GB2155176A

GB2155176A - Sequencing DNA

Info

Publication number: GB2155176A
Application number: GB08500960A
Authority: GB
Inventors: Leroy E Hood; Michael W Hunkapiller; Lloyd M Smith; Tim J Hunkapiller
Original assignee: California Institute of Technology
Current assignee: California Institute of Technology
Priority date: 1984-01-16
Filing date: 1985-01-15
Publication date: 1985-09-18
Also published as: DE3501306C2; DE3501306A1; FR2558262B1; SE8500201D0; SE456348B; SE8500201L; GB2155176B; GB8500960D0; CA1258611A; FR2558262A1

Abstract

A method of sequencing DNA comprises sequencing the DNA by producing DNA fragments therefrom labelled with a colored or fluorescent tag, separating the fragments by gel electrophoresis and determining the relative positions of the fragments on the gel. This method may be effected utilising a system comprising: a source of tagged DNA fragments; a zone for containing an electrophoresis gel; means for introducing the tagged DNA fragments to said zone; and photometric means for monitoring said tagged DNA fragments as they move through said gel.

Description

SPECIFICATION Sequencing DNA The development of reliable methods for sequence analysis of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) has been one of the keys to the success of recombinant DNA and genetic engineering.

When used with the other techniques of modern molecular biology, nucleic acid sequencing allows dissection and analysis of animal, plant, and viral genomes into discrete genes with defined chemical structure. Since the function of a biological molecule is determined by its structure, defining the structure of a gene is crucial to the eventual manipulation of this basic unit of hereditary information in useful ways.

Once genes can be isolated and characterized, they can be modified to produce desired changes in their structure that allow the production of gene products-- proteins--with different properties than those possessed by the original proteins. Microorganisms into which the natural or synthetic genes are placed can be used as chemical "factories" to produce large amounts of scarce human proteins such as interferon, growth hormone, and insulin. Plants can be given the genetic information to allow them to survive harsh environmental conditions or produce their own fertilizer.

The development of modern nucleic acid sequencing methods involved parallel developments in a variety of techniques. One was the emergence of simple and reliable methods for cloning small to medium-sized strands of DNA into bacterial plasmids, bacteriophages, and small animal viruses. This allowed the production of pure DNA in sufficient quantities to allow its chemial analysis. Another was the near perfection of gel electrophoretic methods for the high resolution separation of oligonucleotides on the basis of their size. The key conceptual development, however, was the introduction of methods of generating size-nested sets of fragments cloned, purified DNA that contain, in their collection of lengths, the information necessary to define the sequence of the nucleotides comprising the parent DNA molecules.Two distinct methods of generating these fragment sets are in widespread use, one developed by Sanger; Sanger, F., Nicklen, S. and Coulson,A. R. Proc. Natl.Acad. Sci. USA74, 5463 (1977) and Smith, A. J. H. Methods in Enzymology65, 56-580 (1980); the other by Maxam and Gilbert; Maxam, A. M. and Gilbert, W. methods in Enzymology 65, 499-559 (1980).

The method developed by Sanger is referred to as the dideoxy chain termination method. In the most commonly used variation of this method, a DNA segment is cloned into a single-stranded DNA phage such as M13. These phage DNAs can serve as templates for the primed synthesis of the complementary strand by the Klenowfragment of DNA polymerase I. The primer is either a synthetic oligonucleotide or a restriction fragment isolated from the parental recombinant DNA that hybridizes specifically to a region of the M13 vector near the 3' end of the cloned insert. In each of four sequencing reactions, the primed synthesis is carried out in the presence of enough of the dideoxy analog of one of the four possible deoxynucleotides so that the growing chains are randomly terminated by the incorporation of these "dead-end" nucleotides.The relative concentration of dideoxy to deoxy forms is adjusted to give a spread of termination events corresponding to all the possible chain lengths that can be resolved by gel electophoresis. The products from each of the four primed synthesis reactions are then separated on individual tracks of polyacrylamide gels by the electrophoresis. Radioactive tags incorporated in the growing chains are used to develop an autoradiogram image of the pattern of the DNA in each electrophoresis track. The sequence of the deoxynucleotides in the cloned DNA is determined from an examination of the pattern of bands in the four lanes.

The method developed by Maxam and Gilbert uses chemical treatment of purified DNA to generate size-nested sets of DNA fragments analogous to those produced by the Sanger method. Single or double-stranded DNA, labeled with radioactive phosphate at either the 3' or 5' end, can be sequenced by this procedure. In four sets of reactions, cleavage is induced at one or two of the four nucleotide bases by chemical treatment. Cleavage involves a three-stage process: modification of the base, removal of the modified base from its sugar, and strand scission at that sugar. Reaction conditions are adjusted so that the majority of end-labeled fragments generated are in the size range (typically 1 to 400 nucleotides) that can be resolved by gel electrophoresis.The electrophoresis, autoradiography, and pattern analysis are carried out essentially as is done for the Sanger method. (Although the chemical fragmentation necessarily generates two pieces of DNA each time it occurs, only the piece containing the end label is detected on the autoradiogram.) Both of these DNA sequencing methods are in widespread use, and each has several variations. For each, the length of sequence that can be obtained from a single set of reactions is limited primarily by the resolution of the polyacrylamide gels used for electrophoresis. Typically, 200 to 400 bases can be read from a single set of gel tracks. Although successful, both methods have serious drawbacks, problems associated primarily with the electrophoresis procedure. One problem is the requirement of the use of radiolabel as a tag for location of the DNA bands in the gels.One has to contend with the short half-life of phosphorus-32, and hence the instability of the radiolabeling reagents, and with the problems of radioactive disposal and handling. More importantly, the nature of autoradiography (the film image of a radioactive gel band is broader than the band itself) and the comparison of band positions between four different gel tracks (which may or may not behave uniformly in terms of band mobilities) can limit the observed resolution of bands and hence the length of sequence that can be read from the gels. In addition, the track-to-track irregularities make automated scanning of th autoradiograms difficult--the human eye can presently compensate for these irregularities much better than computers can. This need for manual "reading" of the autoradiograms is time-consuming, tedious and error-prone.Moreover, one cannot read the gel patterns while the electrophoresis is actually being performed, so as to be able to terminate the electrophoresis once resolution becomes insufficient to separate adjoining bands, but must terminate the electrophoresis at some standardized time and wait for the autoradiogram to be developed before the sequence reading can begin.

The present invention addresses these and other problems associated with the electrophoresis step in the DNA sequencing procedures and is believed to represent a significant advance in the art.

Briefly, this invention comprises a novel process for the electrophoretic analysis of DNA fragments produced in DNA sequencing operations wherein a set of four chromophores or fluorophores are used to tag the DNA fragments produced by the sequencing chemistry and permit the detection and characterization of the fragments as they are resolved by electrophoresis through a gel. The detection employs an absorption or fluorescent photometer capable of monitoring the tagged bands as they are moving through the gei.

This invention also includes a novel system for the analysis of DNAfragments comprising: a source of chromophore or fluorescent tagged DNA fragments, said fragments being differently tagged, a zone for containing an electrophoresis gel, means for introducing said tagged DNA fragments to said zone; and photometric means for monitoring or detecting said tagged DNA fragments as they move through and are separated by said gel.

It is an object of this invention to provide a novel process for the sequence analysis of DNA.

It is another object of our invention to provide a novel system for the analysis of DNA fragments.

More particularly, it is an object of this invention to provide an improved process for the sequence analysis of DNA.

These and other objects and advantages of this invention will be apparent from the detailed description which follows taken in conjunction with the accompanying drawings.

Turning to the drawings: Figure 1 is an illustration of one means of end-labeling a DNA fragment with a fluorescent tag. Pst. I and T4 DNA ligase are enzymes commonly used in recombinant DNA research.

Figure 2 is a block diagram of automated DNA sequencer, gel electrophoretic system.

Figure 3 is a schematic diagram of an optical configuration in the detector unit. P, lamp source; L1, objective lens; L2, collimating lens; F1, UV blocking filter; F2, heat blocking filter; F3, band pass excitation filter; F4, long pass emission filter; DM, dichroic mirror; C, polyacrylamide gel; PMT, photomultiplier tube.

Figure 4 is a schematic diagram of another optical configuration in the detector unit. F1 to F4 are bandpass filters centered at the emission maximum of the different dyes. P1 to P4 are photomultiplier tubes. The excitation light is of a wavelength such that it is not transmitted through any of the filters F1 to F4.

Figure 5 is a comparison of the type of data produced by DNA sequencing of the sequence shown in Figure 1.

Figure 6 is a block diagram of a preferred DNA sequencer according to this invention.

Design of tagged primers for use with Sanger method In the previous methods of DNA sequencing, including those based on the Sanger dideoxy chain termination method, a single radioactive label, phosphorus-32, is used to identify all bands on the gels. This necessitates that the fragment sets produced in the four synthesis reactions be run on separate gel tracks and leads to the problems associated with comparing band mobilities in the different tracks. This problem is overcome in the present invention by the use of a set of four chromophores or fluorophores with different absorption or fluorescent maxima, respectively. Each of these tags is coupled chemicaly to the primer used to initiate the synthesis of the fragment strands.In turn, each tagged primer is then paired with one of the dideoxynucleotides and used in the primed synthesis reaction with the Klenowfragment of DNA polymerase.

The primers must have the following characteristics. 1) They must have a free 3' hydroxyl group to allow chain extension by the polymerase. 2) They must be complementary to a unique region 3' of the cloned insert. 3) They must be sufficiently long to hybridize to form a unique, stable duplex. 4) The chromophore or fluorophore must not interfere with the hybridization or prevent 3'-end extension by the polymerase.

Conditions 1,2 and 3 above are satisfied by several synthetic oligonucleotide primers which are in general use for Sanger4ype sequencing utilizing M 13 vectors. One such primer is the 12 mer 5'TCA CGa CGT TGT 3' were A, C, G and T represent the four different nucleoside components of DNA; A, adenosine; C, cytosine; G, guanosine; T,thymidine.

The chemistry for the coupling of the chromophoric or fluorophoric tags is described in Application No.

8432205 filed 20th December 1984. The strategy used is to introduce an aliphatic amino group at the 5' terminus as the last addition in the synthesis of the oligonucleotide primer. This reactive amino group may then readily be coupled with a wide variety of amino reactive chromophores and/or fluorophores. This approach aids compatibility of the labeled primers with condition 4 above.

The four dyes used must have high extinction coefficients and/or reasonably high quantum yields for fluorescence. They must have well resolved absorption maxima and/or emission masima. A representative set of four such amino reactive dyes are: Ex isothiocyanage (FIT, Em fluorescein isothiocyanage (FlTC, A = 495, x = 520, e495 ~ 8 x 104), max max eosin isothiocyanate (EITC, A = 522, Em = 543 E522 = 8 x max max tetramethyl rhodamine isothiocyanate (TMRITC, Am = 550, = 578, E550 = 4 x 104), and max max substituted rhodamine isothiocyanate (XRITC,hEX = 580, A = 604, 580 = 8 x104) max max where A represents the wavelength in nanometers, Ex is excitation, Em is emission, max is maximum, and 8 is the molar extinction coefficient.

These dyes have been attached to the M13 primer and the conjugates electrophoresed on a 20% polyacrylamide gel. The labeled primers are visible by both their absorption and their fluorescence in the gel.

All four labeled primers have identical electrophoretic mobilities. The dye conjugated primers retain their ability to specifically hybridize to DNA, as demonstrated by their ability to replace the underivitized oligonucleotide normally used in the sequencing reactions.

End labeling of DNA for use with Maxam/Gilbert method In the Maxam/Gilbert method of DNA sequencing, the end of the piece of DNA whose sequence is to be determined must be labeled. This is conventionaly done enzymatically using radioactive nucleosides. In order to use the Maxam/Gilbert method in conjunction with the dye detection scheme described in this invention, the DNA piece must be labeled with dyes. One manner in which this may be accomplished is shown in Figure 1. Certain restriction endonucleases generate what is known as a 3' overhang as the product of DNA cleavage. These enzymes generate a "sticky end," a short stretch of single stranded DNA at the end of a piece of double stranded DNA. This region will anneal with a complementary stretch of DNA, which may be covalentlyjoined to the duplex DNA with the enzyme ligase.In this manner one of the strands is covalently linked to a detectable moiety. This moiety may be a dye, an amino group or a protected amino group (which could be deprotected and reacted with dye subsequent to the chemical reactions).

Sequencing reactions The dideoxy sequencing reactions are performed in the standard fashion Smith, A. J. H., Methods in Enzymology 65, 56--580 (1980), except that the scale may be increased if necessary to provide an adequate signal intensity in each band for detection. The reactions are done using a different color primer for each different reaction, e.g., FlTC for the "A" reaction, ElTCforthe "C" reaction, TMRITC for the "G" reaction, and XRITC for the "T" reaction. No radiolabeled nucleoside triphosphate need be included in the sequencing reaction.

The Maxam/Gilbert sequencing reactions are performed in the usual manner, Gill, S. F. Aldrichimica Acta 16(3), 59-61 (1983), except that the end label is either one or four colored dyes, or a free or protected amino group which may be reacted with dye subsequently.

Gel electrophoresis Aliquots of the sequencing reactions are combined and loaded onto a 5% polyacrylamide column 10 shown in Figure 2 from the upper reservoir 12. The relative amounts of the four different reactions in the mixture are empirically adjusted to give approximately the same fluorescence or absorptive signal intensity from each of the dye DNA conjugates. This permits compensation for differences in dye extinction coefficients, dye fluorescence quantum yields, detector sensitivities and so on. A high voltage is placed across the column 10 so as to electrophorese the labeled DNA fragments through the gel. The labeled DNA segments differing in length by a single nucleotide are separated by electrophoresis in this gel matrix.At or nearthe bottom of the gel column 10, the bands of DNA are resolved from one another and pass through the detector 14 (more fully described in the next section). The detector 14 detects the fluorescent or chromophoric bands of DNA in the gel and determines their color, and therefore to which nucleotide they correspond. This information yields the DNA sequence.

Detection There are many different ways in which the tagged molecules which have been separated by length using polyacrylamide gel electrophoresis may be detected. Four illustrative modes are described below. These are i) detection of the fluorescence excited by light of different wavelengths for the different dyes, ii) detection of fluorescence excited by light of the same wavelength for the different dyes, iii) elution of the molecules from the gel and detection by chemiluminescence, and iv) detection by the absorption of light by the molecules. In modes i) and ii) the fluorescence detector should fulfill the following requirements. a) The excitation light beam should not have a height substantially greater than the height of a band. This is normally in the range of 0.1 to 0.5 mm.The use of such a narrow excitation beam allows the attainment of maximum resolution of bands. b) The excitation wavelength can be varied to match the absorption maxima of each of the different dyes or can be a single narrow, high intensity light band that excites all four fluorophores and does not overlap with any of the fluorescence emission. c) The optical configuration should minimize the flux of scattered and reflected excitation light to the photodetector 14.The optical filters to block out scattered and reflected excitation light are varied as the excitation wavelength is varied. d) The photodetector 14 should have a fairly low noise level and a good spectral response and quantum efficiency throughout the range of the emission of the dyes (500 to 600 nm for the dyes listed above). e) The optical system for collection of the emitted fluorescence should have a high numerical aperture. This maximizes the fluorescence signal. Furthermore, the depth of field of the collection optics should include the entire width of the column matrix.

Two illustrative fluorescence detection systems are diagrammed in Figures 3 and 4. The system in Figure 3 is compatible with either single wavelength excitation or multi wavelength excitation. For single wavelength excitation, the filter F4 is one of four band pass filters centered at the peak emission wavelength of each of the dyes. This filter is switched every few seconds to allow continual monitoring of each of the four fluorophores. For multi wavelength excitation, the optical elements F3 (excitation filter), DM (dichroic mirror), and F4 (barrierfilter) are switched together. In this manner both the excitation light and the observed emission light are varied. The system in Figure 4 is a good arrangement for the case of single wavelength excitation.This system has the advantage that no moving parts are required, and fluorescence from all four of the dyes may be simultaneously and continuously monitored. A third approach (iii above) to detection is to elute the labeled molecules at the bottom of the gel, combine them with an agent for excitation of chemiluminescence such as 1,2 dioxetane dione, Gill, S. K. Aidrichimica Acta 16(3), 59-61(1983); Mellbin, G.

J. Liq. Chrom. 6(9), 1603-1616(1983), and and flow the mixture directly into a detector which can measure the emitted light at four separate wavelengths (this detector is similar to that shown in Figure 4, but without a need for an excitation light source). The background signal in chemiluminescence is much lowerthan in fluorescene, resulting in higher signal to noise ratios and increased sensitivity. Finally, the measurement may be made by measurements of light absorption (iv above). In this case, a light beam of variable wavelength is passed through the gel, and the decrease in the beam intensity due to absorption of light by the labeled molecules is measured. By measuring the absorption of light at the different wavelengths corresponding to the absorption maximum of the four dyes, it is possible to determine which dye molecule is in the light path.A disadvantage of this type of measurement is that absorption measurements are inherently less sensitive than fluorescence measurements.

The above-described detection system is interfaced to a computer 16. In each time interval examined, the computer 16 receives a signal proportional to the measured signal intensity at that time for each of the four colored tags. This information tells which nucleotide terminates the DNA fragment of the particular length in the observation window at that time. The temporal sequence of colored bands gives the DNA sequence. In Figure 5 is shown the type of data obtained by conventional methods, as well as the type of data obtained by the improvements described in this invention.

The following example is presented solely to ilustratethe invention.

Example Figure 6 shows a block diagram of a DNA sequenator for use with one dye at a time. The beam (4880 ) from an argon ion laser 100 is passed into the polyacrylamide gel tube (sample) 102 by means of a beamsteerer 104. Fluorescence exited by the beam is collected using a low f-number lens 106, passed through an appropriate set of optical filters 108 and 110 to eliminate scattered excitation light and detected using a photomultiplier tube (PMT) 112. The signal is readily detected on a strip chart recorder. DNA sequencing reactions are carried out utilizing a fluorescein labeled oligonucleotude primer. The peaks on the chart correspond to fragments to fluorescein labeled DNA of varying lengths synthesized in the sequencing reactions and separated in the gel tube by electrophoresis. Each peak contains the order of 10-15to 10-16 moies ffluorescein, which is approximately equal to the amount of DNA obtained per band in an equivalent sequencing gel utilizing radioisotope detection. This proves that the fluorescent tag is not removed or degraded from the oligonucleotide primer in the sequencing reactions. It also demonstrates that the detection sensitivity is quite adequate to perform DNA sequence analysis by this means.

Claims

1. A method of sequencing DNA, which method comprises sequencing the DNA by producing DNA fragments therefrom labelled with a colored or fluorescent tag, separating the fragments by gel electrophoresis and determining the relative positions of the fragments on the gel.

2. A method according to claim 1, wherein the sequencing is effected using the chain termination method.

3. A method according to claim 2, wherein a primer oligonucleotide is labelled with a colored tag.

4. A method according to claim 2, wherein a primer oligonucleotide is labelled with a fluorescent tag.

5. A method according to claim 1, wherein the sequencing is effected using the chemical degradation method.

6. A method according to claim 5, wherein the DNA to be analysed is labelled with a colored tag.

7. A method according to claim 5, wherein the DNA to be analysed is labelled with a fluorescent tag.

8. A method according to claim 5, wherein the DNA to be analysed is provided with an amino group at the 5'-terminus, which group is coupled to a dye molecule subsequent to the sequencing reactions.

9. A method according to claim 5, wherein the DNA to be analysed is provided with a protected amino group at the 5'-terminus, which group is deprotected and coupled to a dye molecule subsequent to the sequencing reactions.

10. A method according to any one of the preceding claims, wherein the four sets of DNA fragments which are produced in sequencing for A, C, G and T respectively carry different colored tags or different fluorescent tags and aliquots from each set are subjected to electrophoresis on the same gel.

11. A method according to claim 10, wherein the aliquots are combined and subjected to electrphoresis on the same gel.

12. A method according to claim 10 or 11, wherein the gel is a polyacrylamide gel.

13. A method according to any one of claims 1 to 4, wherein the sequencing is effected using the chain termination method, the respective primer oligonucleotides used in the sequencing reactions for A, C, G and T have different colored tags and aliquots from the four sequencing reactions are combined and subjected to electrophoresis together on the same polyacrylamide gel.

14. A method according to any one of claims 1 to 4, wherein the sequencing is effected using the chain termination method, the respective primer oligonucleotides used in the sequencing reactions for A, C, G and T have different fluorescent tags and aliquots from the four sequencing reactions are combined and subjected to electrophoresis together on the some polyacrylamide gel.

15. A method according to any one of claims 1 and 5 to 7, wherein the sequencing is effected using the chemical degradation method, the portions of DNA to be analysed in each chemical modification reaction are labelled with different colored tags and aliquots from the sequencing reactions are combined and subjected to electrophoresis together on the same polyacrylamide gel.

16. A method according to any one of claims 1 and 5 to 7, wherein the sequencing is effected using the chemical degradation method, the portions of DNAto be analysed in each chemical modification reaction are labelled with different fluorescent tags and aliquots from the sequencing reactions are combined and subjected electrophoresis together on the same polyacrylamide gel.

17. A method according to claim 8 or 9, wherein the products of each of the different sequencing reactions are coupled with a different color dye and aliquots of each dye-labelled product are combined and subjected to electrophoresis together on the same polyacrylamide gel and detected after their separation on the gel.

18. A method of sequencing DNA using DNA fragments labelled with a colored orfluorescenttag, said method being substantially as hereinbefore described.

19. A method of sequencing DNA substantially as herein before described with reference to any one of Figures 1 to 6 of the accompanying drawings.

20. In the method of DNA sequencing by the chain termination method; the improvement wherein the primer oligonucleotide is labeled with a colored tag.

21. In the method of DNA sequencing by the chain termination method; the improvement wherein the primer oligonucleotide is labeled with a fluorescent tag.

22. In the method of DNA sequencing by the chemical degradation method; the improvement wherein the DNA molecules are labeled with a colored tag.

23. In the method of DNA sequencing by the chemical degradation method; the improvement wherein the DNA molecules are labeled with a fluorescent tag.

24. In the method of DNA sequencing by the chain termination method; the improvement wherein the primer oligonucleotide used in each of the four sequencing reactions, A, C, G and T, has a different colored tag attached to it, and wherein aliquots of the aforesaid sequencing reactions are combined and electrophoresced together on polyacrylamide gel and detected after their separation on the gel.

25. In the method of DNA sequencing by the chain termination method; the improvement wherein the primer oligonucleotide used in each of the four sequencing reactions, A, C, G and T, has a different fluorescent tag attached to it, and wherein aliquots of the aforesaid sequencing reactions are combined and electrophoresed together on polyacrylamide gel and detected after their separation on the gel.

26. In the method of DNA sequencing by the chemical degradation method; the improvement wherein the DNA molecules are labeled with different colored tags, and a different colored DNA is used in each of the chemical modification reactions, and aliquots of the aforesaid sequencing reactions are combined and electrophoresed together on a polyacrylamide gel and detected after their separation of the gel.

27. In the method of DNA sequencing by the chemical degradation method; the improvement wherein the DNA molecules are labeled with different fluorescent tags, and a different fluorescent DNA is used in each of the chemical modificiation reactions, and aliquots of the aforesaid sequencing reactions are combined and electrophoresed together on a polyacrylamide gel and detected after their separation of the gel.

28. In the method of DNA sequencing by the chemical degradation method; the improvement wherein the DNA molecules are labeled with an amino group, which is coupled to a dye molecule subsequent to the sequencing reactions.

29. In the method of DNA sequencing of the chemical degradation method; the improvement wherein the DNA molecules are labeled with a protected amino group which is deblocked and coupled to a dye molecule subsequent to the sequencing reactions.

30. In the method of claim 28, the further improvement wherein the products of each of the different sequencing reactions are coupled with a different color dye, aliquots of the dye labeled reaction are combined and electrophoresed on a polyacrylamide gel and detected after their separation on the gel.

31. In the method of claim 29, the further improvement wherein the products of each of the different sequencing reactions are coupled with a different color dye, aliquots of the dye labeled reaction are combined and electrophoresed on a polyacrylamide gel and detected after their separation on the gel.

32. A novel system for the analysis of DNA fragments comprising: a source of chromophore or fluorescent tagged DNA fragments, a zone for containing an electrophoresis gel, means for introducing said tagged DNA fragments to said zone; and photometric means for monitoring said tagged DNA fragments as they move through said gel.

33. The novel system of claim 32, wherein the photometric means is an absorption photometer.

34. The novel system of claim 32, wherein the photometric means is a fluorescent photometer.

35. The novel system of claim 32, wherein the DNA fragments are labeled with an amino group which is coupled to a dye molecule.

36. The novel system of claim 35 wherein the DNA fragments have different colored tags.

37. Apparatus according to claim 32 substantially as hereinbefore described.

38. Apparatus for the analysis of DNA fragments, said apparatus being substantially as hereinbefore described in any one of Figures 2 to 4 or in Figure 6 of the accompanying drawings.