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WO1993025709A1 - Preparation d'acides nucleiques - Google Patents

Preparation d'acides nucleiques Download PDF

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Publication number
WO1993025709A1
WO1993025709A1 PCT/GB1993/001223 GB9301223W WO9325709A1 WO 1993025709 A1 WO1993025709 A1 WO 1993025709A1 GB 9301223 W GB9301223 W GB 9301223W WO 9325709 A1 WO9325709 A1 WO 9325709A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
complementary
probe
nucleic acid
oligonucleotides
Prior art date
Application number
PCT/GB1993/001223
Other languages
English (en)
Inventor
Trevor Leonard Hawkins
Original Assignee
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
Priority to AU43440/93A priority Critical patent/AU4344093A/en
Publication of WO1993025709A1 publication Critical patent/WO1993025709A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0099—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor comprising robots or similar manipulators
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/005—Beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1081—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices characterised by the means for relatively moving the transfer device and the containers in an horizontal plane
    • G01N35/109—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices characterised by the means for relatively moving the transfer device and the containers in an horizontal plane with two horizontal degrees of freedom

Definitions

  • This invention relates to the preparation of nucleic acids and concerns magnetic particles and their use in the preparation of nucleic acids.
  • M13 phage is used to obtain single stranded DNA for sequencing templates.
  • the traditional methods for M13 DNA purification such as polyethylene glycol (PEG)/phenol procedures (Bankier et al . , [1988], in Wu, R. [ed.] , Methods Enz. 155, 52-93) allow microgram quantities of template to be produced from illilitre volumes.
  • thermally cycled sequencing procedures utilising Taq polymerase (Craxton, [1991], Methods: A Companion to Methods in Enzymology 3_, 20-26) only require 200-500ng of - template DNA per reaction. Therefore traditional methods produce an excess of template for most purposes, which wastes both time and money.
  • the invention provides a composition comprising magnetic particles, each bearing a plurality of oligonucleotides, each oligonucleotide comprising a sequence, acting as a probe, which is complementary to a sequence under investigation, and further comprising a sequence which is non-complementary to the sequence under investigation.
  • the invention provides a method of producing the composition defined above, comprising attaching magnetic particles to an oligonucleotide comprising a complementary probe sequence and further comprising a non-complementary sequence.
  • the magnetic particles take the form of beads. Typically these are about 1-5um in diameter. Suitable magnetic beads are commercially available. For instance, they may be obtained from Dynal or, more preferably, from Promega. Those from Promega are found particularly suitable because they have a pitted surface and therefore a higher surface area, which allows greater ' amounts of oligonucleotides to be carried by the bead.
  • 'Oligonucleotides' as used herein includes both synthetic and native DNA and RNA sequences of any length.
  • the oligonucleotide is preferably single stranded DNA and, conveniently, the complementary probe sequence is substantially 25-45 bases long.
  • the non-complementary sequence is generally 5-15 bases in length, typically about 9 bases long.
  • the non-complementary sequence of the oligonucleotide acts as a 'linker' joining the complementary probe sequence to the magnetic bead.
  • the probe sequence may be attached by either end to the linker oligonucleotide.
  • the oligonucleotide may be attached to the magnetic bead by conventional techniques.
  • the linker oligonucleotide may conveniently be biotinylated at the end region distal to the end joined to the complementary probe sequence, allowing for ready attachment to streptavidin-coated magnetic beads.
  • Other attachment techniques are known to those skilled in the art.
  • the probe sequence can, of course, be selected so as to be complementary to any nucleic acid sequence of interest such that, for example, the sequence of interest may be separated from a complex sample by being bound by the magnetic bead/probe and then separated from the rest of the sample by magnetic attraction.
  • the invention provides a method of separating a nucleic acid sequence from a sample comprising: contacting a complex sample possibly including the sequence of interest with the composition of the present invention; allowing the sequence of interest, if present, to associate with the composition by means of hybridisation with the complementary probe sequence; and separating the composition and any bound nucleic acid sequences from the rest of the sample by magnetic attraction.
  • the complementary probe sequence comprises an oligonucleotide complementary to the sequence of M13.
  • the invention therefore provides a method of preparing template DNA for sequencing.
  • the probe sequence is complementary to a region upstream from the M13 -21 universal primer site.
  • the arrangement described above can confer a number of advantages over prior art methods of purifying nucleic acids.
  • the use of a non-complementary linker sequence reduces the likelihood of steric hindrance between the comparatively bulky magnetic bead and the "target" DNA hybridised to the probe. This allows beads prepared in accordance with the invention to bind far more 'target' DNA than possible hitherto.
  • probe sequences may become separated from magnetic beads of the prior art by shearing and may act as unwanted "false" primers in subsequent sequencing reactions.
  • the non-complementary linker oligonucleotide of the invention ensures that, should the oligonucleotides become separated from the beads, they are far less likely to be able to prime chain-extension reactions. It is a preferred feature therefore that the linker sequence should be at the 3' end of the complementary probe sequence, as this further reduces the risk of non-specific priming.
  • the invention provides a method of preparing a DNA sequence of interest from a complex sample comprising: causing lysis of DNA-containing entities by the action of heat in the presence of a suitable detergent; adding magnetic particles attached to a probe complementary to at least part of the sequence of interest together with hybridisation buffer containing a polymer to cause an increase in the effective nucleic acid concentration; allowing the sequence of interest to hybridise to the complementary probe and then separating the particle/probe complex and any sequences hybridised thereto from the rest of the sample by magnetic attraction.
  • any magnetic particles to which are attached an appropriate probe are suitable for performing the method defined above, including those already known in the art. However, it is preferred that the particles used are those according to the present invention.
  • a suitable detergent is SDS.
  • the DNA-containing entities are typically phages.
  • a suitable polymer is PEG.
  • the use of a polymer to increase the effective concentration of nucleic acid allows smaller volumes of reagents to be used than previously, which in turn enables the method to be performed in a microtitre plate or tray. Similarly, the method is particularly simple and efficient as it employs a target sequence-specific purification step.
  • the invention provides a method of preparing DNA by means of a sequence-specific purification step, capable of being performed in a microtitre plate.
  • the ability to perform the method in a microtitre plate makes the method of the invention particularly amenable to automation.
  • Suitable automated apparatus for performing the method of the invention is described in GB9212164.9 (co-pending Application No PCT/GB93/ ).
  • Figure 1 shows the base sequence of a suitable probe/linker oligonucleotide
  • Figure 2 shows a photograph of gel electrophoresis performed on DNA prepared by the method of the invention.
  • Figure 3 shows a portion of sequencing trace
  • the invention involves an oligonucleotide probe which has been synthesised with a biotin group at the 3' end.
  • the probe is designed to be complementary to a region upstream from the M13 -21 Universal priming site.
  • single plaques may be grown up in culture, the cells harvested and the supernatant collected and lysed to yield free single strands.
  • the bead/probe complex is then added, and the probe allowed to anneal to the target DNA. Once bound, the bead/probe/template complex can be separated from the rest of the sample using magnetic attraction and then washed.
  • the template may then be freed from the bead/probe complex by heating.
  • the procedure is simple and fast. All the post-growth steps can be carried out in microtitre plates with no centrifugation or ethanol precipitations required. In this example, 250 random M13 sub-clones were prepared and se ⁇ uenced using the method outlined below.
  • the amount of template recovered depends directly on the design of the probe.
  • the probe must be specific to the target but must not act as a secondary sequencing primer if free probe is left in solution. Consequently, the probe is 41bp in length and binds to a region upstream from the M13 -21 Universal primer site.
  • the probe has a run of several 'A's at the 3' end together with the biotin group. This acts as a linker arm to prevent steric hindrance between the large streptavidin-coated beads and the binding of the probe to the target. Also the high degree of non-complementarity at the 3 * end would prevent the free probe from acting as a sequencing primer should it shear from the beads.
  • the design of the probe is shown in Figure 1, ('B' represents Biotin).
  • the probe THM13.3 was synthesised on an ABI 380B DNA synthesiser on a 1uMole scale. Biotin phosphoramidite was obtained frm Amersham U.K. The sequence of the probe was: 5' TAT CGG CCT CAG GAA GAT CGC ACT CCA GCC AGC AAA AAA Biotin A 3' (Seq ID No. 1). Following cleavage from the column, the oligonucleotide was deprotected in ammonia at 55°C overnight. A NAP-10 column was used to purify the crude oligonucleotide. Probe/streptavidin bead linkage
  • Promega nucleotide quality beads were used in this example. 1.2ml of Promega beads were used per 12 samples. The beads were washed in 0.1M NaCl three times using a neodymium-iron-boron permanent magnet to separate the beads from the washing solution. 200ul 0.1M NaCl and 1On ol THM13.3 oligonucleotide were then added to 1.2ml( 1.2mg) dry beads and incubated at room temperature for 10 minutes. The beads were then washed 10 times in 0.1M NaCl to remove unbound oligonucleotide. Bead/probe complex was finally taken up in 1.2ml water. Beads may be bound to probe in bulk and stored in storage buffer at 4°C.
  • Random M13 sub-clones with 1-2kb inserts were grown up in 2ml 2xTy medium (16g bacto tryptone, 10g yeast extract, 5g NaCl in 11 water) for 5 hours at 37°C. Cells were spun down at 14,000g for 5 minutes and the supernatant transferred to microtitre plates. 400ul supernatant was used from each sample, using two wells, each containing 200ul supernatant (Falcon 3911 MicroTest Flexible Assay Plate. )
  • the microtitre plate was removed from the cycler and placed on a Dynal MPC-96 magnetic separator. After 30 seconds the supernatant was aspirated, 100ul wash buffer (0.1X SSC) were added to each well and the beads were moved through the wash buffer by repositioning the plate over the magnetic separator three times at intervals of five seconds. This step was repeated a total of three times. Finally, the beads were eluted in 10ul water; using the magnet, beads may be dispersed into this small volume.
  • 100ul wash buffer 0.1X SSC
  • the templates were released from the probe by heating the plate to 80 C for 3 minutes. After heating, the beads were concentrated using the magnet and the supernatant was removed to a fresh microtitre plate. Due to evaporation the final total volume was approximately 8ul from each well, or approximately 16ul per DNA template sample.
  • FIG. 2 The efficacy of the technique is illustrated by Figure 2.
  • Four random M13 subclones were grown as described. The cells were harvested and the supernatant (1200ul) was split three ways to provide three identical samples. A, B and C (400ul each). For each sub clone. Sample A was prepared using the method outlined previously. Sample B was prepared as outlined but leaving out the hybridisation buffer (20% PEG/2.5M NaCl). Sample C was prepared as outlined but using magnetic beads without a probe attached. The samples were then subjected to agarose gel electrophoresis. This showed that when the PEG is removed there is a dramatic reduction in yield, as expected. When beads are used without probe attached the resulting yield is zero. The difference between this control and the method of Alderton et al., (1992), is that in sample C the phage are lysed before addition of PEG.
  • beads may be re-used as outlined below.
  • Beads were collected and pooled in a 1.5ml eppendorf tube. The supernatant was removed and the beads were resuspended in 1ml reuse buffer (0.15M NaOH, 0.001% Tween 20) for 1 minute. The supernatant was removed and the procedure repeated. Finally the beads were washed once in storage buffer and taken up in half the original volume of storage buffer (PBS pH 7.5, 0.1% BSA). Beads were then stored at 4°C.
  • a preferred feature of the method of preparing DNA is to bring about magnetic attraction for the magnetic beads by the use of a "dot magnet".
  • a typical microtitre assay plate generally 130 mm by 85 mm
  • the dot magnet comprises an array of 96 such rare earth magnets which are positioned in a matrix 8 x 12 and are separated by about 9mm from each nearest neighbour (ie. substantially the same spacing as that between the wells of a microtitre plate) thus when the sides of the dot magnet are aligned with the sides of a typical microtitre plate, each rare earth magnet is positioned beneath a well in the microtitre plate.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

L'invention se rapporte à une composition comprenant des particules magnétiques, dont chacune porte une multiplicité d'oligonucléotides, chaque nucléotide comprenant une séquence agissant comme une sonde, qui est complémentaire d'une séquence étudiée, et comprenant en outre une séquence qui n'est pas complémentaire de la séquence étudiée. Un procédé de purification d'acides nucléiques à l'aide d'une composition de l'invention est également décrit, ainsi qu'un procédé de préparation d'un acide nucléique.
PCT/GB1993/001223 1992-06-09 1993-06-09 Preparation d'acides nucleiques WO1993025709A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU43440/93A AU4344093A (en) 1992-06-09 1993-06-09 Preparation of nucleic acids

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9212164.9 1992-06-09
GB929212164A GB9212164D0 (en) 1992-06-09 1992-06-09 Preparation of nucleic acids

Publications (1)

Publication Number Publication Date
WO1993025709A1 true WO1993025709A1 (fr) 1993-12-23

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PCT/GB1993/001223 WO1993025709A1 (fr) 1992-06-09 1993-06-09 Preparation d'acides nucleiques
PCT/GB1993/001222 WO1993025912A2 (fr) 1992-06-09 1993-06-09 Preparation automatique d'acides nucleiques

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Application Number Title Priority Date Filing Date
PCT/GB1993/001222 WO1993025912A2 (fr) 1992-06-09 1993-06-09 Preparation automatique d'acides nucleiques

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AU (2) AU4343993A (fr)
GB (1) GB9212164D0 (fr)
WO (2) WO1993025709A1 (fr)

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WO1993025912A2 (fr) 1993-12-23
AU4343993A (en) 1994-01-04
WO1993025912A3 (fr) 1994-03-17
GB9212164D0 (en) 1992-07-22
AU4344093A (en) 1994-01-04

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