WO2001025479A2 - Sequençage multiplex a fluorescence - Google Patents

Sequençage multiplex a fluorescence Download PDF

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Publication number
WO2001025479A2
WO2001025479A2 PCT/EP2000/009763 EP0009763W WO0125479A2 WO 2001025479 A2 WO2001025479 A2 WO 2001025479A2 EP 0009763 W EP0009763 W EP 0009763W WO 0125479 A2 WO0125479 A2 WO 0125479A2
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Prior art keywords
sequencing
reaction
carried out
different
detection unit
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PCT/EP2000/009763
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German (de)
English (en)
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WO2001025479A3 (fr
Inventor
Wilhelm Ansorge
Vladimir Benes
Josef Stegemann
Robert Ventzki
Elisa Wurmbach
Konrad Faulstich
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Europäisches Laboratorium für Molekularbiologie (EMBL)
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Priority to AU10218/01A priority Critical patent/AU1021801A/en
Publication of WO2001025479A2 publication Critical patent/WO2001025479A2/fr
Publication of WO2001025479A3 publication Critical patent/WO2001025479A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to a method for analyzing nucleic acids which were generated in a nucleic acid sequencing reaction, in particular in an enzymatic sequencing reaction, using fluorescent dyes as a label.
  • a mixture of the dNTPs together with one ddNTP each is usually used in four batches.
  • a statistical incorporation of the chain termination molecules into the growing nucleic acid chains is achieved, and after the insertion of a chain termination molecule the DNA chain can no longer be extended due to the lack of a free 3'-OH group.
  • a large number of DNA fragments of different lengths are therefore created, which statistically contain a chain termination molecule at every possible installation point and end there.
  • DNA sequencing is performed using automated systems that generally use non-radioactive labeling, especially fluorescent labeling (Smith et al., Nature 321 (1 986), 674-679; Ansorge et al., J. Bioehem. Biophys. Meth. 1 3 (1 986), 31 5-323).
  • fluorescent labeling Smith et al., Nature 321 (1 986), 674-679; Ansorge et al., J. Bioehem. Biophys. Meth. 1 3 (1 986), 31 5-323
  • the separation of the nucleic acid fragments e.g. automated by gel electrophoresis, data acquisition and data evaluation.
  • An automated charging system is also available in some devices.
  • the sample preparation in particular the actual sequencing reaction, is carried out outside the system.
  • non-radioactive labeling groups can be introduced via labeled primer molecules, labeled chain termination molecules or as internal labeling via labeled dNTPs.
  • the sequencing reactions are carried out individually in a reaction vessel, so that only one sequence can be determined with a sequencing reaction.
  • the invention thus relates to a method for analyzing nucleic acids, which is characterized in that products of nucleic acid sequencing reactions labeled with at least four different fluorescent dyes are separated together in one lane according to their size and analyzed independently of one another.
  • the analysis is carried out by separating the nucleic acid sequencing products according to their size or length and evaluating the Mark.
  • This separation can be carried out by all methods known in the art, for example by various electrophoretic (for example polyacrylamide electrophoresis) and chromatographic (for example HPLC) methods, gel-electrophoretic separation, in particular in a denaturing gel, being preferred.
  • the separation is particularly preferably carried out in ultra-thin plate gels of 20 to 500 ⁇ m, preferably 1 00 to 300 ⁇ m thick (see, for example, Stegemann et al., Meth. Mol. Cell. Biol. 2 (1 991), 1 82-1 84) or capillaries.
  • the sequencing reactions are preferably evaluated in an automated sequencing device which contains a separate excitation light source and detection unit for each fluorescent dye.
  • Lasers are preferably used as excitation light sources. Examples suitable lasers are neodymium-YAG lasers, argon lasers, helium-neon lasers or semiconductor lasers (laser diodes).
  • spectral residual crosstalk i.e. an intensity ratio of the light originating from the fluorescent dye to be detected with the respective detector to the light of the other fluorescent dyes (to be detected on the other detectors) of 1: ⁇ 0.1, particularly preferably 1: ⁇ 0.01 and most preferably 1 : ⁇ 0.001 can be reached.
  • Suitable bandpass filters are preferably used to increase the detector selectivity, e.g. Absorption or / and interference filter.
  • a light which is essentially perpendicular to the detector with respect to the optical axis and which preferably has a maximum deviation of ⁇ 20 ° C. with respect to the optical axis of the detector can be generated.
  • this essentially perpendicular incidence of light can also be achieved by other optical means, e.g. Apertures etc. can be achieved.
  • the fluorescent dyes used for the method according to the invention are selected so that an independent analysis is possible.
  • the absorption and / or emission maxima of the individual fluorescent dyes are preferably selected such that they are at least 30 nm, preferably at least 50 nm and particularly preferably at least 70 nm apart from one another under the measurement conditions.
  • Dye combinations which contain at least four dyes selected from fluorescein, 5-carboxy-rhodamine 6G, Texas Red, CY2, CY 5, CY 5.5, IRD 700, IRD 800 and CY7 are particularly preferred. Most preferred are (a) a combination of the fluorescent dyes fluorescein, TexasRed, CY 5.5 and IRD 800, (b) a combination of 5-carboxy-rhodamine 6G, TexasRed, CY5.5 and IRD 800, optionally with CY2 or (c) one Combination of 5-carboxy-rhodamine 6G, TexasRed, CY5.5 and CY7 optionally with CY2 or / and IRD 800. The structures of these compounds are shown in Figure 1.
  • the sequencing reactions which lead to the production of products labeled with at least four different fluorescent dyes can be carried out in a conventional manner, ie in separate batches, the batches being combined only shortly before application to the gel web or in succession, if appropriate at intervals of e.g. 1 0 to 20 minutes on the gel sheet.
  • the simultaneous evaluation according to the invention of four or more fluorescent markings in a single lane also allows at least four independent sequencing reactions to be carried out in a single vessel and the resulting nucleic acid fragments labeled with different fluorescent dyes to subsequently be separated in a single lane.
  • the labeling groups are preferably introduced into the sequencing products via differently labeled primers.
  • a nucleic acid preferably a DNA
  • the length of the primer molecule is preferably 1 0 to 1 00, particularly preferably 1 2 to 50 and most preferably 1 2 to 30 nucleotides.
  • an unlabeled primer can also be used if the sequencing reaction is carried out under such Conditions are carried out that only a single type of labeled deoxyribonucleoside triphosphates is added to a primer molecule.
  • the nucleic acid fragments to be analyzed are generated in a single reaction vessel using four different labeled primers and four mutually independent DNA matrices.
  • the tandem duplex reaction also uses four differently labeled primers, but two each bind to complementary strands of a DNA template, i.e. there are a total of two DNA matrices.
  • the sequence gap filling reaction four primers and a DNA template are used, the primers each binding to different regions on a strand of the DNA template.
  • the competitive tandem duplex reaction uses four primers and a DNA template, with two primers binding to one strand and two other primers to the opposite strand of the template.
  • sequencing protocols can also be performed using more than four fluorescent dyes in a single reaction vessel, e.g. using five, six or seven fluorescent dyes, i.e. with a corresponding number of fluorescence-labeled primers or nucleotides.
  • the sequencing reaction is preferably carried out according to the enzymatic method.
  • Enzymes can be, for example, the Klenow fragment of E. coli DNA polymerase I, unmodified T7 DNA polymerase, modified T7 DNA polymerase (Sequenase), T4 DNA polymerase or thermostable DNA polymerases such as the Taq DNA polymerase Bst DNA polymerase or modifications thereof can be used.
  • the sequencing can be carried out as a "thermocycling" reaction. This reaction corresponds to a normal sequencing reaction, but - like a PCR - is carried out in several cycles.
  • the reaction mixture contains the nucleic acid matrix, the primer molecules, the dNTPs and the corresponding chain termination molecules as well as the thermostable polymerase. In this way, a certain amount of the labeled nucleic acid fragments is always synthesized per cycle, it being possible to generate large amounts of labeled fragments in several cycles.
  • the nucleic acid to be sequenced can be used both in single-stranded and in double-stranded form. Good results are obtained if the nucleic acid to be sequenced is on a double-stranded DNA vector, for example a plasmid, cosmid, bacteriophage, a viral vector or an artificial chromosome.
  • Another object of the invention is a reagent kit for carrying out the method according to the invention, which contains at least four different fluorescent dye labels in addition to other components required for sequencing, each label being intended for the determination of a different sequence.
  • the labels can be in the form of primers, dNTPs or chain termination molecules, for example ddNTPs. Different labeled primers are preferably used.
  • the reagent kit can contain additional reagents, e.g. enzymes, buffer solutions, unlabeled dNTPs, primers, terminators etc.
  • Figure 1 shows the chemical structure of the fluorescent dyes FITC, CY 5.5, 5-carboxy-rhodamine 6G, TexasRed, IRD 800, CY 5, IRD 700 and CY 2.
  • Either standard primers (UPO, UP-40, RPO, T7, T7term) or the specific primers 1 to 8 specified below were used as primers.
  • Suitable vectors with inserts to be sequenced were used as matrices for the respective primer.
  • the fluorescent dyes fluorescein isothiocyanate (FITC), TexasRed (TR), CY 5.5 and IRD 800 were used as labels.
  • thermocycling reactions (Murray et al., Nucleic Acids Res. 17 (1 989), 8889) using a thermal cycler (MJ Research, PTC-200) with the following profile: 25 or 40 cycles at 97 ° C for 1 5 sec, 55 ° C for 30 sec, 68 ° C for 60 sec or 30 sec.
  • 3 ⁇ l DNA template 1 (1 ⁇ g / ⁇ l) were mixed with 2 ⁇ l p DNA template 2 (0.5 ⁇ g / ⁇ l) and 3 ⁇ l each of the primers FITC-T7term (4 ⁇ M), TR-T7, CY 5.5-RPO and IRD 800-UPO (2 ⁇ M each), 2.1 ⁇ l 1x 0x cycling buffer, 3 ⁇ l AmpliTaqFS (Applied Biosystems) and 1.9 ⁇ l water mixed. The mixture was divided into four aliquots (5 ul each). 3 ⁇ l of the respective termination mixture (A, C, G, T) were added to the individual aliquots. Then thermal cycling was started. After 25 cycles, 1 ul stop solution was added to each reaction and the volume reduced by about half during denaturation at 95 ° C for about 10 minutes. Then the batch was put on hold.
  • DNA template 1 1.5 ⁇ l DNA template 1 (0.7 ⁇ g / ⁇ l) were mixed with 2 l each of primers 5 to 8 (2 ⁇ M each), 2, 1 ⁇ l 10 X cycling buffer, 3 ⁇ l AmpliTaqFS, 1, 5 ⁇ l DMSO and 4 , 9 ul water mixed. The mixture was divided into four aliquots (each
  • DNA template 1 4 ⁇ l of DNA template 1 (1 ⁇ g / ⁇ l) were each with 2 ⁇ l primers (FITC-RP, TR-40, RPO-CY 5.5, IRD 800-40, 2 A / M each), 2.0 ⁇ l 1 0 X Cycling buffer, 3 ⁇ l AmpliTaqFS and 3 ⁇ l water mixed. The mixture was divided into four aliquots (5 ⁇ l each) and 3 ⁇ l of the respective termination mixture (A, C, G, T) were pipetted into the individual aliquots. Then thermal cycling was started. After 40 cycles (extension at 68 ° C. for 60 seconds), 1 ⁇ l of stop solution was added to each reaction mixture and the volume was reduced to about half during a denaturation at 95 ° C. for about 10 minutes. Then the approaches were put on hold.
  • primers FITC-RP, TR-40, RPO-CY 5.5, IRD 800-40, 2 A / M each
  • 2.0 ⁇ l 1 0 X Cycling buffer 3 ⁇ l
  • the lasers were coupled into the gel via a single coupling plate at four different positions with a vertical distance of 10 mm between the beams.
  • the beam position was fine-tuned individually for all four lasers using a motorized optical stage.
  • optical bandpass filters were selected for the optimal transmission of the respective emission light spectra and the maximum suppression of scattered excitation laser light and of the infrared background due to glass and gel fluorescence.
  • the high selectivity of the transmission profile was achieved by a combination of absorption and multilayer interference filters (Schott, Mainz, Germany).
  • the light was collected and focused on the detectors by an optical system that provides a spatially continuous and upright image, high resolution and a high numerical aperture (i.e. minimal photon loss).
  • a gradient index lens array (Nippon Sheet Glass Company, Japan) was used to collect and image fluorescent light. 1 .3 Gel electrophoresis
  • the products of the sequencing reactions were separated on 5.0% or 4.3% Hydrolink Long Ranger denaturing gels (AT Bioehem, Malvern, PA, USA) with 42% urea.
  • Running and gel buffers were 1, 2 x TBE.
  • 100 ml of gel solution were polymerized by adding 84.5 ⁇ l of N, N, N ′, N′-tetramethylethylenediamine (TEMED) and 650 ⁇ l of 10% ammonium peroxodisulfate (APS) solution in water.
  • the gels were 0.30 mm thick.
  • the temperature was 45 ° C.
  • the separation was carried out over a distance of approximately 50 cm using 60 cm long and 34 cm wide glass plates.
  • the electrophoresis was carried out with a power of 60 W and an initial voltage of approximately 1,500 V.
  • Combs made of porous filter material and a robotic system were used to handle the large number of samples and the simultaneous loading of the 1 20 traces of a gel.
  • This method is described in detail by Erfle et al. (Nucleic Acids Res. 25 (1 997), 2229-2230) and includes loading the wells of a polyacrylic (Plexiglas) block with the sample suspension by a pipetting robot.
  • the porous material absorbs the 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 the electric field is applied, the samples are pulled out of the comb and into the gel.
  • the four lasers were switched on and off during an electrophoresis run and the associated change in the background was measured quantitatively.
  • the fluorescence quadruplex sequencing method allows the use of different strategies.
  • Four different matrices were thus prepared in one reaction vessel using four differently labeled primers (4-4-1), two different matrices with a tandem duplex sequence reaction in two directions and four differently labeled primers in one vessel (4-2-1). and sequence gap filling reactions using four "walking primers" to simultaneously close four gaps on the same matrix in a vessel (4-1-1).
  • sequence gap filling reactions using four "walking primers” to simultaneously close four gaps on the same matrix in a vessel (4-1-1).
  • With a resolution of 1000 bases per strand about 4000 bases per sequencing reaction could be determined by the techniques described. This means that 1 20 sequencing reactions can be separated on a single gel, thereby obtaining 1 20 kb information per gel.
  • the excitation of the fluorescent dyes and the collection of emission light took place spatially continuously over the width of the gel and temporally continuously over the entire duration.
  • the accuracy and readable sequence length showed no difference compared to standard sequencing reactions.
  • Each dye could be selectively excited and selectively detected.
  • a quantitative measurement of the background light showed that the influence of the lasers on the non-associated detectors was surprisingly extremely small.
  • a maximum increase in the background signal of 2.9% was measured with the IRD 800 detector due to the laser light used to excite CY 5.5. All other laser / detector combinations showed lower values. This insignificant signal increase remains constant during the electrophoresis run and is automatically subtracted from the measurement signal.
  • the fluorescence quadruplex sequencing method is also suitable for inner labels or dye-labeled terminators.
  • the dye FITC used in Example 1 was replaced by 5-carboxy-rhodamine 6G.
  • a neodymium-YAG laser with an excitation wavelength of 532 nm and a power of 5 to 10 mW was used.
  • selective excitation and selective detection of four dyes on a gel sheet was possible.
  • dye CY2 was used.
  • a neodymium YAG laser with a wavelength of 473 nm and a power of 5 to 10 mW was used as the excitation light source.

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Abstract

La présente invention concerne un procédé d'analyse d'acides nucléiques, qui sont générés dans une réaction de séquençage d'acides nucléiques, notamment dans une réaction de séquençage enzymatique au moyen de colorants fluorescents utilisés en tant que marqueurs.
PCT/EP2000/009763 1999-10-07 2000-10-05 Sequençage multiplex a fluorescence WO2001025479A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU10218/01A AU1021801A (en) 1999-10-07 2000-10-05 Fluorescent multiplex-sequencing

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19948260A DE19948260A1 (de) 1999-10-07 1999-10-07 Fluoreszenz-Multiplex-Sequenzierung
DE19948260.8 1999-10-07

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WO2001025479A2 true WO2001025479A2 (fr) 2001-04-12
WO2001025479A3 WO2001025479A3 (fr) 2002-06-20

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034114A1 (fr) * 1995-04-27 1996-10-31 Europäisches Laboratorium für Molekularbiologie (EMBL) Sequencage simultane d'acides nucleiques
WO1997007245A1 (fr) * 1995-08-14 1997-02-27 Ely Michael Rabani Procedes et dispositifs de sequençage multiplex parallele de polynucleotides
WO1997040184A1 (fr) * 1996-04-18 1997-10-30 Visible Genetics Inc. Procede, appareil et kits pour la mise en sequence d'acides nucleiques a l'aide de plusieurs colorants
WO1997041259A1 (fr) * 1996-05-01 1997-11-06 Visible Genetics Inc. Procede de sequencage de polymeres d'acide nucleique
US5861287A (en) * 1995-06-23 1999-01-19 Baylor College Of Medicine Alternative dye-labeled primers for automated DNA sequencing
WO1999002726A1 (fr) * 1997-07-11 1999-01-21 Brax Group Limited Caracterisation de l'acide nucleique

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034114A1 (fr) * 1995-04-27 1996-10-31 Europäisches Laboratorium für Molekularbiologie (EMBL) Sequencage simultane d'acides nucleiques
US5861287A (en) * 1995-06-23 1999-01-19 Baylor College Of Medicine Alternative dye-labeled primers for automated DNA sequencing
WO1997007245A1 (fr) * 1995-08-14 1997-02-27 Ely Michael Rabani Procedes et dispositifs de sequençage multiplex parallele de polynucleotides
WO1997040184A1 (fr) * 1996-04-18 1997-10-30 Visible Genetics Inc. Procede, appareil et kits pour la mise en sequence d'acides nucleiques a l'aide de plusieurs colorants
WO1997041259A1 (fr) * 1996-05-01 1997-11-06 Visible Genetics Inc. Procede de sequencage de polymeres d'acide nucleique
WO1999002726A1 (fr) * 1997-07-11 1999-01-21 Brax Group Limited Caracterisation de l'acide nucleique

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LI L ET AL: "An estimate of the crosstalk matrix in four-dye fluorescence-based DNA sequencing " ELECTROPHORESIS, vol. 20, no. 7, June 1999 (1999-06), pages 1433-1442, XP001053279 *
STEGEMANN J: "System for simultaneous sequencing of 240 clones with 5 dyes" FASEB JOURNAL, vol. 11, no. 9, 1997, page A1131 XP001053229 *
YAGER T ET AL: "High performance DNA sequencing, and the detection of mutations and polymorphisms, on the Clipper sequencer" ELECTROPHORESIS, vol. 20, no. 6, June 1999 (1999-06), pages 1280-1300, XP001053278 *

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DE19948260A1 (de) 2001-04-12
AU1021801A (en) 2001-05-10
WO2001025479A3 (fr) 2002-06-20

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