FR2558262A1 - METHOD AND SYSTEM FOR ELECTROPHORESIS ANALYSIS OF DNA FRAGMENTS OBTAINED BY SEQUENCING - Google Patents

Abstract

THE INVENTION CONCERNS A PROCESS FOR THE ELECTROPHORESIS ANALYSIS OF DNA FRAGMENTS PRODUCED IN DNA SEQUENCING OPERATIONS, IN WHICH A SET OF FOUR CHROMOPHORES OR FLUOROPHORES IS USED TO MARK DNA FRAGMENTS PRODUCED BY THE CHEMISTRY OF SEQUENCING, WHICH ALLOWS THE DETECTION AND CHARACTERIZATION OF FRAGMENTS WHEN THEY ARE SOLVED BY ELECTROPHORESIS ON A GEL. THE INVENTION ALSO RELATES TO A SYSTEM FOR THE ANALYSIS OF DNA FRAGMENTS COMPRISING: A SOURCE OF DNA FRAGMENTS MARKED WITH A CHROMOPHORINE OR A FLUOROPHORINE; ZONES FOR CONTAINING THE GEL FOR ELECTROPHORESIS; A DEVICE FOR THE INTRODUCTION OF TRADEMARKED DNA FRAGMENTS IN THE SAID AREA; AND A PHOTOMETRIC DEVICE TO CONTROL MARKED DNA FRAGMENTS AS THEY MOVE THROUGH THE GEL.

Description

The development of reliable methods for the analysis of DNA sequences

  (deoxyribonucleic acid) and RNA (ribonucleic acid) was one of the keys to the success of recombinant DNA technology and genetic engineering. When used with other techniques of modern molecular biology, the sequencing of a

  nucleic acid allows the dissection and analysis of

  animal, plant and viral forms in discrete genes with a defined chemical structure. Since the function of a biological molecule is determined by its structure, defining the structure of a gene is crucial for

  the possible manipulation of this basic unit of information

  heredity, in a useful way. As soon as genes

  can be isolated and characterized, they can be mod-

  they are designed to produce desired changes in their structures, allowing the production of gene products - proteins - with properties different from those possessed by the original proteins. The

  microorganisms in which the natural or syn-

  theories are placed, can be used as chemical "factories" to produce large quantities of proteins

  such as interferon, the growth hormone

  and insulin. Plants can receive the

  genetic training that allows them to survive in harsh environmental conditions or to produce their

own fertilizer.

  The development of modern methods of

  nucleic acid quenches has involved

  parallels in various techniques. One of these is the development of simple and reliable methods for the cloning of small to medium sized DNA cords into bacterial plasmids, bacteriophages and small animal viruses. This allows the production of pure DNA in sufficient quantities for its chemical analysis. Another of these developments is the perfection approach for gel electrophoresis methods, useful for resolution separation of oligonucleotides from

  their dimension. But the main development is the introduction

  production of suitably sized assemblies of cloned, purified DNA fragments which contain, in their length collection, the necessary information

  to define the sequence of nucleotides comprising the

  parent DNA lecules. Two separate methods of producing these sets of fragments are of general use, one is developed by Sanger (Sanger F., Nicklen S. and Coulson AR, Proc Natl Acad Sci USA 74, 5453 (1977)). and Smith AJH, Methods in Enzymology 65, 56-580 (1980)), the other by

  Maxam and Gilbert (Maxam A.M. and Gilbert W., Methods in Enzy-

65, 499-559 (1980)).

  The process developed by Sanger is called the dideoxy chain termination method. In the most common variation of this method, a segment of DNA is cloned into a single-stranded phage DNA such as M13. These phage DNAs can serve as copies for the primed synthesis of the complementary bead by the Klenow fragment of DNA

  I. The initiator is either a synthetic oligonucleotide

  This is a restriction fragment isolated from the parental recombinant DNA that specifically hybridizes with a region of the M13 vector near the 3 'end of the insert.

  clone. In each of the 4 sequencing reactions, the syn-

  initiated thesis is carried out in the presence of a sufficient quantity

  dideoxy analogue of one of the 4 deoxynucleotides

  possible, so that the longer chains are terminated

  born by the incorporation of these nucleotides "fer-

  The relative concentration of dideoxy deoxy forms

  is adjusted to provide a range of eventualities of

  corresponding to all chain lengths pos-

  sibles that can be solved by gel electrophoresis.

  The products of each of the 4 primed synthesis reactions are then separated on individual plots of polyacrylamide gels by electrophoresis. Radioactive markers incorporated into the stretching chains are used to develop an autoradiographic image of the DNA type in

  each electrophoretic trace. The sequence of deoxynoxides

  cleotides in ulcerated DNA is determined from an examination

  the type of bands in the 4 trajectories.

  The process developed by Maxam and Gilbert uses

  reads a chemical treatment of purified DNA to produce

  appropriate size sets of analogous DNA fragments

  to those produced by the Sanger process. This mode

  allows the sequencing of single or double-stranded DNA,

  labeled with a radioactive phosphate at the 3 'or 5' end.

  In four reaction sets, one or two of the four nucleotide bases are cleaved by chemical treatment. The cut involves a process in three stages: modification of the base, elimination of the modified base to party. of its sugar and splitting of the chain on this sugar. The reaction conniments are adjusted from

  most of the fragments marked at their extremes.

  mitted, generated, in a range of dimensions (typical-

  from 1 to 400 nucleotides) which can be solved by electrophoresis on ge.L electrophoresis, autoradiography

  and trace analysis are performed essentially

  me in the process of Sanger. (Although fragmentation

  chemically generates two DNA fragments each

  that once it takes place, only the fragment containing the

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  end marker is detected on the autoradiogram).

  These two methods of DNA sequencing are widely used and each has several variations. For each of them, the length of the sequence that can be obtained from a simple set of reactions, is mainly

  limited by the resolution of the polyacrylamide gels used

  electrophoresis. Typically from 200 to 400 bases

  can be read from a simple set of gel plots.

  Although satisfactory, these two methods have serious drawbacks, mainly associated problems.

  in the procedure of electrophoresis. One of the problems

  This is because of the need to use a radiolabel as a tracer for the location of the DNA bands in the gels. The short half-life period must be taken into account

  phosphorus 32 and consequently the instability of the

  radioactive labeling, and the problems posed by

  disposal and handling of radioactive products.

  More importantly, the nature of autoradiography (the

  age on film of a band of radioactive gel is wider than

  the band itself) and the comparison of the positions of the

  between the four different gel patterns (which may or may not behave in a uniform manner with respect to band mobilities) may limit the observed resolution of the bands and hence the length of the sequence that can be read from the gels. In addition, irregularities

  tracing can make it difficult to scan the car radio.

  grams - the human eye can currently better compensate

  these irregularities that computers do. This requirement

  Manual "reading" of autoradiograms takes time, is painful and prone to errors. In addition, it is not possible to read the traces of the gels during the course of the electrophoresis, in order to be able to finish the electrophoresis when the resolution becomes insufficient to separate the adjoining bands, but we must finish

  electrophoresis at a specific moment and wait for the development

  Development of the autoradiogram before you can read the sequence. The invention relates to these and other problems associated with the electrophoresis operation in DNA sequencing methods and is considered to be representative of

  significant progress in the art.

  In summary, the invention provides a novel

  assigned for electrophoresis analysis of DNA fragments

  products in DNA sequencing operations, in the-

  a set of 4 chromophores or fluorophores are used to mark the DNA fragments produced by the sequencing chemistry and to allow detection and characterization of the fragments when resolved by gel electrophoresis. Detection involves the use of an absorption or fluorescent photometer capable of controlling the bands

  marked moving through the gel.

  The invention also includes a new system

  DNA fragment analysis system, consisting of: a source of DNA fragments labeled with a chromophore or a fluorescent agent, these fragments being labeled differently,

  an area for containing an electrophobic gel

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  a mechanism for the introduction of frag-

  labeled DNA in said area, and a photometric device for monitoring or detecting labeled DNA fragments as they move

in and are separated by the gel.

  The object of the invention is to provide a new

  method for the analysis of the DNA sequence.

  The invention also aims to provide

  a new system for the analysis of DNA fragments.

  More specifically, the object of the invention is to

  to provide an improved method for the analysis of the

quence of DNA.

  Other goals and benefits will appear at the

  reading of the following description, which refers to the

  FIG. 1 is an illustration of one of the ways of marking the end of a DNA fragment with a fluorescent marker. Pst I and T4 DNA ligase are enzymes commonly used in the study of recombinant DNA

  Figure 2, an overall diagram of a sequence

  automated DNA probe, with gel electrophoresis system; FIG. 3 is a diagram of an optical configuration in the detection unit. P, light source; Ll, lens of the lens; L2, collimator lens; F1, filter stopping UV; F2, filter stopping heat; F3, bandpass excitation filter; F4, emission filter

  a long passage; DM, dichroic mirror; C, poly-gel

  acrylamide; PMT, photomultiplier tube; FIG. 4 is a diagram of another optical configuration in the detection unit. F1 to F4 are band pass filters, centered at the maximum emission of

  different dyes. P1 to P4 are photomultiplier tubes

  ers. The excitation light has a wavelength that is not transmitted by any of the filters F1 to F4; Figure 5 is a comparison of the type of data produced by DNA sequencing of the sequence illustrated in Figure 1;

  Figure 6, an overall diagram of a sequence

  preferred DNA core according to the invention.

  Concepts of labeled primers usable with the Sanger process. In prior 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 implies that the sets of fragments produced in the 4 synthesis reactions are processed on separate gel plots and causes problems associated with the comparison of band mobilities in the different plots. This problem is solved in the invention using a set of 4 chromophores or fluorophores with

  different absorption or fluorescence maxima,

  tively. Each of these markers is chemically coupled to the initiator used to initiate the synthesis of the fragment chains. Each marked initiator is in turn paired

  with one of the dideoxynucleotides and used in the reaction

  synthetic synthesis initiated with the Klenow fragment of DNA polymerase. Primers must have the following characteristics. 1) They must have a 3 'hydroxyl group for

  allow extension of the chain by the polymerase.

  2) They must be complementary to a single region 3 'of the cloned insert. 3) They must be long enough to hybridize to form a single, stable duplex. 4) The chromophore or fluorophore should not interfere with hybridization or prevent extension at the 3 'end by

polymerase.

  Conditions 1, 2 and 3 above are fulfilled by several synthetic oligonucleotide primers which are generally used for Sanger sequencing using M13 vectors. Such an initiator is 12 mer 'TCA CGA CGT TGT 3' in which A, C, G and T represent the 4 different nucleotide constituents of the DNA:

  A, adenosine; C, cytosine; G, guanosine; T, thymidine.

  Chemistry for the coupling of chromo-

  phores or fluorophores is described in the patent application

  No. 565,010, filed December 20, 1983. The technique employs

  is to introduce an aliphatic amino group at i. The 5 'end as the last addition in the synthesis of the oligonucleotide initiator. This reactive amino group can then be easily coupled with a wide variety of chromophores and / or amino-reactive fluorophores. This means allows the labeled primers to fulfill condition 4 above.

  The 4 dyes used must have coeffi-

  high quenching ficients and / or reasonably high quantum yields for fluorescence. They must have maximum absorption and / or maximum emission

  well resolved. As representatives of a game of four of these

  amino-reactive agents, include: fluorescein isothio-

  cyanate (FITC, kEx = 495, Em = 520, e × 104) max eosin isothiocyanate eosin (EITC, XEx = 522, λm = 543, max 4 max 8x10 4), tetramethyl rhodamine isothiocyanate (TMRITC, Emax = 550maxEm = 578, 55x105 and the Max max 550 'rhodamine isothiocyanate substituted (XTITC, λex = 580 Em max 4 Emax = 604, λ580 X 8x10) in which A represents the length wave in nanometers, Ex is the excitation, Em is the emission, max is the maximum and ó is the coefficient

molar extinction.

  These dyes are attached to the M13 initiator and the conjugate product is electrophoresed on a gel

  at 20% polyacrylamide. The marked initiators are visi-

  both by their absorption and their fluorescence in

  the freeze. The four marked initiators have electricity mobilities

  identical trophoretics. Primers conjugated with a

  dyes retain their ability to hybridize specifically

  with DNA, as demonstrated by their ability to

  place the non-derivatized oligonucleotide, normally used

  in sequencing reactions.

  Marking a DNA end for use in the

Maxam / Gilbert process.

  In the DNA sequencing method of Maxam / Ä358262

  Gilbert, the end of the DNA fragment that is to be

  undermine the sequence must be marked. This is conventionally carried out enzymatically using radioactive nucleotides. To use the Maxam / Gilbert method with the dye detection scheme described in this invention, the DNA fragment must be labeled with dyes. One way to achieve the labeling is shown in Figure 1. Some restriction endonucleases generate what is known as a protrusion

  3 ', as a DNA cleavage product. These enzymes

  produce a "cohesive end", a short extension of single-stranded DNA at the end of a bi-catenary DNA fragment. This region will form an annular structure with complementary extension of DNA, which can be covalently joined to the duplex DNA with the enzyme ligase. In this way, one of the chains is covalently linked to a detectable unit. This unit can be a dye, an amino group or a protected amino group (which could be deprotected and react with a dye after

chemical reactions).

Sequencing reactions.

  The dideoxy sequencing reactions are carried out

  killed in a standard way, Smith A.J.H., Methods in Enzy-

  mology 65, 56-580 -1980), except that the scale may be increased if necessary to provide adequate signal strength in each band for detection. The reactions are conducted using a different colored initiator for each of the reactions, for example FITC for reaction "A", EITC for reaction "C", TMRITC for reaction "G" and XRITC for reaction "T". No radiolabelled nucleoside triphosphate needs to be included in the

sequencing reaction.

  Maxam / Gilbert sequencing reactions are routinely performed, Gill SF, Aldrichimica et al., Acta 16 (3), 59-61 (1983), except that the end marker is either one of the 4 dyes, either a free or protected amino group which can subsequently react with the dye. Gel electrophoresis. Aliquots of the reactions are combined

  sequencing and loaded into a column of poly-

  5% acrylamide, from the upper reservoir 12, as shown in FIG. 2. The relative amounts of the 4 different reactions in the mixture are adjusted empirically to give about the same intensity of

  fluorescence or absorption signal for each of the

  DNA / dye conjugates. This makes it possible to compensate for the differences in the extinction coefficients of the dyes, the fluorescence quantum yields of the dyes, the sensitivities of detection and others. We apply a tension

  column 10 so as to subject the fragments to

  DNA fragments electrophoresed through the gel.

  Labeled DNA segments differing in length from a single nucleotide are separated by electrophoresis in this gel matrix. In the bottom or near the bottom of the gel column 10, the DNA strips are resolved one through the other and pass through the detector 14 (described in more detail in the following paragraph). The detector 14 detects the fluorescent or chromophoric bands of DNA in the gel and determines their color and therefore which nucleotide they correspond to. This information gives the DNA sequence. Detection. There are different ways of detecting labeled molecules that have been separated according to their length

  by means of polyacrylamide gel electrophoresis.

  Four illustrative modes are described below. These are i) the detection of the fluorescence excited by a light of different wavelength for the different dyes, ii) the detection of the fluorescence excited by a light of the same wavelength for the different dyes, iii) l 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 must fulfill the following conditions. (a) The excitation light beam shall not have a height substantially greater than the height of the strip. This is normally between 0 and 0.5 mm. The use of such a narrow excitation beam makes it possible to obtain a maximum resolution of the bands. b) The

  excitation wavelength can be modified to correct

  respond to the absorption maxima of each of the different dyes or may be a narrow, high intensity, light band that excites the four fluorophores and

  does not cover any fluorescence emission. (c) The confi-

  optical gating must minimize the excision light

  diffuse and reflected to the photodetector.

  optical filters to stop the scattered excitation light

  and reflect reflect at the same time as the excitation wavelength. d) The phiotodetector 14 should have a fairly low noise level and a good spectral response, with quantum efficiency over the entire emission range of

  dyes (from 500 to 500 μm for the dyestuffs indicated below).

  above. e) The optical system for the collection of the

  fluorescence emitted must have a high numerical aperture

  Vee. This maximizes the fluorescence signal. In addition, the

  depth of field these collection optics must

  take all the lar (geur of the matrix of the column.

  Two fluorescence detection systems are illustrated in FIGS. 3 and 4. The system shown in FIG. 3 is compatible with single-wavelength excitation or with multiple-length excitation.

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  wave. For single wavelength excitation, the

  filter F4 is one of the four band pass filters,

  at the emission wavelength of the peak of each of the

  dyes. This filter is switched every few seconds

  to allow continuous monitoring of each of the four fluorophores. For multi-wavelength excitation, the optical elements F3 (excitation filter), DM (mirror

  dichroque) and F4 (barrier filter) are switched together.

  In this way, the excitation light and the emission light observed are modified. The system shown in Figure 4 is a good arrangement for the case of single wavelength excitation. This system presents

  the advantage of not requiring any moving parts and the fluo-

  rescence of the four dyes can be monitored simultaneously

  continuously. A third mode (iii above) of detection consists in eluting the labeled molecules in

  the bottom of the gel, combine them with an agent for excitement

  chemiluminescence such as 1,2-dioxethane-dione, Gill S.K., Aldrichimica Acta 16 (3), 59-61 (1983); Mellbin G.J., Liq. Chrom. 6 (9), 1603-1616 (1983), and send the mixture directly into a detector that can measure the light emitted for the four separate wavelengths (this detector is similar to that shown in FIG.

  4, but without requiring a source of excitation light).

  The background signal in chemiluminescence is much lower than in fluorescence, which results in higher signal-to-noise ratios and increased sensitivity. Finally, the determination can be obtained by light absorption measurements (iv above). In this case, a light beam of variable wavelength passes through the gel and the decrease in beam intensity is measured.

  due to the absorption of light by the labeled molecules.

  By measuring the absorption of light at different

  wavelengths corresponding to the maximum absorption of

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  four dyes, it is possible to determine the molecule

  dye in the path of light.

  A disadvantage of this type of measurement is that the absorption measurements are inherently less sensitive than the fluorescence measurements. The detection system described above is connected to a computer 16. In each time interval examined, the computer 16 receives a signal proportional to the intensity of the signal measured each time for each of the

  four colored markers. This information indicates the number

  cleotide that ends the DNA fragment of the length par-

  particular in the observation window at this given moment.

  The time sequence of the stained bands gives the DNA sequence. FIG. 5 shows the type of data obtained by conventional methods, as well as the type of data obtained by the improvements described in the invention.

  The following example is intended only to illustrate

the invention.

Example.

  Figure 6 is a block diagram of a DNA sequencer usable with a dye each time. The beam (4880 A) of an argon ion laser 100 passes through the polyacrylamide gel (sample) tube 102 by means of a beam guide 104. The fluorescence excited by the beam is collected using a crystal lens.

  low focal length 106, passes through an appropriate set of threads

  very optics 108 and 110 to eliminate the excitation light

  diffuse and is detected by means of a photomulti

  112. The signal is easily detected on a tape recorder. DNA sequencing reactions are performed using an oligonucleotide initiator

  labeled with fluorescein. The peaks on the diagram correspond to

  fluorescein-labeled DNA fragments of different lengths, synthesized in

  sequencing and separated in the gel tube by electrophoresis.

  Each peak contains 10-15 to 10-16 moles of fluorescein,

  which is approximately equal to the amount of DNA obtained

  band-like in an equivalent sequencing gel using a radioisotope detection. This proves that the marker

  fluorescent is not separated or degraded from the amorphous

  oligonucleotide in sequencing reactions. This-

  it also demonstrates that the sensitivity of the detection is quite adequate to perform a sequence analysis

of DNA by this method.

  The invention having been described in detail, it is

  obvious that we can make changes without

  shot from his mind, nor get out of his frame.

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Claims (17)

  1. Procea. of DNA sequencing by the technique   termination device, characterized in that the oligonucleotide   initiator cleotide is labeled with a colored tracer.   2. Method of sequencing DNA by the technique   terminating agent, characterized in that the oligonucleotide   initiator cleotide is labeled with a fluorescent tracer.   3. Method of sequencing by the chemical degradation technique, characterized in that the molecules   of DNA are labeled with a colored tracer.   4. A method of sequencing DNA by the chemical degradation technique, characterized in that the molecules   of DNA are labeled with a fluorescent tracer.   5. Method of sequencing by the technique of   chain termination; characterized in that the oligonucleotide   tide initiator uses in each of the four reactions of   Sequencing, A, C, G c 'T, has a different colored tracer.   fixed on him, e. in that aliquots of the above-described sequence reactions are combined and subjected together to polyacrylamide gel electrophoresis and   detected after their repair on the gel.   6. Sequencing procedure by the technique of   chain termination, characterized in that the oligonucleotide   The initiator used in each of the four sequencing reactions A, C, G and T has a different fluorescent tracer attached to 11X and aliquots of the above sequencing reactions are combined.   subjected to polyacryl gel electrophoresis   amide and detected stopped their separation on the gel.   7. DNA sequencing method by the chemical degradation technique, characterized in that the DNA molecules are labeled 7-sec different color tracers, and a different colored DNA is used in each of the reactions   chemical modification, and aliquots of the reaction   sequencing procedures above are combined and submitted   appears to be electrophoresis on a polyacrylamide gel and   detected after their separation from the gel.   8. A method of sequencing DNA by the chemical degradation technique, characterized in that the DNA molecules are labeled with different fluorescent tracers, and a different fluorescent DNA is used in each   chemical modification reactions, and   quantizes of the above sequencing reactions are combined and electrophoresed together on a   polyacrylamide and detected after their separation from the gel.   9. A method of sequencing DNA by the chemical degradation technique, characterized in that the DNA molecules are labeled with an amino group which is coupled with   a dye molecule after the sequencing reactions.   A method of sequencing DNA by the chemical degradation technique, characterized in that the DNA molecules are labeled with a protected amino group which is unblocked and coupled with a dye molecule after the sequencing reactions.   11. The method of claim 9, characterized   rised that the products of each of the different   sequencing sequences are coupled with a different dye,   aliquots of the labeled reaction with a colo-   are combined and electrophoresed on a polyacrylamide gel and detected after separation on the freeze.   12. Process according to claim 10, characterized   in that the products of each of the different reactions   sequencing rents are coupled with a different dye   Aliquots of the dye-labeled reaction are combined and subjected to polyacrylamide gel electrophoresis and detected after separation. on the gel.   2z58262 A novel system for analyzing DNA fragments comprising: a source of DNA fragments labeled with a chromophore or a fluorescent agent; a zone for containing the gel for electrophoresis, a device for introducing the labeled DNA fragments into said zone; and a photometric device for monitoring the labeled DNA fragments as they move through the gel. The novel system of claim 13, wherein the photometric device is a photometer absorption.   15. New system according to claim 13, wherein the photometric device is a photometer fluorescence.   The novel system of claim 13, wherein the DNA fragments are labeled with a group   amino which is coupled with a dye molecule.   17. The novel system of claim 16, wherein the DNA fragments have different color tracers.