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WO1992003575A1 - Procede de preparation, d'isolation et de sequencage de polynucleotides - Google Patents

Procede de preparation, d'isolation et de sequencage de polynucleotides Download PDF

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Publication number
WO1992003575A1
WO1992003575A1 PCT/GB1991/001379 GB9101379W WO9203575A1 WO 1992003575 A1 WO1992003575 A1 WO 1992003575A1 GB 9101379 W GB9101379 W GB 9101379W WO 9203575 A1 WO9203575 A1 WO 9203575A1
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WO
WIPO (PCT)
Prior art keywords
sequencing
strand
preparing
fragment
conducted
Prior art date
Application number
PCT/GB1991/001379
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English (en)
Inventor
David Stephen Charnock Jones
Julian Paul Schofield
Mark Vaudin
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Medical Research Council
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Medical Research Council filed Critical Medical Research Council
Publication of WO1992003575A1 publication Critical patent/WO1992003575A1/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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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 of preparing, isolating and sequencing polynucleotides.
  • the method is particularly useful for isolating and sequencing polynucleotide fragments prepared by a modified polymerase chain reaction (PC ) technique that is disclosed in International patent application PCT/GB91/00803.
  • PC polymerase chain reaction
  • PCR polymerase chain reaction
  • Higuchi depends on the phosphorylation of one of the PCR primers which then targets the strand to be digested, following PCR, by ⁇ exonuclease III.
  • Other workers have used P end-labelled PCR or internal primers prior to the sequencing step ( rischnik et al ['1987] NAR .15 529, Wong et al [1987] Nature 330 384) .
  • direct sequencing from low-melt agarose has been conducted (Kretz et al [1989] NAR 12 5864). Accordingly, current techniques for the preparation, isolation and sequencing of nucleic acid fragments include a first step of amplifying a target nucleic acid fragment
  • the Hultman method was devised for the sequencing of polynucleotide fragments.
  • the method yields a purified polynucleotide fragment attached to a solid support, which can then be readily sequenced by standard Sequenase (trade mark) or Taquenase (trade mark) methodology.
  • the Hultman method uses streptavidin coated magnetic beads to immobilise biotin labelled polynucleotide fragments. Once the fragments are immobilised, any unwanted products and artifacts can be easily removed by washing the beads. The fragments can thus be isolated and purified by a straightforward and simple procedure.
  • the Hultman method of immobilisation includes the following 6 steps:
  • preparingmagneticbeads containingcovalently coupled streptavidin e.g. Dynabeads M280- Streptavidin, which can be obtained from Dynal AS, Norway
  • preparing a target double stranded (ds) DNA fragment e.g. from a plasmid or a sample of genomic DNA
  • the binding of the biotin label can be performed by either a filling-in of nucleotide overhangs of a sticky- ended fragment with biotin-dUTP and polymerase (i.e. extension of the target strand) or by using a biotinylated primer that anneals to a universal cassette that is ligated to the target strand during an in vivo amplification operation (e.g. during an exponential PCR amplification) .
  • the fragments prepared by the alternative biotin binding methods have different orientations when immobilised on the beads.
  • the filling-in step using biotin-dUTP produces a biotin label at the 3 1 end of a strand of the target fragment. Therefore, when the target strand is immobilised on the beads, the 5' end of this target strand is distanced away from the beads.
  • the use of a biotin labelled primer that anneals to a ligated universal cassette produces a biotin label at the 5 ' end of a template strand of the target fragment. Therefore, when this target strand is immobilised, the 3' end of the target strand is distanced away from the beads.
  • the fragments to which the biotin labels bind can be prepared, for example, by PCR exponential amplification. After PCR amplification, the biotin labelled strands of the ' fragments can be bound to the beads.
  • the PCR products can be easily purified because the non-biotinylated strands of the fragments can be selectively removed by alkaline treatment. The purified polynucleotide fragment strand can then be sequenced.
  • the immobilised biotinylated polynucleic acid fragment strand is sequenced by a procedure including the following 12 steps:
  • the typical nucleotide concentrations and mixtures used in step 6 are 0.080 mM of each dNTP . 0.0063 mM of the respective ddNTP, 50 mM NaCl and 40 mM Tris-HCl (pH 7.5) .
  • the method requires large quantities of Taq polymerase, biotinylated primer and streptavidin beads.
  • the sequencing stage requires a labelled primer and a purified template.
  • Our method also allows the direct addition of ⁇ phage, ml3 phage or bacterial cells to the PCR reaction without any additional purification of the PCR template. Thus it is possible to circumvent the need for bacterial growth and DNA template preparation. This makes the whole process very amenable to automation.
  • the preparing step, the immobilising step and the (or at least part of the) treating step are conducted in the same vessel.
  • all of the sequencing reaction is conducted in the same vessel and the product is then sequenced on an automated machine, such as Du Pont's Genesis 2000 machine. This can be achieved by use of fluorescent dideoxy chain terminators.
  • the products of the treating step need only then be run on one gel in order to establish the sequential order of the nucleotides in the immobilised strand.
  • the polynucleic acid target ' fragment is prepared by a PCR amplification step,
  • the PCR amplification is conducted in a volume of between 5 ⁇ l and 20 ⁇ l, preferably lO ⁇ l.
  • one of the prirr.ers used in the step has attached thereto the separating label.
  • the separating label is attached to the 5' end of the primer.
  • the primer with the attached label is a primer that anneals to a specific region on the target fragment. This ensures that only the target fragment is isolated.
  • the label is biotin and the support matrix is a magnetic bead having streptavidin covalently coupled thereto.
  • streptavidin beads are particularly advantageously because harsh conditions (e.g. strong alkaline solutions) can be used to clean the immobilised fragments without the immobilised fragments becoming unbound from the beads.
  • the beads are added in the form of an aqueous dispersion to the amplification mixture.
  • a typical aqueous dispersion has a volume of 15 ⁇ l.
  • the labelled target fragment is a double stranded fragment, it can be denatured while it is bound to the support matrix.
  • the target fragment can be denatured prior to the addition of the support matrix.
  • a typical denaturation solution is 20 ⁇ l of 0.15M NaOH.
  • the fragments bound to the beads can then be washed with, approximately, 20 ⁇ l solutions of 0.15M NaOH and 40 ⁇ l solutions of H 2 0.
  • the purified fragments attached to the beads are dispersed in approximately 5-10 ⁇ l H 2 0 ( preferably 7.5 ⁇ l) and sequenced using approximately "5-12 ⁇ l of sequencing solution (preferably 7-10 ⁇ l) .
  • a typical sequencing solution comprises approximately 0.5 ⁇ l of lO ⁇ M forward primer, 2 ⁇ l of 5x Sequenase buffer, l ⁇ l of lOOmM DTT, 2 ⁇ l of labelling mix (radiolabelled dideoxy terminators or fluorescent dideoxy terminators), 0.5 ⁇ l of 5 S dATP and 2 ⁇ l of Sequenase; 4 ⁇ l of which is mixed with 2.5 ⁇ l of termination labelling mix.
  • the present method is suitable for a simple kit so that the method can be conducted on a laboratory workbench.
  • the present method can also be conducted by an automated machine, particularly as the amounts of reagents required are low. Also, such an automated machine could be supplied in kit form.
  • the present method has a considerable number of advantages over existing methods. It is quicker than conventional methods, it yields better quality sequences using either 35S and autoradiography or fluorescent di-deoxy chain terminators (e.g. with the Du Pont Genesis 2000TM sequencing machine) and it allows the rapid sequencing of any nucleic acid fragment, for example genomic DNA that can be, but need not be, cloned in a vector.
  • the present method makes it possible to prepare the template DNA, perform the sequencing reactions and run a sequencing gel in one working day.
  • the data obtained using the present method are of higher quality than those obtained from either conventional double or single stranded DNA sequencing.
  • the isolated target fragment template produced is extremely clean and therefore the sequence produced is also very clean. This is true both of 35 S-labelled DNA and fluorescently labelled fragments.
  • Step 1 Preparation by PCR amplification
  • the target fragment to be sequenced (which may be contained within ⁇ phage, M13 phage, yeast or bacterial cells) is added to the lO ⁇ l reaction mixture, which comprises l ⁇ l of lOx Cetus buffer, 0.5 ⁇ l of lO ⁇ M biotinylated reverse primer, l ⁇ l of 2mM dNTPs and 0.04 units of Taq polymerase. H 2 0 is then added so that the total volume is lO ⁇ l.
  • the mixture is overlayed with 10-2O ⁇ l light paraffin oil, then heated at 95°C for 0.3 min, 55°C for 0.5 min and 72°C for 1 min for a total of 35 cycles, followed by heating at 72°C for 3 min.
  • the beads are washed four times ith TE/NaCl.
  • the beads are incubated at room temperature for 5 min with 20 ⁇ l of 0.15M NaOH. The beads are then washed with 20 ⁇ l of 0.15M NaOH (if desired, the paraffin can be wholly or partially removed at this stage) and then three times with 40 ⁇ l of H 2 0.
  • the beads are resuspended in a total volume of 7.5 ⁇ l of H 2 0, to which suspension is added 0.5 ⁇ l of lO ⁇ M forward primer and 2 ⁇ l of 5x Sequenase buffer. The mixture is incubated at 65°C for 2 min, then ice cooled for 1 min. Next, I ⁇ l of lOOmM DTT, 2 ⁇ l of diluted labelling mix (1:5 for longer sequences, 1:20 for shorter ones), 0 5 ⁇ l of 35 SdATP (0.5 ⁇ Ci) and 2 ⁇ l of diluted Sequenase (1:8) are added.
  • the mixture is then incubated at room temperature for 5 min. 4 ⁇ l is transferred to the termination mixes (2.5 ⁇ l). The mixture is incubated at 37°C for 5 min and then washed with 2O ⁇ l TE. Afterwards, the supernatant is removed and 4 ⁇ l of formamide dye mix is added. Following incubation at 80°C for 5 min, the mixture is spun and the supernatant removed. A sample (e.g. 3-4 ⁇ l) of the supernatant is then loaded on a buffer gradient sequencing gel.
  • a 96-prong replica-plated comb transfers bacterial cells into the wells of a thermostable polycarbonate microtiter plate.
  • a programmed robotic workstation dispenses lO ⁇ l of PCR mixture (200 ⁇ M dNTP's, 1.5mM MgCl 2 , 50mM Tris-HCl pH 8.3, 0.01% gelatin, l ⁇ M each of M13 Universal forward and reverse sequencing primers, 0.8 Units Taq polymerase USB W ) into the microtiter wells and, following plating, overlays the solution with 40 ⁇ l light mineral oil.
  • the plate is incubated on a modified programmable ther ocycler adapted to accommodate such plates.
  • the target DNA is then exposed by heating the solution to 95°C for 2 minutes prior to 30 cycles of amplification (95°C - 0.5 min, 55°C - 0.5 min, 72°C - 1 min).
  • This present method allows the presence and size of inserts in 96 clones to be determined within 4 hours.
  • the present method includes the small-scale amplification of DNA template in which one of the PCR primers has a biotin group at its 5' prime end. This allows a single strand of DNA to be purified by binding it to streptavidin-coated magnetic beads and washing with alcohol. Because the reactions are carried out on a small scale, the amount of Taq polymerase enzyme required is kept to a minimum, thus reducing cost.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Molecular Biology (AREA)
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  • Physics & Mathematics (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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Abstract

Procédé de préparation, d'isolation et de séquençage ordonnés d'un brin d'acide polynucléotidique, lequel procédé consiste: (1) à préparer une solution d'un fragment cible d'acide polynucléique auquel est fixé un marqueur de séparation; (2) à mélanger la solution avec une matrice de support à laquelle est fixé un groupe apte à former une liaison coopérative avec le marqueur de séparation; (3) à immobiliser le fragment d'acide polynucléique sur la matrice de support par la formation d'une liaison entre le marqueur de séparation et ledit groupe; (4) à purifier le brin immobilisé; et (5) à traiter ce brin immobilisé pour en permettre le séquençage. Les étapes de préparation et d'immobilisation, ainsi qu'au moins une partie de l'étape de traitement s'effectuent dans un même vase.
PCT/GB1991/001379 1990-08-14 1991-08-14 Procede de preparation, d'isolation et de sequencage de polynucleotides WO1992003575A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9017788.2 1990-08-14
GB909017788A GB9017788D0 (en) 1990-08-14 1990-08-14 Method for preparing,isolating and sequencing polynucleotides

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WO1992003575A1 true WO1992003575A1 (fr) 1992-03-05

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5622824A (en) * 1993-03-19 1997-04-22 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
WO1998031833A1 (fr) * 1997-01-15 1998-07-23 Incyte Pharmaceuticals, Inc. Sequençage d'acides nucleiques a l'aide de terminateurs pouvant etre captures en phase solide
US6194144B1 (en) 1993-01-07 2001-02-27 Sequenom, Inc. DNA sequencing by mass spectrometry
US6949633B1 (en) 1995-05-22 2005-09-27 Sequenom, Inc. Primers useful for sizing nucleic acids
US6991903B2 (en) 1992-11-06 2006-01-31 Sequenom, Inc. Solid phase sequencing of double-stranded nucleic acids
US7005518B2 (en) 2002-10-25 2006-02-28 Li-Cor, Inc. Phthalocyanine dyes
US7198893B1 (en) 1996-11-06 2007-04-03 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US7319003B2 (en) 1992-11-06 2008-01-15 The Trustees Of Boston University Arrays of probes for positional sequencing by hybridization
EP1967592A1 (fr) 1995-06-07 2008-09-10 Solexa, Inc. Procédé d'amélioration de l'efficacité de séquençage de polynucléotide
USRE41005E1 (en) * 1996-11-06 2009-11-24 Sequenom, Inc. Beads bound to a solid support and to nucleic acids
US9255293B2 (en) 2010-11-01 2016-02-09 Gen-Probe Incorporated Integrated capture and amplification of target nucleic acid for sequencing
EP3091053A1 (fr) 2006-05-19 2016-11-09 Li-Cor, Inc. Imagerie optique fluorescente utilisant des colorants de cyanine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0371437A2 (fr) * 1988-11-29 1990-06-06 Orion-Yhtymà„ Oy Procédé et combinaison de réactif pour déterminer des séquences nucléotides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0371437A2 (fr) * 1988-11-29 1990-06-06 Orion-Yhtymà„ Oy Procédé et combinaison de réactif pour déterminer des séquences nucléotides

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOTECHNIQUES vol. 9, no. 1, July 1990, EATON PUBL. CO.,NATICK,MA,USA pages 32 - 36; B.F. KOOP ET AL.: 'Sequencing reactions in microtiter plates' see the whole document *
CLINICAL CHEMISTRY. vol. 35, no. 11, November 1989, WINSTON US pages 2196 - 2201; L.J.MCBRIDE ET AL.: 'Automated DNA sequencing methods involving Polymerase Chain Reaction' see the whole document esp. abstract *
DNA SEQUENCE vol. 1, no. 4, 1991, HARWOOD ACADEMIC PUBLISHERS, UK pages 279 - 283; D.S.C. JONES ET AL.: 'Fluorescent and radioactive solid phase dideoxy sequencing of pcr products in microtiter plates' see the whole document *
NUCLEIC ACIDS RESEARCH. vol. 17, no. 13, 1989, ARLINGTON, VIRGINIA US pages 4938 - 4946; T. HULTMAN ET AL.: 'Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support' cited in the application see the whole document *
NUCLEIC ACIDS RESEARCH. vol. 17, no. 22, 25 November 1989, ARLINGTON, VIRGINIA US page 9498; J.P. SCHOFIELD ET AL.: 'A rapid semi-automated microtiter plate method for analysis and sequencing by PCR from bacterial stocks' see the whole document *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6991903B2 (en) 1992-11-06 2006-01-31 Sequenom, Inc. Solid phase sequencing of double-stranded nucleic acids
US7319003B2 (en) 1992-11-06 2008-01-15 The Trustees Of Boston University Arrays of probes for positional sequencing by hybridization
US6194144B1 (en) 1993-01-07 2001-02-27 Sequenom, Inc. DNA sequencing by mass spectrometry
US5622824A (en) * 1993-03-19 1997-04-22 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US5851765A (en) * 1993-03-19 1998-12-22 Sequenon, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US5872003A (en) * 1993-03-19 1999-02-16 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6949633B1 (en) 1995-05-22 2005-09-27 Sequenom, Inc. Primers useful for sizing nucleic acids
EP1967592A1 (fr) 1995-06-07 2008-09-10 Solexa, Inc. Procédé d'amélioration de l'efficacité de séquençage de polynucléotide
US7198893B1 (en) 1996-11-06 2007-04-03 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US7501251B2 (en) 1996-11-06 2009-03-10 Sequenom, Inc. DNA diagnostics based on mass spectrometry
USRE41005E1 (en) * 1996-11-06 2009-11-24 Sequenom, Inc. Beads bound to a solid support and to nucleic acids
USRE44693E1 (en) 1996-11-06 2014-01-07 Sequenom, Inc. Beads bound to a solid support and to nucleic acids
WO1998031833A1 (fr) * 1997-01-15 1998-07-23 Incyte Pharmaceuticals, Inc. Sequençage d'acides nucleiques a l'aide de terminateurs pouvant etre captures en phase solide
US7005518B2 (en) 2002-10-25 2006-02-28 Li-Cor, Inc. Phthalocyanine dyes
EP3091053A1 (fr) 2006-05-19 2016-11-09 Li-Cor, Inc. Imagerie optique fluorescente utilisant des colorants de cyanine
US9255293B2 (en) 2010-11-01 2016-02-09 Gen-Probe Incorporated Integrated capture and amplification of target nucleic acid for sequencing

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Publication number Publication date
GB9017788D0 (en) 1990-09-26

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