; 112 a),
  the spectral filter and radiation guide arrangement deflecting the emission radiation emitted by the excited fluorescent markers onto receiving surfaces or receiving surface sections ( 104 a) of the detector field ( 102 a), which are arranged at least approximately orthogonally to the direction of molecular propagation (M).

50. Device according to Anpruch 49 , characterized in that the detector field ( 102 a) is arranged on a carrier board ( 114 a) which is arranged at least approximately orthogonally to the molecular propagation direction (M). 51. Device according to claim 49 or 50, characterized in that in the molecular propagation direction one behind the other a plurality of laser beams preferably assigned different wavelengths associated detector fields ( 102 a) each with the molecular propagation direction (M) at least approximately orthogonally arranged receiving surfaces or receiving surface sections ( 104 a) are, which are each preferably arranged on a separate carrier board ( 114 a). 52. Device according to one of claims 49 to 51, characterized in that the spectral filter and radiation guide arrangement ( 112 a) at least one deflection prism arrangement associated with a respective detector field ( 102 a) and extending essentially parallel to this detector field, possibly a continuous deflection prism ( 110 a). 53. Device according to claim 52, characterized in that the deflecting prism arrangement ( 110 a) is arranged on an output side of a lens arrangement, possibly a gradient index lens field ( 108 a), of the spectral filter and radiation guide arrangement. 54. Device according to one of claims 49 to 53, characterized in that the deflecting prism arrangement ( 110 a) is formed from a highly refractive optical material for adapting an image-side optical path between a / the lens arrangement, possibly a / the gradient index lens field ( 108 a) , the spectral filter and radiation guide arrangement on the one hand and the receiving surfaces ( 104 a) on the other hand to an image-side focal length of the lens arrangement. 55. Device according to one of claims 52 to 54, characterized in that the deflecting prism arrangement ( 110 a) is part of an integral optical module ( 112 a) which has a / the lens arrangement ( 108 A) and at least one spectral filter ( 106 a, 107 a) includes. 56. Device according to claim 55, characterized in that the deflecting prism arrangement ( 110 a), the lens arrangement ( 108 a) designed as a gradient index lens field, the at least one spectral filter ( 106 a, 107 a) and, if desired, a carrier rail for producing the integral optical module ( 112 a) are glued together. 57. Device according to claim 56, characterized in that on an optical input side of the gradient index lens field ( 108 a) and / or on an optical output side of the gradient index field ( 108 a), optionally between the gradient index lens field and the deflection prism arrangement ( 110 a), at least one Spectral filter ( 106 a) is glued on. 58. Device according to claim 56 or 57, characterized in that at least one spectral filter ( 107 a) of the optical module ( 112 a) is designed as an interference filter or comprises an interference filter. 59. Device according to one of claims 56 to 58, characterized in that at least one spectral filter ( 107 a) is designed as a combination of an absorption filter and an interference filter. 60. Device according to claim 59, characterized in that at least one spectral filter ( 107 a) of the optical module comprises an absorption filter, possibly colored glass filter, which serves as a carrier substrate for thin-film films of an associated interference filter. 61. Device according to one of claims 56 to 60, characterized in that the optical module ( 112 a) for adjusting an optical path in a module carrier in one of the laser beam sections intersecting the gel electrophoresis tracks ( 12 ) and the direction of molecular propagation in substantially orthogonal direction is slidably disposed. 62. Device according to one of claims 55 to 61, characterized in that a plurality of detector fields ( 102 a), a plurality of optical modules ( 112 a) each assigned to one of the detector fields as well as detection electronics ( 116 a) assigned to the detector fields in a detector module ( 90 ) are integrated with a common housing, the detector module being able to be handled as a unit, and preferably by displacement in a laser beam section intersecting the gel electrophoresis tracks and to the direction of molecular propagation (M), which is essentially orthogonal, a common adjustment of associated object-side optical Path lengths for all optical modules ( 112 a) of the detector module enabled. 63. Device according to one of claims 49 to 62, characterized by the remaining features of at least one of the preceding Claims 1 to 48. 64. Electrophoresis device ( 10 ) for analyzing fluorescent marker-labeled molecules, in particular biological molecules, comprising:

• a plurality of gel electrophoresis tracks ( 12 ) which run essentially parallel to one another and along a molecular propagation direction (M),
• an energy source ( 20 ) for applying an electrical potential to a start region and an end region of the gel electrophoresis tracks,
• a laser arrangement ( 50 , 52 , 54 , 56 ) for exciting a plurality of different fluorescent markers assigned to the molecules,
  wherein the laser arrangement in the electrophoresis mode generates laser radiation which, as laser beams ( 60 , 62 , 64 , 66 ) of different wavelengths which run essentially transversely to the direction of molecular propagation and essentially parallel to one another and are offset with respect to one another in the direction of molecular propagation, has at least several of the gel electrophoresis tracks ( 12 ) cuts in order to excite fluorescent markers of the molecules present in a detection area of the respective gel electrophoresis path,
• a detection arrangement ( 90 ) for the gel electrophoresis web-resolved and fluorescence marker-resolved detection of emission radiation emanating from the excited fluorescence markers,
  wherein the detection arrangement comprises a plurality of detector fields ( 102 ; 10 a) which are offset from one another in the molecular propagation direction and preferably extend essentially parallel to the laser beams, each of which is assigned to a specific one of the laser beams and is designed for this purpose, preferably along a respective one of the laser beams to detect at least a part of the essentially parallel detection section of the emission radiation which is generated in the detection areas due to the excitation by the respective laser beam and which is guided to the detector field via an associated spectral filter and radiation guide arrangement ( 106 , 108 ; 112 a),

wherein the laser arrangement has at least one laser ( 50 , 52 ) with an optical laser resonator, which is arranged at least approximately parallel to the detector fields ( 102 ; 102 a) with respect to a resonator axis which characterizes the beam path of the laser beam ( 60 or 62 ) emerging from the resonator The laser beam ( 60 or 62 ) emerging from the laser resonator is deflected by means of a beam guiding arrangement ( 70 , 72 ), so that a laser beam section running transversely to the molecular propagation direction intersects the gel electrophoresis tracks ( 12 ). 65. Device according to claim 64, characterized in that a plurality of lasers ( 50 , 52 ) with at least approximately parallel resonators with respect to a respective resonator axis are provided with the detector fields, the laser resonators with respect to the resonator axes at least approximately corresponding to the offset of the gel Electrophoresis tracks ( 12 ) intersecting laser beam sections in the molecular propagation direction (M) are offset from one another. 66. Electrophoresis device ( 10 ), optionally according to claim 64 or 65, for analyzing fluorescence marker-labeled molecules, in particular biological molecules, comprising:

• a plurality of gel electrophoresis tracks ( 12 ) which run essentially parallel to one another and along a molecular propagation direction (M),
• an energy source ( 20 ) for applying an electrical potential to a start region and an end region of the gel electrophoresis tracks ( 12 ),
• a laser arrangement ( 50 , 52 , 54 , 56 ) for exciting a plurality of different fluorescent markers assigned to the molecules,
  wherein the laser arrangement in the electrophoresis mode generates laser radiation which, as laser beams ( 60 , 62 , 64 , 66 ) of different wavelengths which run essentially transversely to the direction of molecular propagation and essentially parallel to one another and are offset with respect to one another in the direction of molecular propagation, has at least several of the gel electrophoresis tracks ( 12 ) cuts in order to excite fluorescent markers of the molecules present in a detection area of the respective gel electrophoresis path,
• a detection arrangement ( 90 ) for the gel electrophoresis web-resolved and fluorescence marker-resolved detection of emission radiation emanating from the excited fluorescence markers,
  wherein the detection arrangement comprises a plurality of detector fields ( 102 ; 102a ) which are offset from one another in the direction of molecular propagation and preferably extend essentially parallel to the laser beams, each of which is assigned to a specific one of the laser beams and is designed for this purpose, preferably along one associated with the respective laser beam to detect at least a part of the essentially parallel detection section of the emission radiation which is generated in the detection areas due to the excitation by the respective laser beam and which is guided to the detector field via an associated spectral filter and radiation guide arrangement ( 106 , 108 ; 112 a),

wherein the laser arrangement has at least two lasers ( 54 , 56 ) with a respective laser resonator, each with an angle to the detector fields ( 102 ; 102 a) based on a resonator axis that characterizes the beam path of the laser beam ( 64 or 66 ) emerging from the respective resonator ) are preferably arranged at least approximately orthogonally to the detector fields ( 102 ; 102 a), the laser beam ( 64 or 66 ) emerging from the respective laser resonator being deflected by means of a beam guiding arrangement ( 72 ), so that a beam extending transversely to the direction of molecular propagation Laser beam section intersects the gel electrophoresis tracks ( 12 ) and the laser resonators are offset with respect to the resonator axes at least approximately in accordance with the offset of the laser beam sections cutting the gel electrophoresis tracks in the direction of molecular propagation (M) and different lateral spacing vo n have the gel electrophoresis sheets ( 12 ). 67. Device according to claim 66, characterized in that the Resonator axes at least approximately in a respective, for Molecular propagation direction (M) lie orthogonal plane. 68. Device according to one of claims 64 to 67, characterized by a stepped optical bench ( 80 ) arranged on the side of the gel electrophoresis tracks ( 12 ) with an angle / to the direction of the molecular propagation (M), preferably at least approximately orthogonal to the direction of molecular propagation (M) extending steps ( 82 ), which increase with increasing lateral distance from the gel electrophoresis tracks ( 12 ) opposite to the molecular propagation direction (M) at least approximately in accordance with the offset of the laser beam sections cutting the gel electrophoresis tracks ( 12 ) and a respective one the lasers are assigned to hold a respective beam guiding arrangement ( 70 , 72 or 72 ) assigned to the laser and / or the laser resonator ( 54 , 56 ) which extends at least approximately along the step with its resonator axis. 69. Device according to claim 68, characterized in that the respective beam guiding arrangement is an optical fiber, preferably single-mode fiber, and / or at least one deflecting mirror ( 72 ) and / or at least one optical element for focusing the laser beam and / or adjusting a laser beam waist in relation to the gel electrophoresis tracks, optionally an optical lens ( 74 ), an optical objective or an optical telescope, and / or an optical element for conditioning, possibly spatial filtering, the laser beam. 70. Device according to claim 69 , characterized in that at least one deflecting mirror ( 72 ) and / or the optical element ( 74 ) for adjusting the beam path of the respective laser beam is adjustable, preferably adjustable by motor. 71. Device according to claim 70, characterized in that a deflecting mirror ( 72 ), from which the laser beam section intersecting the gel electrophoresis tracks, emanates at least approximately in a direction orthogonal to the molecular propagation direction (M) and the laser beam section, preferably at least approximately longitudinally the respective stage ( 82 ) of the optical bench ( 80 ), can be displaced for adjusting the laser beam section cutting the gel electrophoresis tracks ( 12 ). 72. Device according to claim 70 or 71, characterized in that at least one optical element ( 74 ) is at least approximately displaceable in the molecular propagation direction (M) for adjusting the laser beam section intersecting the gel electrophoresis tracks ( 12 ). 73. Electrophoresis device ( 10 ), optionally according to one of claims 64 to 72, for analyzing molecules marked with fluorescent markers, in particular biological molecules, comprising:

• a plurality of gel electrophoresis tracks ( 12 ) which run essentially parallel to one another and along a molecular propagation direction (M),
• an energy source ( 20 ) for applying an electrical potential to a start region and an end region of the gel electrophoresis tracks ( 12 ),
• a laser arrangement ( 50 , 52 , 54 , 56 ) for exciting a plurality of fluorescence markers assigned to the molecules,
  the laser arrangement in electrophoresis mode generating laser radiation which, as at least one laser beam ( 60 , 62 , 64 , 66 ) running essentially transversely to the molecular propagation direction, cuts at least several of the gel electrophoresis tracks ( 12 ) in order to detect in a detection area of the respective gel To excite fluorescent markers of the molecules present there, assigned to the electrophoresis path,
• a detection arrangement ( 90 ) for the gel electrophoresis web-resolved and fluorescence marker-resolved detection of emission radiation emanating from the excited fluorescence markers,

wherein the detection arrangement has at least one detector field ( 102 ; 102 a) which preferably extends essentially parallel to the laser beams and which is designed to along at least a portion of the in the detection areas along a detection path assigned to the laser beam, preferably essentially parallel to this To detect excitation caused by the assigned laser beam, using an assigned spectral filter and radiation guide arrangement ( 106 , 108 ; 112 a), to detect emission radiation guided to the detector field in a spatially resolved manner,
wherein the laser arrangement has at least one laser, the laser beam of which is coupled into an optical fiber, preferably a single-mode fiber, in order to guide the laser beam to the gel electrophoresis tracks and / or to condition the laser beam, or / and
wherein the laser arrangement has at least one laser, the laser beam of which is guided through at least one optical element for conditioning, possibly spatial filtering, the laser beam. 74. Device according to one of claims 69 to 73, characterized in that the laser beam possibly having a non-Gaussian profile beforehand is conditioned in such a way that it is at least approximately a Gaussian profile and / or a reduced divergence or / and one reduced beam waist diameter or / and a reduced, with the product of a divergence angle describing the divergence angle and the beam waist diameter increasing collimation factor, possibly so-called M ² factor. 75. Device according to one of claims 64 to 74, characterized in that at least one of the laser beams ( 60 , 62 , 64 , 66 ) is guided and possibly focused in such a way that a laser beam region of minimal laser beam diameter, possibly one / the beam waist, is along of the assigned detector field ( 102 ; 102 a) is arranged approximately in the region of a central one of the gel electrophoresis tracks ( 12 ) cut by the laser beam. 76. Device according to one of claims 64 to 75, characterized by the remaining features of at least one of the preceding Claims 1 to 63. 77. Adjustment method for adjusting the course of at least one laser beam ( 60 , 62 , 64 , 66 ) with respect to associated gel electrophoresis tracks of an electrophoresis device, the at least one excitation laser for generating the laser beam and at least one detector field ( 102 ; 102 a ) for recording excited fluorescent markers having emission radiation, characterized in that the adjustment is carried out on the basis of detection signals recorded by means of a detector field ( 102 ; 102 a) which generate background radiation induced by the laser beam, possibly background fluorescence or / and scattered light , represent. 78. Adjustment method according to claim 77, characterized in that in the course of the adjustment at least one optical element ( 72 , 74 ) assigned to the laser beam, optionally a lens ( 74 ), a lens, a telescope or a deflecting mirror ( 72 ), for receiving a background radiation profile is adjusted, and the optical element is then adjusted such that a background radiation is subsequently detected at least approximately in accordance with a maximum and / or a center of the profile. 79. adjustment method according to claim 77 or 78, characterized in that first a gel electrophoresis webs (12) intersecting the laser beam process and then to the gel electrophoresis lanes (12) at least approximately orthogonal laser beam profile or / and to the associated detector array ( 102 ; 102 a) at least approximately parallel laser beam path is set. 80. Adjustment method according to one of claims 77 to 79, characterized in that a laser beam path intersecting the gel electrophoresis tracks is primarily based on at least one detector field section at one end, preferably at that of a laser beam coupling point into / which the gel electrophoresis Gel matrix having webs next to the end of the detector field ( 102 ; 102 a) is set and a laser beam path at least approximately orthogonal to the gel electrophoresis paths or / and a laser beam path at least approximately parallel to the detector field is primarily based on at least one detector field section on the other Detected signals recorded at the end of the detector field is set. 81. Adjustment method according to one of claims 77 to 80, characterized characterized in that the adjustment by motor and preferably automated.   82. Adjustment method according to one of claims 77 to 81, which is applied to a device ( 10 ) according to one of claims 1 to 76. 83. Electrophoresis device ( 10 ) for analyzing labeled molecules, in particular biological molecules, comprising:

• - a plurality of substantially parallel to each other and along a molecule propagation direction (M) running gel electrophoresis tracks (12) extending in a gel matrix (12), possibly a slab gel (12),
• - at least one thermostatic plate ( 16 ; 16 a; 16 c; 16 d) in heat exchange connection with a gel matrix surface,
• an energy source ( 20 ) for applying an electrical potential to a start region and an end region of the gel electrophoresis tracks ( 12 ),

wherein the thermostating plate ( 16 ; 16 a; 16 c; 16 d) is suitable for producing and / or maintaining a uniform temperature distribution in the gel matrix in the cross-cutting direction of the gel electrophoresis webs,
wherein the at least one thermostatic plate ( 16 c) is further suitable for in the gel matrix ( 12 ) a temperature gradient in the direction of molecular propagation (M) or / and in a thickness of the gel matrix ( 12 ) associated with the thermostatic plate ( 16 d) substantially orthogonal Set direction, or / and is suitable for setting a temperature profile with a local temperature increase or a local temperature decrease in at least one transverse region of the gel matrix,
in the case of a thermostatic plate ( 16 c) through which a heat exchanger means flows, this is suitable for setting the relevant temperature gradient or the temperature profile in the state of continuous flow through the thermostatic plate ( 16 c) with the heat exchanger means. 84. Device according to claim 83, characterized in that two thermostatting plates ( 16 d1, 16 d2) are provided, between which the gel matrix ( 12 d) is arranged. 85. Device according to claim 83 or 84, characterized in that a temperature profile (T) with in the direction of molecular propagation (M) decreasing or increasing temperature is adjustable. 86. Device according to one of claims 83 to 85, characterized in that, based on the direction associated with the thickness of the gel matrix ( 12 d), a temperature profile (T) can be set with a temperature maximum lying in a central region of the gel matrix ( 12 d) . 87. Device according to one of claims 83 to 86, characterized characterized in that a local temperature rise in one an initial area of the gel electrophoresis webs corresponding transverse areas of the gel matrix can be set. 88. Device according to one of claims 83 to 87, characterized in that a laser arrangement ( 50 , 52 , 54 , 56 ) is provided for exciting a plurality of fluorescent markers assigned to the molecules,
the laser arrangement in electrophoresis mode generating laser radiation which, as at least one laser beam ( 60 , 62 , 64 , 66 ) running essentially transversely to the molecular propagation direction, cuts at least several of the gel electrophoresis tracks ( 12 ) in order to detect in a detection area of the respective gel To excite fluorescent markers of the molecules present there, assigned to the electrophoresis path,
wherein a local temperature increase can be set in a transverse region of the gel matrix that encloses the respective detection region. 89. Device according to one of claims 83 to 88, characterized by the remaining features of at least one of the preceding Claims 1 to 76. 90. A method for operating an electrophoresis device ( 10 ) for analyzing labeled molecules, in particular biological molecules, which comprises:

• - a plurality of substantially parallel to each other and along a molecule propagation direction (M) running gel electrophoresis tracks (12) extending in a gel matrix (12), possibly a slab gel (12),
• - At least one with a gel matrix surface in heat exchange connection, through which a heat exchanger flowable thermostatic plate ( 16 b), which is suitable for producing a uniform temperature distribution in the gel matrix ( 12 ) in the gel electrophoresis pathways cutting transverse direction to the molecular propagation direction (M) or / and maintain,
• an energy source ( 20 ) for applying an electrical potential to a start region and an end region of the gel electrophoresis tracks ( 12 ),

characterized by the step of interrupting the flow of the heat exchanger means through the thermostat plate ( 16 b) preheated by the heat exchanger means after reaching a predetermined / predeterminable gel temperature or / and a predetermined / predeterminable gel temperature distribution. 91. The method according to claim 90, characterized in that after interrupting the flow in the gel matrix ( 12 ) there is a temperature gradient in the direction of molecular propagation (M) and / or in a thickness of the gel matrix associated with the thermostatic plate ( 16 b) substantially orthogonal direction sets and / or that a temperature profile with a local temperature increase or a local temperature decrease occurs in at least one transverse region of the gel matrix. 92. The method according to claim 90 or 91, characterized in that two thermostatic plates ( 16 d1, 16 d2) are provided, between which the gel matrix ( 12 d) is arranged. 93. The method according to claim 89 or 90, characterized in that a temperature profile (T) with in the direction of molecular propagation (M) decreasing or increasing temperature is set. 94. The method according to any one of claims 91 to 93, characterized in that, based on the direction associated with the thickness of the gel matrix ( 12 d), a temperature profile (T) is set with a temperature maximum lying in a central region of the gel matrix ( 12 d) . 95. The method according to any one of claims 91 to 94, characterized characterized in that a local temperature rise in one an initial area of the gel electrophoresis webs corresponding cross-section of the gel matrix is set.   96. The method according to any one of claims 91 to 95, characterized in that a laser arrangement ( 50 , 52 , 54 , 56 ) is used to excite a plurality of fluorescent markers assigned to the molecules,
the laser arrangement in electrophoresis mode generating laser radiation which, as at least one laser beam ( 60 , 62 , 64 , 66 ) running essentially transversely to the molecular propagation direction, cuts at least several of the gel electrophoresis tracks ( 12 ) in order to detect in a detection area of the respective gel To excite fluorescent markers of the molecules present there, assigned to the electrophoresis path,
wherein a local temperature increase is set in a transverse region of the gel matrix that encloses the respective detection region. 97. The method according to any one of claims 90 to 96, that is applied to a device ( 10 ) according to one of claims 1 to 76 with a thermostatic plate ( 16 b) through which a heat exchanger means can flow. 98. A method for analyzing nucleic acids, characterized in that products of nucleic acid sequencing reactions labeled with at least four different fluorescent markers are separated together using a device ( 10 ) according to one of claims 1 to 76 and 83 to 89 according to their size and are analyzed independently of one another, if desired including the method according to one of claims 77 to 82 or / and the method according to one of claims 90 to 97.